University of Southern California Dissertations and Theses

Breast cancer susceptibility gene 1: A role in transcriptional regulation

(USC Thesis Other)

Breast cancer susceptibility gene 1: A role in transcriptional regulation

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content INFORMATION TO USERS
This manuscript has been reproduced from the microfilm master. U M I films
the text directly from the original or copy submitted. Thus, some thesis and
dissertation copies are in typewriter face, while others may be from any type of
computer printer.
The quality of this reproduction is dependent upon the quality of the
copy submitted. Broken or indistinct print, colored or poor quality illustrations
and photographs, print bleedthrough, substandard margins, and improper
alignment can adversely affect reproduction.
In the unlikely event that the author did not send UM I a complete manuscript
and there are missing pages, these will be noted. Also, if unauthorized
copyright material had to be removed, a note will indicate the deletion.
Oversize materials (e.g., maps, drawings, charts) are reproduced by
sectioning the original, beginning at the upper left-hand comer and continuing
from left to right in equal sections with small overlaps.
ProQuest Information and Learning
300 North Zeeb Road, Ann Arbor, M l 48106-1346 USA
800-521-0600
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
BREAST CANCER SUSCEPTIBILITY GENE 1 : A ROLE IN
TRANSCRIPTIONAL REGULATION
by
John Jungha Park
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PATHOBIOLOGY)
May 2002
Copyright 2002 John Jungha Park
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
UM I Number: 3073832
_ _ ____ _ _ (g )
UMI
UMI Microform 3073832
Copyright 2003 by ProQuest Information and Learning Company.
All rights reserved. This microform edition is protected against
unauthorized copying under Title 17, United States Code.
ProQuest Information and Learning Company
300 North Zeeb Road
P.O. Box 1346
Ann Arbor, Ml 48106-1346
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
UNIVERSITY OF SOUTHERN CALIFORNIA
T he G raduate School
U niversity Park
LOS ANGELES, CALIFORNIA 90089-1695
This dissertation, w ritten b y
■TToH nJ 3 v * * » o 6> H A P a o K __________________
Under the direction o f A.lS.. Dissertation
Committee, and approved b y all its members,
has been presented to and accepted b y The
Graduate School, in partial fulfillm ent o f
requirements for the degree o f
DOCTOR OF PHILOSOPHY
o f Graduate Studies
DaCe -H ay-H h-2002
DISSERTATION COMMITTEE
2 . . . fj f 4
Chairperson
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
John Jungha Park Michael F. Press, M.D.,Ph.D
ABSTRACT
BREAST CANCER SUSCEPTIBILITY GENE 1: A ROLE IN
TRANSCRIPTIONAL REGULATION
Breast cancer is the second leading cause of cancer-related death among women
in the United States. It is estimated that approximately 5-10% of all cases of breast
cancers are associated with inheritable genetic factors, or genes. The recent discoveries
of two genes, breast cancer susceptibility genes 1 and 2, have shed some light into the
genetics of hereditary breast cancer, but little is known as to the functions of these two
gene products. In the current work, we describe a novel function for the BRCA1
protein product in steroid hormone signaling, suggesting a possible role in
transcriptional regulation. In chapter 1, we provide a general overview of hereditary
breast cancer and conclude with a brief introduction of BRCA1 and its putative
functions. In chapter 2, we describe a novel role for BRCA1 in androgen receptor
signaling, both alone and in the presence of other nuclear receptor (NR) coactivators,
such as the pl60 family of NR coactivators. In chapter 3, we demonstrate that BRCA1
functions as a general coactivator of steroid receptors, enhancing both the progesterone
and estrogen receptors under physiologic conditions. In chapter 4, we show that
BRCA1 may function in chromatin remodeling via recruitment of a novel class of
1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
histone methylating proteins, or protein methyltransferases (PMTs), during steroid
hormone signaling. In conclusion, the studies summarized in this dissertation describe a
novel role for BRCA1 in transcriptional regulation pathways, specifically in response to
steroid hormone signaling, and also provide one possible mechanism by which BRCA1
may function as a coactivator through the recruitment of specific histone remodeling
proteins, such as the PMTs. Finally, all the work presented here was conceived and
performed by the author and is believed to be novel in nature.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
TABLE OF CONTENTS
LIST OF FIGURES
CHAPTER 1. INTRODUCTION
BACKGROUND 1
GENETIC FACTORS ASSOCIATED WITH INHERITED BREAST CANCER
The Breast-Ovarian Cancer Syndromes (BRCA1, BRCA2, BRCAX) 3
The Breast Cancer Susceptibility Gene I, BRCA1 3
The Breast Cancer Susceptibility Gene 2, BRCA2 5
Other Candidate Breast Cancer Susceptibility
Genes, BRCAXs 6
Other Hereditary Syndromes that also Features Breast Cancer
(p53, pTEN, ATM) 7
Li-Fraumeni Syndrome (LFS) 8
Cowden Syndrome 8
Ataxia Telangiectasis 9
Other Genes 10
THE FUNCTION OF BRCA1
Structural evidence gives minimal clues to BRCA1 function 1 1
BRCA1 is involved in DNA repair 13
BRCA1 and cell-cycle regulation 17
BRCA1 andapoptosis 19
BRCA1 in transcription regulation 21
CONCLUDING REMARKS 22
REFERENCES 25
CHAPTER 2. BRCA1 IS A NOVEL C O A C TIV A TO R OF THE
ANDROGEN R EC EPTO R (AR) IN TRANSIENTLY
TRANSFECTED CELLS
ABSTRACT 37
INTRODUCTION 38
MATERIALS AND METHODS
Mammalian expression vectors and plasmid construction 40
Bacterial expressionpPlasmids 41
Tissue culture and transfections 41
Chloramphenicol acetyltransferase (CAT) assays 42
Glutathione-S-Transferase (GST) Pull-Downs 43
RESULTS
BRCA1 Enhances AR Signaling 43
Exogenous BRCA1 does not increase AR stability 44
ii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
BRCA1 enhances AF-1 of the AR 44
Potentiation of pl60 coactivation by BRCA1 45
BRCA1 enhances GRIP 1-mediated coactivation of AR LBD 46
BRCA1 interacts with the N-terminus of AR and the
C-terminus of GRIP1 46
Both AR and GRIP I interact with the N-terminus of BRCA1 47
BRCA1 coimmunoprecipitates with GRIP1, but not
with GRIP 1A AD 2 47
BRCA1 synergy with GRIP I is dependent upon an intact
AD 2 domain 48
DISCUSSION 49
REFERENCES 73
CHAPTER 3. AN EM ERGING ROLE FOR BRCA1 IN ESTROGEN
R E C E P T O R AND PR O G E ST E R O N E R E C E P T O R
REGULATION: CLEARING UP THE CONTROVERSY
ABSTRACT 81
INTRODUCTION 83
Circumstantial evidence for the importance of proliferative activity
in breast cancer development and the role of ovarian hormones 84
The estrogen receptor and progesterone receptor 87
Estrogen Receptor: Two Subtypes, ER-a and ER-fi 88
Progesterone Receptors: PR-A, PR-B, and PR-C 90
The Structure o f ER and PR 91
Ovarian hormones and BRCA1 regulation 94
BRCA1 regulation of hormone function 95
MATERIALS AND METHODS
Mammalian expression vectors and plasmid construction 98
Bacterial expression plasmids 100
Tissue culture and transfections 100
Chloramphenicol acetyltransferase (CAT) assays 101
Luciferase (LUC) assays 101
Glutathione-S-Transferase (GST) Pull-Downs 102
Preparation of whole cell extracts 102
Coimmuniprecipitation and western immunoblotting 103
RESULTS
BRCA1 Enhances ER signaling through potentiation of GRIP1 103
BRCA1 function in ER signaling is dependent on nuclear
receptor dose 105
BRCA1 coactivation depends on ER levels 106
BRCA1, GRIP1, and BRCA1-GRIP1 enhancement of ER also
depends on NR levels 107
BRCA1 and progesterone receptor regulation 108
iii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
DISCUSSION
REFERENCES
109
138
CHAPTER 4. RECRUITMENT OF PROTEIN METHYL-TRANSFERASES,
CARM1 AND PRMT1, BY BRCA1 IN STEROID RECEPTOR
SIGNALING
INTRODUCTION 152
MATERIALS AND METHODS
Mammalian expression vectors and plasmid construction 156
Bacterial expression plasmids 157
Tissue culture and transfections 157
Luciferase (LUC) assays 157
Glutathione-S-Transferase (GST) Pull-Downs 158
Preparation of whole cell extracts 158
Coimmuniprecipitation and wester immunoblotting 159
RESULTS
BRCA1 recruits protein methyltransferases in the enhancement
of androgen receptor signaling 159
Recruitment of PMT activity by BRCA1 in progesterone and
estrogen receptor signaling pathways 161
BRCA1 and the protein methyltransferases interact in vitro 161
Interaction of BRCAl and HA-PRMT1 in mammalian cells 162
Enhancement of AR signaling by CARM1 and PRMT1 is not
synergistic with bRCAl, unlike with GRIP1 163
Synergistic enhancement of androgen receptor signaling by BRCAl,
GRIP I, and C ARM 1 or PRMT1 163
Synergy by BRCAl, GRIP1, and CARM1 is dose-dependent
throughout a linear range of exogenous methyltransferase 164
Synergistic enhancement of ER and PR signaling by BRCAl,
GRIP1, and the protein methyltransferases 165
Synergy among GRIP1, BRCAl, and CARM1 requires the AD2
interaction domain of GRIPl 166
DISCUSSION
BRCAl and chromatin remodeling 167
Physiological significance of coactivator synergy 169
REFERENCES 198
GLOBAL BIBLIOGRAPHY 203
iv
»
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
IJST OF FICIIRF.S_________________________
Figure 1-1. BRCAl is a nuclear protein implicated in multiple pathways. 24
Figure 2-1. Wild-type BRCAl coactivates AR transactivation in PC-3
prostate cancer cells. 52
Figure 2-2. AR is not stabilized by BRCAl coexpression. 54
Figure 2-3. BRCAl works through AR AF-1 in PC-3 cells. 56
Figure 2-4. Synergistic coactivation of AR signaling by BRCAl and
members of the pl60 family of nuclear receptor coactivators. 58
Figure 2-5. Potentiation of AR signaling by BRCAl occurs in both prostate-
and breast-derived cell lines. 60
Figure 2-6. BRCAl potentiates GRIP I-mediated coactivation of AR AF-2
on the MMTV promoter. 62
Figure 2-7. Schematic diagrams of AR, GRIP1, and BRCAl showing
the locations of various functional domains. 64
Figure 2-8. Full-length BRCAl interacts with the N-terminal domain
of the androgen receptor. 66
Figure 2-9. Full-length BRCAl interacts with the C-terminus of GRIP1. 68
Figure 2-10. Autoradiographs showing the localization of the AR NTD
and GRIPlc interactions on BRCAl. 70
Figure 2-11. BRCAl coimmunoprecipitates with GRIP1, but not
with GRIP1AAD2. 72
Figure 2-12. Synergy with GRIP1 is dependent upon an intact AD2 domain. 74
Figure 3-1. Schematic illustration of the estrogen receptor (ER)
genome and messenger RNA with protein structure. 115
Figure 3-2. Schematic illustration of the progesterone receptor (PR)
genome and messenger RNA with protein structure. 117
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3-3. Functional domains of the steroid hormone receptors. 119
Figure 3-4. BRCAl does not coactivate or repress ER-a transactivation
in PC-3 cells. 121
Figure 3-5. BRCAl potentiates pl60-mediated ER-a signaling in
both prostate and breast cell lines. 123
Figure 3-6. BRCAl regulation of ER depends upon receptor levels. 125
Figure 3-7. Dose-dependent kinetics of ER activity. 127
Figure 3-8. BRCAl is a coactivator at low-dose ER. 129
Figure 3-9. BRCAl inhibits ER signaling at high receptor levels. 131
Figure 3-10. BRCA1-GR1P1 synergy depends on transfected ER levels. 133
Figure 3-11. BRCAl is a general coactivator of the steroid receptors. 135
Figure 3-12. BRCAl potentiates GRIP1 enhancement of PR signaling. 137
Figure 4-1. Recruitment of CARM1 and PRMT1 by BRCAl in AR signaling. 175
Figure 4-2. Recruitment of CARM1 and PRMT1 by BRCAl in PR signaling. 177
Figure 4-1. Recruitment of CARM1 by BRCAl in ER signaling. 179
Figure 4-4. BRCAl and the protein methyltransferases interact in vitro. 181
Figure 4-5. Interaction of BRCAl and HA-PRMT1 in mammalian cells. 183
Figure 4-6. GRIP1 recruits CARM1 and PRMT1 during steroid
receptor signaling. 185
Figure 4-7. Synergistic enhancement of NR function by CARM1,
PRMT1, and GRIPl. 187
Figure 4-8. Lack of synergistic enhancement of NR function by
CARM1, PRMT1, and BRCAl. 189
Figure 4-9. Synergistic enhancment of AR signaling by BRCAl,
GRIPl and CARM1 or PRMT1. 191
vi
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4-10. CARM1 dose and it role in coactivator synergy. 193
Figure 4-11. Synergistic enhancement of ER and PR signaling by BRCAl,
GRIPl and the protein methyltranferases, CARM1 and PRMT1. 195
Figure 4-12. Synergy among BRCAl, GRIPl and CARM1 requires the
AD2 interaction domain of GRIP 1. 197
vii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
CHAPTER 1. INTRODUCTION
BACKGROUND
Breast cancer is the second leading cause of cancer-related death among women
in the United States. According to the American Cancer Society, one in eight American
women is expected to develop breast cancer during her lifetime (Landis S et al., 1998).
Breast cancer, as with other cancers, is a genetic disease arising from an accumulation
of mutations that result in increased tumor cell survival over normal cells. Although the
vast majority of mutations discovered in breast cancers are somatic in origin, it is
estimated that approximately 5-10% of all cases of breast cancers are associated with
inheritable genetic factors, or genes (Duncan et al., 1998). The existence of an
inheritable genetic predisposition for breast cancer is not a recent concept. As early as a
century ago, it was suggested that some breast cancers were familial in origin, though
the exact factors involved were unknown (Broca; Szabo and King, 1995). Only within
the past few decades were actual breast cancer susceptibility genes known and isolated.
Among these, two genes, the breast cancer susceptibility genes BRCAl and BRCA2,
comprise the majority of hereditable breast cancers (Miki et al., 1994; Wooster et al.,
1995).
Hereditary breast cancers can be separated into two classes, as determined by
the inherited susceptibility factors involved (Rebbeck, 1999). The first class is
comprised of a small subset of genes that confer high breast cancer risk, but are
1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
relatively rare in the general population. Examples of these genes are BRCAl, BRCA2,
p53, and pTEN. The second class of inherited factors are genes that confer relatively
low risk, but germline alterations exist in a larger population. Hence, though their
relative risk is low, their overall contribution to hereditary breast cancer is large.
Examples of genes that confer low to moderate risk are ATM, hRASl, and CYP1A1.
Though it is not clear what makes genes in one class more penetrant, or likely to result
in phenotypic cancer, than genes in another class, it is likely that other factors, such as
additional genetic alterations and/or environment, play a significant role in tumor
etiology. Furthermore, these other factors often appear to be individualistic as similar
mutations in different individuals often behave differently (ie. regarding penetrance and
organ specificity, especially in the case of cancer syndromes).
Although sporadic breast cancers are more common than hereditary breast
cancers, they are usually caused by mutations within the affected individual’s somatic
cells and are not passed on to progeny. Hereditary cancers, on the other hand, are
germline by definition, and cancer-predisposing mutations are passed on from
generation to generation, provided carriers survive to reproductive age. Since
individuals who are carriers for germline mutations only have one normal allele of the
affected gene, it only takes one “hit”, according to Knudson’s two-hit hypothesis, to
result in a genetic “knock-out” of the gene’s function, compared to the two hits that are
required in sporadic breast cancers. Hence, hereditary breast cancers typically present
at an unusually young age. Furthermore, there is a higher incidence of multifocal
development of cancer in a single affected organ, or bilateral development of cancer in
2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
paired organs. Lastly, patients with hereditary breast cancer commonly develop
multiple primary cancers affecting different organs, making them syndromes. Below is
a summary and discussion of a few genes that are associated with major syndromes of
which breast cancer is a common cancer.
GENETIC FACTORS ASSOCIATED WITH INHERITED BREAST
CANCER
The Breast-Ovarian Cancer Syndromes {BRCAl, BRCA2, BRCAX)
The Breast Cancer Susceptibility Gene 1, BRCAl
BRCAl is found to be mutated in approximately 52% of all hereditary cancers
with site-specific breast cancer (Ford et al., 1998). It is located on chromosome 17q21
and spans a region of over 100 kilobases. It is a large gene, composed of 24 exons, of
which 22 are coding. The encoded protein product is 1863 amino acids in length.
However, despite BRCAl’s large size, it shows little sequence homology with other
sequences of known function, making its initial characterization difficult. The mutation
spectrum for BRCAl reveals a vastly heterogeneous distribution. To date, over 600
distinct disease-causing mutations have been isolated. Interestingly, the majority of
mutations (95%) result in premature termination of the protein product. The types of
mutations range from deletions (70%) and insertions (10%) to point-mutations (10%)
3
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
and splicing errors (5%) (Rahman and M.R., 1998). Due to the preponderance of
disease-associated truncation mutations, it is likely that having full-length BRCAl is
vital in maintaining normal, physiologic function. Even a relatively minor mutation in
the C-terminus of BRCAl resulting in a 10 amino acid truncation (5677insA) has been
shown to be associated with an increase in breast cancer susceptibility, which argues
that this terminal region may play an important role in cancer prevention.
Though the types of BRCAl mutations and their distribution along the gene
appear to be heterogeneous, a few mutations have been found with increased frequency
in some populations. Because it is believed that the higher incidence in these
populations is the result of genetic isolationism, these mutations have been called
“founder” mutations. Among these, the 185delAG and the 5382insC mutations, or
“Ashkenazi” Jewish mutations due to their higher carrier frequency within this
population, are the most prevelant. It is estimated that the proportion of individuals in
the general population who carry BRCAl mutations is probably between 1:2000 and
1:500 (Ford et al., 1994). In contrast, the carrier frequency of the 185delAG mutation
in the Ashkenazi population is approximately 1%, or 1:100 and the 5382insC is 0.13%
(Rao et al., 1996).
In the United States, it is estimated that 1 in 8 women will develop breast cancer
by the age of 70. In contrast, the early estimates of penetrance for BRCAl mutations
were proposed to be as high as 87%. Though recent studies seem to place the true
penetrance within a range of 56-87% by age 70 (Ford et al., 1998; Struewing et al.,
1997), it is clear that women with BRCAl mutations are at significantly greater risk for
4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
developing breast cancer than the general population, with the risk being relatively
much greater at earlier ages (CASH study, 33% of all breast cases 20-29 years of age
were due to dominant genetic susceptibility genes and only 2% over the age of 70)
(Claus et al., 1996).
As part of the breast and ovarian cancer syndrome, BRCAl associated cancers
also include prostate cancer, with a relative risk of 3.3 (8% by age 70) and colon cancer,
with a relative risk of 4.1 (6% by age 70) (Ford et al., 1994), in addition to the increased
risks for both breast and ovarian cancer.
The Breast Cancer Susceptibility Gene 2, BRCA2
The second breast cancer susceptibility gene, BRCA2, was discovered in 1994
(Wooster et al., 1994), and took little more than a year before the gene was isolated and
cloned in 1995 (Wooster et al., 1995). The entire gene was later cloned the following
year (Tavtigian et al., 1996). BRCA2 is estimated to be mutated in approximately 32%
of all hereditary breast cancers (Ford et al., 1998). The BRCA2 gene is located on
chromosome 13ql2-13 and is composed of 27 coding exons. Like BRCAl, the BRCA2
protein product is a large protein, containing 3418 amino acids. Yet, despite its large
size, it too lacks significant sequence homology with other known proteins. Hence,
progress in elucidating BRCA2’s function has been slow.
As with BRC Al, the mutation profile of BRCA2 is quite heterogeneous.
According to the Breast Cancer Information Core (BCIC), there are over 400 distinct
5
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
disease-causing mutations discovered thus far. Among these mutations are deletions,
insertions, and point mutations, with the vast majority of mutations resulting in
premature truncation products. However, unlike BRCAl, no splicing mutations have
been recorded for BRCA2. A founder effect involving a 6174delT mutation was seen in
Ashkenazi Jewish women (Neuhausen et al., 1996). This mutation is estimated to have
a carrier frequency of 1.5% within this population (Rao et al., 1996) and is believed to
be present in 8% of Ashkenazi women diagnosed with breast cancer before the age of
42 (Berman et al., 1996).
Early estimates on penetrance initially placed BRCA2 mutations as high as 84%
by age 70. However, the true penetrance may range from as low as 37 to as high as
84%, depending upon the population studied (Ford et al., 1998; Thorlacius et al., 1998).
Therefore, though the absolute risk for breast cancer conferred by BRCA2 is not known,
it is likely that the overall risk is very similar to that of BRCAl.
Other Candidate Breast Cancer Susceptibility Genes, BRCAX
In a study performed under the breast cancer linkage consortium (BCLC), it was
estimated that mutations in BRCAl and BRCA2 comprise up to 84% of families with 4
or more cases of breast cancer (either male or female), irrespective of ovarian cancer
status (Ford et al., 1998). In that same study, of families with 4 or more cases of breast
cancer and only a single case of ovarian cancer, 69% were attributed to BRCAl and
21% to BRCA2. In contrast, in families with 2 or more cases of ovarian cancer, 91%
6
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
was estimated to be due to BRCAl and only 9% to BRCA2. Therefore, in families
where both breast and ovarian cancers are present (breast and ovarian cancer
syndrome), BRCAl and BRCA2 compose nearly all cases. This is in contrast to families
with only site-specific breast cancer, irrespective of the number of cases. In these
families, only 58% were attributed to either BRCAl or BRCA2. This suggests the
existence of other breast cancer susceptibility genes (BRCAX), which most likely
predispose primarily for breast cancer. It is not known if this BRCAX gene is a single
gene, or a set of genes, but it will be interesting to see what other candidates are
discovered in the coming years.
Other Hereditary Syndromes that also Features Breast Cancer (p53, pTEN, ATM)
Breast cancer is also found in certain rare, genetic syndromes. Though their
overall contribution to hereditary breast cancer is small, these other syndromes are
notable in that they typically feature breast cancer among the many types of primary
cancers that describe them. Among these other syndromes include Li-Faumeni
syndrome, Cowden disease, and ataxia telangiectasis. The underlying genetic lesions
that cause these syndromes are germline mutations in p53, pTEN, and ATM,
respectively.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Li-Fraumeni Syndrome (LFS)
LFS is caused by underlying mutations in the tumor suppressor gene, TP53,
which is located on chromosome I7pl3.1. LFS is believed to be transmitted in an
autosomal dominant mode of inheritance and is characterized by a spectrum of cancers,
including soft tissue sarcomas, breast cancer, brain tumors, osteosarcomas, leukemia,
and adrenocorticol carcinoma. Affected individuals develop cancer at unusually early
ages and often present with multiple primary tumors. Although the risk for developing
any invasive cancer (other than skin cancer) is estimated to approach 50% by age 30,
compared to 1% in the general population, and 90% by age 70, germline mutations in
TP53 is believed to account for only 1% of breast cancers diagnosed under the age of 35
(Malkin et al., 1990). Hence, its involvement in hereditary breast cancer is minimal.
Cowden Syndrome
Cowden Syndrome is a genetic disease that predisposes for the development of
multiple hamartomatous lesions, including th skin, mucus membranes, colon, breast,
and thyroid. In addition, facial trichilemmomas are very common in affected
individuals. The underlying lesion in the vast majority of Cowden Syndrome families
involves the tumor suppressor gene, pTEN. This gene is located on chromosome
10q22-23 (Nelen et al., 1996) and is believed to be mutated in approximately 80% of
Cowden Syndrome families (Liaw et al., 1997). Like LFS, Cowden Syndrome follows
8
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
an autosomal dominant mode of inheritance. Though the incidence for the disorder is
unknown, it is estimated that 30% of female carriers will develop breast cancer.
Therefore, although Cowden Syndrome is relatively rare and comprise only a small
percentage of hereditary breast cancers, its penetrance for female breast cancer is high.
Ataxia Telangiectasis
Ataxia telangiectasis (A-T) is an autosomal recessive disorder characterized by
cerebellar ataxia (present in 100% of cases) and telangiectasia, usually affecting sun-
exposed areas and the cunjunctiva. Immune deficiency, with increased risk for
developing infections and cancer, is also common with this disorder. Affected
individuals usually do not survive past 30 and approximately 90% of A-T related deaths
are the result of cancer or infection. The gene affected in A-T is the ataxia
telangiectasis mutated (ATM) gene, which is located on chromosome 1 lq22.3 and is
thought to play a role in DNA repair. Because A-T is an autosomal recessive disorder,
affected individuals must inherit two mutated alleles of the ATM gene, one from each
parent, in order to exhibit clinically apparent disease. Approximately one-third of all A-
T patients will develop cancer during their lifetime, and 15% will die of their cancer.
The contribution of A-T in hereditary breast cancer remains controversial. In one study,
it was estimated that women who were heterozygous for ATM mutations may account
for up to 6.6% of all breast cancers diagnosed in the United States (Althma et al., 1996).
In a separate study, however, this figure was reduced to only 0.5% of women diagnosed
9
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
under the age of 40, whereas the controls in the same study were shown to have a
frequency of 1% (FitzGerald et al., 1997). To further increase the controversy, it was
initially estimated that the relative risk for breast cancer in women who were A-T
heterozygotes was as high as 5.1 (Swift et al., 1987). However, a second study found
no evidence of linkage in familial breast cancer families to the A-T locus, suggesting
that the contribution of the A-T gene in familial cancer is minimal. Yet, despite these
conflicting data, it is likely that ATM plays an important role in some hereditary breast
cancers, though how significant a role will depend upon future studies further defining
the prevalence and penetrance of A-T mutations in the general population, as well as
determining common risk factors involved.
Other Genes
There are many other genetic disorders that may present with clinical breast
cancer as part of their syndromes. However, aside from the two known breast cancer
susceptibility genes, none of these other loci, including TP53, pTEN, or ATM, appear to
play a significant role in hereditary breast cancer. Furthermore, there are clearly some
familial clustering that cannot be attributed to either BRCAl or BRCA2, suggesting the
existence of additional breast cancer susceptibility genes. As future studies look to
isolate these highly-penetrant genes, it will be interesting to if any of these susceptibility
genes play a role in sporadic cancer. Given the high predisposition for breast cancer
that these susceptibility genes confer, it is reasonable to believe that they may somehow
10
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
contribute to sporadic breast cancer as well, though mutation analysis alone has yet to
find a link with germline mutations in either BRCAl or BRCA2. Perhaps other
mechanisms, such as promoter methylation, loss of expression, or loss of
downstream/upstream mediators are involved.
THE FUNCTION OF BRCAl
Structural Evidence Gives Minimal Clues to BRCAl Function
The BRCAl gene encodes a 1863-amino acid protein with an estimated
molecular weight of approximately 210 kDa. It is a nuclear phosphoprotein with
putative roles in transcription regulation, cell cycle control, apoptosis induction, and
DNA repair. Yet, despite its large size, BRCAl appears to have no known intrinsic
enzymatic function. Structural analysis shows little sequence homology with other
proteins, and even between human and mouse BRCAl there is only 58% homology,
making initial characterization of the protein product difficult. Yet, despite these initial
setbacks, some structural clues existed. The protein contains a highly conserved NH2 -
terminal RING finger domain, two putative nuclear localization signal (NLS)
sequences, and a C-terminal acidic transcriptional activation domain (Irminger-Finger et
al., 1999). The C3 HC4 RING finger domain is a cysteine-rich, zinc-binding motif
believed to be important in protein-protein interactions. Although it is possible that this
domain is also involved in DNA-binding, there is currently no evidence to support this
11
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
role. Recently, a BRCAl Associated Ring Domain (BARD) protein was isolated using
a yeast-two hybrid screen with the BRCAl Ring Domain as bait (Wu et al., 1996). This
novel protein was also found to contain a RING domain, which is believed to be
important for BRCAl-BARD interactions. Other proteins also contain RING-like
domains, but it is not known if they too can interact with BRCAl (Borden, 2000).
The two NLS sequences are located on exon 11 (Wilson et al., 1997) and are
thought to function in nuclear redistribution by interacting with the importin-a subunit
of the nuclear transport signal receptor (Chen et al., 1996). Disruption of this region
results in aberrant localization of the mutated BRCAl protein within the cytoplasm.
Wilson et al has isolated and characterized a major splice variant of BRCAl in which a
part of exon 11 containing the NLS was deleted. This splice variant was found to have
a molecular weight of 110 kDa (estimated molecular weight is 85.7kDa) and was
localized to the cytoplasm. Hence, both molecular and physiological evidence suggest
that BRCAl is normally a nuclear protein, and the putative NLS sequences are
important in correct redistribution of newly translated proteins.
The C-terminal acidic transcriptional activation domain contains two, tandemly
repeated sequences which are found to be conserved in a number of other proteins, such
as mammalian p53 binding protein, 53BP1, and the yeast DNA-repair protein, RAD9
(Callebaut and Momon, 1997). This BRCAl C-terminal (BRCT) domain, is found in a
number of other proteins involved in DNA repair and cell-cycle regulatory pathways,
suggesting that BRCAl may also function in these pathways. Additionally, the C-
terminus of BRCAl has been shown to activate transcription when fused to a
12
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
heterologous GAL4 DNA-binding domain. Though this supports a putative function in
transcription regulation, it remains to be seen if this activation domain has any relevant
physiologic function.
BRCA1 is Involved in DNA Repair
There is compelling evidence to support a role for BRCA1 in DNA repair and in
the maintenance of genome integrity. Firstly, BRCA1 has been shown to localize in the
centrosomes of cells during M phase (Hsu and White, 1998), and subsequently,
BRCAl'" mouse embryo fibroblasts (MEFs) with partial loss of function phenotypes
exhibited chromosomal abnormalities associated with a defective G2-M cell cycle
checkpoint and centrosome amplification (Xu et al., 1999b). Additionally, other studies
have demonstrated that cells carrying mutations in BRCA1 were hypersensitive to
ionizing radiation and oxidative damage (Foray et al., 1999), suggesting an increased
susceptibility to genomic alterations in these cells. This hypersensensitivity to DNA
damaging agents, however, was reversed by the introduction of wild-type BRCA1,
which resulted in an increased resistance to DNA damage through the activation of
BRCAl-mediated transcription-coupled DNA repair (Abbott et al., 1999; Gowen et al.,
1998). In this repair process, DNA damage is repaired more rapidly in transcriptionally
active DNA than in non-active genomic DNA, linking BRCA1 function in DNA repair
to another putative function in transcription regulation (refer to below). In addition to
transcription-coupled repair, BRCA1 appears to function through other repair pathways
13
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
via its interaction with various other DNA repair proteins. The first of these BRCA1-
associated DNA repair proteins discovered was hRAD51. In brief, hRAD51 is
homologous with the bacterial RecA recombination and repair protein and is believed to
catalyze ATP-dependent DNA strand exchange reactions, functioning in normal meiotic
and mitotic recombination, DNA damage repair, and chromosome segregation
(Baumann and West, 1998; Ivanov and Haber, 1997). Immunocytochemical studies
revealed that hRAD51 accumulates at discrete foci on chromosomal DNA during
meiotic prophase (Barlow et al., 1997; Haaf et al., 1995; Plug et al., 1996), and also
forms nuclear foci in somatic cells following DNA damage (Haaf et al., 1995).
Similarly, BRCA1 has recently been shown to form characteristic nuclear foci,
colocalizing with hRAD51 both in discrete S phase foci in normal cells, and on the axial
elements of developing synaptonemal complexes in primary human spermatocytes,
supporting a role in meiotic recombination (Scully et al., 1997). Though it is unclear if
BRCA1 and hRAD5l interact directly, the two proteins have been shown to be
complexed in extracts of human cells, suggesting potential physiological association.
Furthermore, BRCAl-hRAD51 S phase foci were found to be labile in response to
DNA damaging agents, relocalizing to replicating DNA structures. In particular,
following either hydroxyurea (HU) treatment or low-dose UV-irradiation, both BRCA1
and hRAD51 were found to relocate onto subnuclear regions containing proliferating
cell nuclear antigen (PCNA), a marker of replication, suggesting a common
physiological role in DNA repair response (Scully et al., 1997). Whether this response
is linked to transcription, as described above in the transcription-coupled repair
14
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
pathways, is not known, though intriguingly, both BRCA1 and hRAD51 are
components of the RNA polymerase H holoenzyme complex (Maldonado et al., 1996;
Scully et al., 1997). Given BRCAl’s putative role in transcription regulation (refer to
below), it is possible that, in addition to functioning in repair complexes associated with
replicating DNA, both BRCA1 and hRAD51 may also function by upregulating
pathways involving the transcriptional activation of DNA repair and/or cell growth
regulatory genes through their association with the transcription initiation complex and
other factors. Clearly BRCAl’s ability to carry out transcription-coupled repair
supports this hypothesis.
In addition to hRADSl, BRCA1 was also found to be associated within a
separate complex of DNA repair proteins, hMREl l-hRAD50-p95/nibrin (Zhong et al.,
1999). This multiprotein complex has been shown to carry out two different processes
of double-strand chromosome break repair, homologous recombination and non-
homologous end-joining (NHEJ), and is also thought to play a prominent role in
telomere maintenance. Furthermore, evidence suggest that the hMREl l-hRAD50-
p95/nibrin complex functions in checkpoint response to the presence of newly-formed
double-strand breaks (DSBs), making it a key regulator and effector of genome integrity
(Haber, 1998). In normal cells, assembly of this complex into nuclear foci occurs
following DNA damage, with BRCA1 colocalizing with hMREl l-hRAD50-p95
following y-irradiation, HU-, or UV-treatment. However, in cells deficient for BRCA1
function, this damage-induced assembly process is reduced and the cells are more
sensitive to genotoxic agents such as MMS, suggesting that BRCA1 functions in the
15
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
recruitment process and formation of these macromolecular DNA repair protein
complexes. In support of this putative physiologic role, transfection with wild-type
BRCA1, but not mutant BRCAls, results in partial restoration of complex formation, as
well as increased resistance to MMS. Hence, in addition to hRAD51, BRCA1 appears
to function in at least one other DNA repair pathway, though the signals that determine
how and when a particular pathway is activated is unclear. It is possible that other
mechanisms are involved.
A number of proteins have been shown to phosphorylate BRCA1 in response to
DNA damage. ATM, which is found to be mutated in ataxia-telangiectsia, was shown
to phosphorylate BRCA1 following exposure of cells to Y-irradiation (Cortez et al.,
1999). This phosphorylation appears to play a physiologically relevant role in BRCA1-
mediated response to DNA damage as mutations of the two phosphorylated sites, serine
1423 and serine 1524, abrogates the ability of the mutant BRCAI to rescue the radio
sensitivity of a BRCAI-deficient cell line. Similarly, phosphorylation of serine 988 by
the human Cdsl kinase (hDdsl/Chk2) in response to DNA-damage has also been shown
to be important in regulating BRCAI DNA repair function (Lee et al., 2000). BRCAI
and hCdsl were found to colocalize within discrete nuclear foci, but separated after y-
irradiation. Furthermore, when transfected into a BRCAI-mutated cell line, only
phosphorylated wild-type BRCAI with an intact serine 988 was able to restore survival
after DNA damage. Therefore, since BRCAI is phosphorylated in a differential manner
by many different regulatory proteins, it is tempting to speculate that covalent
modification, such as phosphorylation, plays an important role in specifying BRCAI
16
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
function, possibly by modifying protein-protein interfaces and interaction domains,
thereby, effectively increasing the modularity of the BRCAI protein. Given the
numerous different cell cycle dependent, DNA damage induced, and possible growth
factor mediated (Altiok et al., 1999) phosphorylation states of BRCAI, this may explain
one possible, though highly speculative, mechanism by which BRCAI is able to
participate in so many divergent pathways.
Taken together, these evidence support a general role for BRCAI in the
maintenance of genome integrity through active surveillance of DNA alterations and
participation in DNA damage response pathways.
BRCAI and Cell-Cycle Regulation
Whether as part, or separate, of its ability to function in DNA repair, BRCAI
also appears to function in cell-cycle control. Early evidence of this role stemmed from
cell line studies in which BRCAI expression was altered by either forced
overexpression or lost by antisense therapy. In brief, whereas overexpression of
BRCAI resulted in significant inhibition of cell growth and tumor formation ability in
transfected cells, reduced expression by antisence BRCAI mRNA resulted in
morphologically flattened cell phenotype and increased proliferative rate, suggesting a
general role for BRCAI in cell-cycling. Recently, BRCAI was shown to upregulate
p2iW A F i/ciP i gene expression, presumably through enhancement of p53-dependent
transcription activation (Somasundaram et al., 1997). However, p53 nullizygous MEFs
17
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
also showed increased p21 induction by exogenous BRCAI, suggesting other cellular
factors may also be able to recruit BRCAI in p21 regulation. The p2 l W A F I /C I P 1 protein is
a universal cell-cycle inhibitor that binds specifically to cyclin-CDK (cyclin dependent
kinase) complexes and PCNA, thereby, serving as a growth inhibitor and effector of
cell-cycle checkpoints (Somasundaram et al., 1997). When BRCAI was mutated, the
altered BRCAI protein was unable to induce p21 expression, resulting in loss of cell-
cycle inhibition. However, transfection with wild-type BRCAI resulted in increased
p21 induction, suggesting that induction of BRCAl-mediated p21 gene expression
requires fully-functional BRCAI.
In addition to regulating p21 expression, BRCAI was also shown to interact
directly with the tumor suppressor pRb (Aprelikova et al., 1999), resulting in growth
suppression in cells overexpressing BRCAI. This growth suppression was pRb-
dependent as inactivation of pRb by HPV E7 protein resulted in abrogation of BRCAl-
mediated arrest. Furthermore, BRCAI was unable to suppress BrdUrd uptake in
primary fibroblasts that were rb '\ and to a lesser extent rbw\ Together, these results
point out the requirement for functional pRb in BRCAl-mediated growth arrest with,
perhaps, pRb serving as a direct downstream effector of multiple pathways associated
with growth suppression. One such potential pathway may involve pRb’s role in the
suppression of E2F-responsive gene transcription, presumably through its association
with histone deacetylases. With the recent finding that BRCAI is also a component of
the histone deacetylase complex (Yarden and Brody, 1999), it is not hard to imagine
BRCAI functioning as a converging factor for pRb and the histone deacetylase complex
18
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
during checkpoint regulation or DNA repair response, resulting in the inhibition of
specific genes responsible for cellular growth and proliferation. Hence, whether
through activation of p21 gene expression, or through recruitment of pRb, BRCAI
appears to play a significant role in cell-cycle control.
Finally, it is know that BRCAI mRNA expression is cell-cycle dependent, rising
in late G1 phase and remaining so until late G2 phase (Gudas et al., 1996; Vaughn et al.,
1996). This accumulation of mRNA is correlated with protein expression (Chen et al.,
1999) and is thought to function in the regulation of proliferation. The phosphorylation
status of BRCAI also mirrors its expression pattern, becoming first detectable by late
G1 phase, peaking in S phase, and remaining high thoughout M phase (Chen et al.,
1999; Ruffner et al., 1999). BRCAI has been shown to be phosphorylated by kinases
associated with cyclins D and A, as well as CDK2, through in vitro studies (Chen et al.,
1996). In a subsequent study, it was shown, again by in vitro analysis, that BRCAI
may associate with cell cycle factors cyclins A, D1 and Bl, cdc2, cdk-2, cdk4 and E2F-
4. Taken together, these results give compelling evidence that BRCAI plays a role in
cell-cycle regulation.
BRCAI and Apoptosis
In addition to DNA repair and cell-cycle control, BRCAI has been shown to
mediated apoptosis through the induction of the DNA damage response gene, GADD45.
The growth arrest DNA damage induced gene product, GADD45, is a stress-inducible
19
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
protein that was initially identified as a gene whose mRNA is rapidly induced by agents
that cause DNA damage, such as UV, N-acetoxy-2-acetylaminofluorene, MMS, and
H2 0 2 (Fomace et al., 1988; Papathanasiou et al., 1991). The DNA damage induced
transcription of GADD45 is mediated by both p53-dependent and p53-independent
mechanisms (Takekawa and Saito, 1998). Once induced, Gadd45 binds and activates
MTK MAPKKK, which then activates p38 and JNK MAPK cascades, resulting in the
activation of other pathways, including apoptosis. It has been hypothesized that p53
may induce apoptosis through activation of the GADD45-mediated MK1 MAPKKK
signaling cascade. However, although p53-dependent activation of GADD45-mediated
apoptosis only occurs as a consequence of DNA damage, p38 and JNK/MAPK
pathways can also be activated by a number of other environmental stimuli that result in
p53-independent activation. Among some of these other inducers are osmotic shock
and proinflammatory cytokines, which mediate their effects through a host of other
upstream activators. Hence, with these examples, other p53-independent mechanisms
of p38 and JNK/MAPK activation may exist. BRCAI, though a coactivator of p53-
dependent gene expression, has recently been shown to mediate apoptosis through
upregulation of GADD4S and activation of p38 and JNK/SAPK. This induction was
found to be p53-independent, suggesting that BRCAI may also function as a key
checkpoint control mechanism during stress-response under conditions that are not
normally regulated by p53-dependent pathways.
20
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
BRCAI in Transcription Regulation
Over the past few years, compelling evidence has been accumulating to support
a role for BRCAI in transcription regulation. It has already been discussed how
BRCAI may function in both DNA repair and cell-cycle control by regulating the
transcriptional activation of specific genes related to those functions. Furthermore,
numerous studies have implicated BRCAI as playing a direct role in various aspects of
transcription control. Protein purification studies have shown that BRCAI coelutes
with the RNA polymerase II holoenzyme complex (Scully et al., 1997), possibly
through its interaction with RNA helicase A (Anderson et al., 1998). BRCAI was also
found to interact with various other transcriptional regulatory proteins, including p53
(Ouchi et al., 1998), CtIP (Yu et al., 1998), c-myc (Wang et al., 1998), and most
recently, p300/CBP (Pao et al., 2000), suggesting a general role in transcription control.
The C-terminus of BRCAI has been shown to mediate transcription when fused
to a heterologous DNA-binding domain (Chapman and Verma, 1996; Monteiro et al.,
1996). Though the lack of its own DNA-binding domain precludes the possibility that
this activation function is directly involved in transcriptional activation, it is possible
that the C-terminus participates in other types of associations which, in turn, mediate
BRCAl’s ability to regulate transcription. The fact that BRCAI interacts with both the
RNA Pol II holoenzyme complex and the p300/CBP histone acetyltransferases through
this C-terminus supports this role.
21
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
In addition to regulating p21 gene expression by p53-independent pathways,
BRCAI has also been shown to be a coactivator of p53, enhancing p53-dependent gene
expression of both in vitro and in vivo targets (Ouchi et al., 1998; Somasundaram et al.,
1997; Zhang et al., 1998). Furthermore, BRCAI has recently been described by
transient transfection assays as being a repressor of estrogen receptor a signaling
pathways. Therefore, these data suggests a prominent role for BRCAI in transcriptional
regulation through mechanisms that may involve various aspects of its other roles in
DNA damage response and surveillance, DNA repair and apoptosis, or during general
housekeeping functions that may also be cell-cycle dependent.
CONCLUDING REMARKS
Mutations in BRCAI predisposes women to breast and ovarian cancers, and are
also implicated in prostate and colorectal cancers. Although BRCAI appears to have no
known intrinsic enzymatic activity, its C-terminus has been shown to function as an
activator of transcription when fused to a GAL4 DNA-binding domain. However, the
physiologic relevance of this property yet remains in question. BRCAI is known to
interact with various regulatory proteins involved in multiple cellular pathways.
Among these pathways are DNA repair, cell-cycle control, apoptosis, and
transcriptional regulation (Figure 1-1). Through the many interactions that BRCAI
participates in, it is now becoming apparent that BRCAI functions as a large scaffold
upon which other proteins of divergent pathways are recruited in response to different
environment stimuli, as well as specific temporal/spatial events, such as in response to
22
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
DNA damage, or through the specific phosphorylation of key residues by various
regulatory factors. Regardless of the mode of activation, it is clear that BRCAI plays a
critical role in multiple pathways, and may in fact function as a converging point for
these seemingly unrelated pathways. Yet, despite the myriad interactions and functions
that are already associated with BRCAI, the possibility that new interactions, and
hence, new functions with therapeutic targets being discovered remains great.
23
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Ill
cc
3
(D
C O
>»
C O
£
£
C O
Q .
fl)
a
■ o
a )
c o
a
■—
Q .
E
o>
o
l a
Q .
la
C O
0)
C O
C O
<
O
CC
C D
z *
in
24
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
REFERENCES
Abbott, D. W., Thompson, M. E., Robinson-Benion, C., Tomlinson, G., Jensen, R. A.,
and Holt, J. T. (1999). BRCAI expression restores radiation resistance in BRCA1-
defective cancer cells through enhancement of transcription-coupled DNA repair. J Biol
Chem 274, 18808-12.
Althma, P., Rappaport, R., and Swift, M. (1996). Molecular genotyping shows that
ataxia-telangiectasia heterozygotes are predisposed to breast cancer. Cancer Genet
Cytogenet 92, 130-134.
Altiok, S., Batt, D., Altiok, N., Papautsky, A., Downward, J., Roberts, T. M., and
Avraham, H. (1999). Heregulin induces phosphorylation of BRCAI through
phosphatidylinositol 3-Kinase/AKT in breast cancer cells. J Biol Chem 274, 32274-8.
Anderson, S. F., Schlegel, B. P., Nakajima, T., Wolpin, E. S., and Parvin, J. D. (1998).
BRCAI protein is linked to the RNA polymerase II holoenzyme complex via RNA
helicase A. Nature genet 19, 254-256.
Aprelikova, O. N., Fang, B. S., Meissner, E. G., Cotter, S., Campbell, M., Kuthiala, A.,
Bessho, M., Jensen, R. A., and Liu, E. T. (1999). BRCAI-associated growth arrest is
RB-dependent. Proc Natl Acad Sci U S A 9 6 ,11866-71.
25
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Barlow, A. L., Benson, F. E., West, S. C., and Hulten, M. A. (1997). Distribution of the
Rad51 recombinase in human and mouse spermatocytes. Embo J 16, 5207-15.
Baumann, P., and West, S. C. (1998). Role of the human RAD51 protein in homologous
recombination and double-stranded-break repair. Trends Biochem Sci 23, 247-51.
Berman, D. B., Costalas, J., Schultz, D. C., Grana, G., Daly, M., and Godwin, A. K.
(1996). A common mutation in BRCA2 that predisposes to a variety of cancers is found
in both Jewish Ashkenazi and non-Jewish individuals. Cancer Res 56, 3409-3414.
Borden, K. L. B. (2000). RING domains: master builders of molecular scaffolds? J Mol
Biol 295, 1103-1112.
Broca, P. Traite des Tumeurs 1, 1886-1889.
Callebaut, I., and Momon, J. P. (1997). From BRCAI to RAP1: a widespread BRCT
module closely associated with DNA repair. FEBS Lett 400, 25-30.
Chapman, M. S., and Verma, I. M. (1996). Transcriptional activation by BRCAI.
Nature 382,678-679.
26
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Chen, C.-F., Li, S., Chen, Y., Chen, P.-L., Sharp, Z. D., and Lee, W.-H. (1996). The
nuclear localization sequences of the BRCAI protein interact with the importin-a
subunit of the nuclear transport signal receptor. J Biol Chem 271, 32863-32868.
Chen, J.-J., Silver, D., Cantor, S., Livingston, D. M., and Scully, R. (1999). BRCAI,
BRCA2, and Rad51 operate in a common DNA damage response pathway. Cancer Res
59, 1752s-1756s.
Chen, Y., Farmer, A. A., Chen, C. F., Jones, D. C., Chen, P. L., and Lee, W. H. (1996).
BRCAI is a 220-kDa nuclear phosphoprotein that is expressed and phosphorylated in a
cell cycle-dependent manner [published erratum appears in Cancer Res 1996 Sep
1;56(17):4074]. Cancer Res 56, 3168-72.
Claus, E. B., Schildkraut, J. M., Thompson, W. D., and Risch, N. J. (1996). The genetic
attributable risk of breast and ovarian cancer. Cancer 77, 2318-2324.
Cortez, D., Wang, Y., Qin, J., and Elledge, S. J. (1999). Requirement of ATM-
dependent phosphorylation of brcal in the DNA damage response to double-strand
breaks [see comments]. Science 286, 1162-6.
Duncan, J. A., Reeves, J. R., and Cooke, T. G. (1998). BRCAI and BRCA2 proteins:
roles in health and disease. Mol Pathol 51, 237-247.
27
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FitzGerald, M. G., Bean, J. M., and Hegde, S. R. e. a. (1997). Heterozygous ATM
mutations do not contribute to early onset of breast cancer. Nat Genet 15, 307-310.
Foray, N., Randrianarison, V., Marot, D., Perricaudet, M., Lenoir, G., and Feunteun, J.
(1999). Gamma-rays-induced death of human cells carrying mutations of BRCAI or
BRCA2. Oncogene 18,7334-7342.
Ford, D., Easton, D. F., Bishop, D. T., Narod, S. A., and Goldgar, D. E. (1994). Risks of
cancer in BRCAl-mutation carriers. Lancet 343, 692-695.
Ford, D., Easton, D. F., Stratton, M., Narod, S., Goldgar, D., and Devilee, P. e. a.
(1998). Genetic heterogeneity and penetrance analysis of the BRCAI and BRCA2
genes in BC families. Am J Hum Genet 62, 676-689.
Fomace, A. J. J., Alamo, I. J., and Hollander, M. C. (1988). DNA damage-inducible
transcripts in mammalian cells. Proc Natl Acad Sci USA 85, 8800-8804.
Gowen, L. C., Avrutskaya, A. V., Latour, A. M., Roller, B. H., and Leadon, S. A.
(1998). BRCAI required for transcription-coupled repair of oxidative DNA damage.
Science 281, 1009-1012.
28
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Gudas, J. M., Li, T., Nguyen, H., Jensen, D., Rauscher, F. J., 3rd, and Cowan, K. H.
(1996). Cell cycle regulation of BRCAI messenger RNA in human breast epithelial
cells. Cell Growth Differ 7,717-23.
Haaf, T., Golub, E. I., Reddy, G., Radding, C. M., and Ward, D. C. (1995). Nuclear foci
of mammalian Rad51 recombination protein in somatic cells after DNA damage and its
localization in synaptonemal complexes. Proc Natl Acad Sci U S A 92, 2298-302.
Haber, J. E. (1998). The many interfaces of Mrel 1. Cell 95, 583-586.
Hsu, L.-C., and White, R. L. (1998). BRCAI is associated with the centrosome during
mitosis. Proc Natl Acad Sci 95, 12983-12988.
Irminger-Finger, I., Siegel, B. D., and Leung, W. C. (1999). The functions of breast
cancer susceptibility gene 1 (BRCAI) product and its associated proteins. Biol Chem
380,117-128.
Ivanov, E. L„ and Haber, J. E. (1997). DNA repair: RAD alert. Curr Biol 7, R492-5.
Landis S, Murray T, Bolden S, and Wingo P (1998). Cancer Statistics, 1998. CA Cancer
J CUn 48,6-30.
29
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Lee, J. S., Collins, K. M., Brown, A. L„ Lee, C. H., and Chung, J. H. (2000). hCdsl-
mediated phosphorylation of BRCAI regulates the DNA damage response. Nature 404,
201-4.
Liaw, D., Marsh, D. J., and Li, J. e. a. (1997). Germline mutations of the PTEN gene in
Cowden disease, and inherited breast and thyroid cancer syndrome. Nat Genet 16, 64-
67.
Maldonado, E., Shiekhattar, R., Sheldon, M„ Cho, H., Drapkin, R., Rickert, P., Lees, E.,
Anderson, C. W., Linn, S., and Reinberg, D. (1996). A human RNA polymerase II
complex associated with SRB and DNA-repair proteins [published erratum appears in
Nature 1996 Nov 28;384(6607):384]. Nature 381, 86-9.
Malkin, D., Li, F. P., Strong, L. C., Fraumeni, J. F. J., Nelson, C. E., and Kim, D. H. e.
a. (1990). A cancer family syndrome in twenty-four kindreds. Science 250,1233-1238.
Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P. A., and Harshman, K. e. a.
(1994). A strong candidate for the breast and ovarian cancer susceptibility gene
BRCAI. Science 266, 66-71.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Monteiro, A. N. A., August, A., and Hanafusa, H. (1996). Evidence for a transcriptional
activation function of BRCAI C-terminal region. Proc Natl Acad Sci USA 93, 13595-
13599.
Nelen, M. R., Padberg, G. W., and Peeters, E. A. e. a. (1996). Localization of the gene
for Cowden disease to chromosome 10q22-23. Nat Genet 13, 114-116.
Neuhausen, S. L., Gilewski, T., Norton, L., Tran, T., and McGuire, P. e. a. (1996).
Recurrent BRCA2 6174delT mutations in Ashkenazi Jewish women affected by breast
cancer. Nat Genet 13, 126-128.
Ouchi, T., Monteiro, A. N., August, A., Aaronson, S. A., and Hanafusa, H. (1998).
BRCAI regulates p53-dependent gene expression. Proc Natl Acad Sci U S A 95, 2302-
6.
Pao, G. M., Janknecht, R., Ruffner, H., Hunter, T., and Verma, I. M. (2000). CBP/p300
interact with and function as transcriptional coactivators of BRCAI. Proc Natl Acad Sci
U S A 97, 1020-5.
Papathanasiou, M. A., Kerr, N. C., Robbins, J. H., McBride, O. W., Alamo, I., Jr.,
Barrett, S. F., Hickson, I. D., and Fomace, A. J., Jr. (1991). Induction by ionizing
31
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
radiation of the gadd45 gene in cultured human cells: lack of mediation by protein
kinase C. Mol CeU Biol 11, 1009-16.
Plug, A. W., Xu, J., Reddy, G., Golub, E. I., and Ashley, T. (1996). Presynaptic
association of Rad51 protein with selected sites in meiotic chromatin. Proc Natl Acad
Sci U S A 93, 5920-4.
Rahman, N., and M.R., S. (1998). The genetics of breast cancer susceptibility. Annu
Rev Genet 32, 95-121.
Rao, B. B., Boyd, A. A., Volcik, K., and Richards, C. S. (1996). Ashkenazi Jewish
population frequencies for common mutations in BRCAI and BRCA2. Nat Genet 14.
Rebbeck, T. R. (1999). Inherited genetic predisposition in breast cancer: A population-
based perspective. Cancer Supplement 36, 1673-1681.
Ruffner, H., Jiang, W., Craig, A. G., Hunter, T., and Verma, I. M. (1999). BRCAI is
phosphorylated at serine 1497 in vivo at a cyclin-dependent kinase 2 phosphorylation
site. Mol Cell Biol 19,4843-54.
32
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Scully, R., Anderson, S. F., Chao, D. M., Wei, W., Ye, L., Young, R. A., Livingston, D.
M., and Parvin, J. D. (1997). BRCAI is a component of the RNA polymerase II
holoenzyme. Proc Natl Acad Sci U S A 94, 5605-10.
Scully, R., Chen, J., Ochs, R. L., Keegan, K., Hoekstra, M., Feunteun, J., and
Livingstion, D. L. (1997). Dynamic changes of BRCAI subnuclear location and
phosphorylation state are initiated by DNA damage. Cell 90.
Scully, R„ Chen, J., Plug, A., Xiao, Y., and Weaver, D. e. a. (1997). Association of
BRCAI with Rad5l in Mitotic and Meiotic Cells. Cell 88, 265-275.
Somasundaram, K., Zhang, H., Zeng, Y. X., Houvras, Y„ Peng, Y., Wu, G. S., Licht, J.
D., Weber, B. L., and El-Deiry, W. S. (1997). Arrest of the cell cycle by the tumour-
suppressor BRCAI requires the CDK-inhibitor p21WAFl/CiPl. Nature 389, 187-90.
Struewing, J. P., Hartge, P., Wacholder, S., Baker, S. M., Berlin, M., McAdams, M.,
Timmerman, M. M., Brody, L. C., and Tucker, M. A. (1997). The risk of cancer
associated with specific mutations of BRCAI and BRCA2 among Ashkenazi Jews. N
Engl J Med 20,1401-1408.
Swift, M., Reitnauer, P. J., Morrell, D., and Chase, C. L. (1987). Breast and other
cancers in families with ataxia-telangiectasia. N Engl J Med 316,1289-1294.
33
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Szabo, C., and King, M.-C. (1995). Inherited breast and ovarian cancer. Human
Molecular Genetics 4, 1811-1817.
Takekawa, M., and Saito, H. (1998). A family of stress-inducible GADD45-like
proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell
95, 521-30.
Tavtigian, S. V., Simard, J., Rommens, J., Couch, F., and Shattuck-Eidens, D. e. a.
(1996). The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds.
Nat Genet 72, 333-337.
Thorlacius, S., Struewing, J. P., Hartge, P., Olafsdottir, G. H., Sigvaldason, H., and
Tryggvadottir, L. e. a. (1998). Population-based study of risk of breast cancer in carriers
of BRCA2 mutation. Lancet 352, 1337-1339.
Vaughn, J. P., Davis, P. L., Jarboe, M. D., Huper, G., Evans, A. C., Wiseman, R. W.,
Berchuck, A., Iglehart, J. D., Futreal, P. A., and Marks, J. R. (1996). BRCAI expression
is induced before DNA synthesis in both normal and tumor-derived breast cells. Cell
Growth Differ 7,711-5.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Wang, Q„ Zhang, H., Kajino, K., and Greene, M. I. (1998). BRCAI binds c-Myc and
inhibits its transcriptional and transforming activity in cells. Oncogene 17, 1939-48.
Wilson, C. A., Payton, M. N., Elliott, G. S., Buaas, F. W., Cajulis, E. E., Grosshans, D.,
Ramos, L., Reese, D. M„ Slamon, D. J., and Calzone, F. J. (1997). Differential
subcellular localization, expression and biological toxicity of BRCAI and the splice
variant BRCAl-deltal lb. Oncogene 14, 1-16.
Wooster, R., Bignell, G., Lancaster, J., Swift, S., and Seal, S. e. a. (1995). Identification
of the breast cancer susceptibility gene BRCA2. Nature 378.
Wooster, R., Neuhasen, S. L., Mangion, J., Quirk, Y., and Ford, D. e. a. (1994).
Localisation of a breast cancer susceptibility gene, BRCA2, to chromosome 13ql2-13.
Science 265,2088-2090.
Wu, L. C., Wang, Z. W., Tsan, J. T., Spillman, M. A., Phung, A., Xu, X. L., Yang, M.-
C. W., Hwang, L.-Y., Bowcock, A. M., and Baer, R. (1996). Identification of a RING
protein that can interact in vivo with the BRCAI gene product. Nat Genet 14,430-440.
Xu, X., Weaver, Z., Linke, S. P., Li, C., Gotay, J., Wang, X. W., Harris, C. C., Ried, T.,
and Deng, C. X. (1999b). Centrosome amplification and a defective G2-M cell cycle
35
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
checkpoint induce genetic instability in BRCAI exon 11 isoform-deficient cells. Mol
Cell 3, 389-395.
Yarden, R. I., and Brody, L. C. (1999). BRCAI interacts with components of the
histone deacetylase complex. Proc Natl Acad Sci U S A 96,4983-8.
Yu, X., Wu, L. C., Bowcock, A. M., Aronheim, A., and Baer, R. (1998). The C-terminal
(BRCT) domains of BRCAI interact in vivo with CtIP, a protein implicated in the CtBP
pathway of transcriptional repression. J Biol Chem 273, 25388-92.
Zhang, H., Somasundaram, K., Peng, Y., Tian, H., Bi, D., Weber, B. L., and El-Deiry,
W. S. (1998). BRCAI physically associates with p53 and stimulates its transcriptional
activity. Oncogene 16, 1713-21.
Zhong, Q., Chen, C. F., Li, S., Chen, Y., Wang, C. C., Xiao, J., Chen, P. L., Sharp, Z.
D., and Lee, W. H. (1999). Association of BRCAI with the hRad50-hMrell-p95
complex and the DNA damage response. Science 285, 747-50.
36
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
CHAPTER 2. BRCAI is a Novel Coactivator of the Androgen
Receptor in Transiently Transfected Mammalian Cells
ABSTRACT
Germiine mutations in the BRCAI tumor suppressor gene have been implicated in
hereditary breast and ovarian cancers. Since BRCAI-associated cancers typically involve
steroid hormone responsive tissues, it is possible that BRCAI plays a role in steroid
receptor signaling. Using transient transfection assays, BRCAI was found to enhance
androgen signaling via activation function-1 (AF-1) of the androgen receptor (AR).
Furthermore, coexpression of BRCAI with pl60 nuclear receptor coactivators SRCla,
GRIP1, or AIB1 resulted in synergistic potentiation of androgen receptor and estrogen
receptor (ER-a) signaling in both breast and prostate cells. The N-terminal subdomain of
BRCAI physically interacted with both the N-terminal domain of the AR and the C-
terminal domain of the pl60 nuclear receptor coactivator, GRIP1, by in vitro protein
binding and mapping assays. Though further in vivo studies are necessary to determine
the physiological significance of these interactions, these results suggest that BRCAI may
play a role hormone regulation by directly modulating nuclear receptor-pl60 coactivator
interactions.
37
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
INTRODUCTION
BRCA1 is a tumor suppressor gene found to be mutated in 30-45% of hereditary
breast and ovarian cancers (Rahman and M.R., 1998). It encodes a nuclear
phosphoprotein with putative roles in DNA repair, cell-cycle control and transcriptional
regulation (Chen et al., 1999; Duncan et al., 1998; Irminger-Finger et al., 1999). While
mutations in BRCAl confer increased risk for both early-onset breast and ovarian
cancers, their role in prostate cancer is controversial (Hubert et al., 1999; Johannsson et
al., 1999; Struewing et al., 1997). Moreover, since the same germline BRCAl mutation
exists in all tissues of an affected individual, it is not clear why breast and ovarian tissues
are preferentially affected by neoplastic disease. The recent finding that BRCAl inhibits
estrogen receptor (ER-a) signaling (Fan et al., 1999) suggests one mechanism by which
loss of BRCAl function may lead to increased breast cancer risk in some carriers, but it
does not explain all aspects of the disease seen in affected women.
The androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily
of transcriptional regulators, which includes the steroid, thyroid hormone and retinoic
acid receptors. These receptors function in enhancing promoter-specific gene expression
through the recruitment of multiple coregulatory proteins (ie. coactivators/corepressors)
involved in various aspects of transcriptional control (McKenna et al., 1999; Xu et al.,
1999). The AR shares a basic structural homology with the other NRs containing: an N-
terminal transactivation domain (NTD), a highly-conserved DNA-binding domain (DBD),
and a C-terminal ligand-binding domain (LBD) (Mangelsdorf et al., 1995). Both the
NTD and the LBD contain separate activation functions (AF-1 and AF-2, respectively)
that mediate the transcriptional action of the receptor. However, unlike other Class I
steroid hormone receptors, AR also contains two, N-terminal poly-amino acid stretches
38
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
encoded by polymorphic trinucleotide repeats. One of these is a highly-variable CAG-
microsatellite which encodes polyglutamine (poly-Q). Although deleterious expansion of
the microsatellite (> 40 CAGs) does occur, resulting in a rare, neurodegenerative disorder
called spinal and bulbar muscular atrophy (SBMA), or Kennedy’s disease, the normal
CAG size-range in the general population is between 9 to 29 CAG repeats (Edwards et
al., 1992; Irvine et al., 1995) and averages about 20, depending on the population ethnic
group (Giovannucci et al., 1997). Variation in CAG-repeat length inversely modulates
AR activity (Chamberlain et al., 1994; Irvine et al., 1995), and shorter CAGs are
correlated with both increased risk and earlier age-of-onset for prostate cancer (Hardy et
al., 1996), supporting the hypothesis that androgens play a direct role in promoting
prostate cancer development (Ross et al., 1998). Yet, despite this role in the prostate,
androgens may play a protective role in breast, possibly through AR-mediated inhibition
of breast epithelial cell proliferation (Hackenberg et al., 1993a; Szelie et al., 1997). This
is supported by the fact that some mutations in the AR leading to loss of function or
expression have been correlated with breast cancer and earlier age-of-onset in some
individuals, though the frequency of such alterations are rare and not well-characterized
(Lobaccaro et al., 1993; Munoz de Toro et al., 1998; Wooster et al., 1992; Zhu et al.,
1997). Recently, a correlation between CAG-repeat length and risk for breast cancer
development was observed in a cohort of women with known BRCAl mutations
(Rebbeck et al., 1999). In that study, women who were BRCAl mutation carriers and
had at least one AR allele with > 28 CAG repeats were more likely to develop breast
cancer earlier than similar age-matched controls. In a separate study of women who
developed sporadic breast cancer by age of 40 years, no significant correlation with CAG
repeat size and cancer risk was found (Spurdle et al., 1999). Therefore, since variation in
39
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
AR activity, as determined by CAG repeat size, affected -associated breast cancer
risk, but not sporadic breast cancer risk, it is possible that BRCAl may function either
directly, or indirectly, in regulating AR signaling. Together, these observations suggest a
likely functional connection between the AR and BRCAl.
MATERIALS AND METHODS
Transient transfection assays were performed using a chloramphenicol-acetyl
transferase (CAT) assay system. Mammalian cells were transfected with plasmids
encoding various transcription factors and tested for reporter gene activation. In some
cases, protein expression from transfected plasmids were checked by
immunofluorescence. Protein binding assays were performed using glutathione-S-
transferase (GST) fusion proteins and in vitro transcribed and translated protein targets.
Mammalian Expression Vectors and Plasmid Construction
Plasmids pCMV-hAR (Tilley et al., 1989), pSG5-ERa (Green et al., 1988),
pSG5-GRIPl and pSG5-SRC-la (Ma et al., 1999), pcDNA3.1-AIBl (Anzick et al.,
1997), ARR3 tk-CAT (Snoek et al., 1998), ERE-C0U 6O-CAT (Webb et al., 1995), and
MMTV-CAT (Giguere et al., 1986) were described previously. To construct the vector
pcDNA-AR (NTD-DBD), an Nhel-BamHI fragment was PCR amplified from pcDNA-
hAR (Irvine et al., 2000) plasmid DNA using AR (NTD-DBD) primer pairs SI and AS1
(Table 1) and inserted into the reciprocal restriction sites of pcDNA3.1 (+). Vector
pcDNA-AR (DBD-LBD) was constructed in sequential cloning steps. First, an Nhel-
40
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Kpnl PCR fragment containing the AR Kozak sequence was amplified using primers SI
and AS2 and inserted into the corresponding sites of pcDNA3.1 (+). Second, a Kpnl-
EcoRI PCR fragment was amplified using primers S2 and AS3 (Table 1) and inserted into
the restored Kpnl site and the downstream EcoRI site of the pcDNA3.1 (+) multiple
cloning site. BRCAl mammalian expression plasmid pcDNA-BRCAl was constructed
by inserting a 5’ Notl-Xhol 3’-treated BRCAl insert derived from pBSK-lhFL plasmid
(Chen et al., 1999) into corresponding endonuclease restriction sites of a
pcDNA3.1/mycHisC(-) vector (Invitrogen).
Overlapping KpnI-XhoI BRCAl fragments were amplified by PCR using the
indicated primer pairs (Table I). Forward primers contain engineered Kpnl restriction
endonuclease sites followed by a SV40 T antigen nuclear localization signal (NLS).
Reverse primers contain a hemagluttinin A (HA) tag followed by a novel Xhol site.
Elongase PCR (Life Technologies, Rockville, MD) amplified BRCAl fragments were
purified by gel extraction (Qiagen, Valencia, CA), double-digested with KpnI-XhoI and
inserted into a pcDNA3.1 (Kozak) vector.
Bacterial Expression Plasmids
Bacterial expression plasmids encoding GST, GST-AR and GST-GRIP1
fragments were previously described (Chen et al., 1999; Ma et al., 1999).
Tissue culture and transfections
Cells obtained from the American Type Culture Collection (Manassas, VA) were
maintained in RPMI (PC-3, DU-145, and HBL-100) or DMEM (MCF-7) medium that
41
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
contained 10% fetal bovine serum (FBS). Approximately 24 h prior to transfection, 106
(PC-3, DU-145, and HBL-100) or 5 x 10s (MCF-7) cells were seeded into each 60-mm
dish. Cells were transfected in serum-free conditions with Lipofectamine reagent (Life
Technologies, Rockville, MD) according to the manufacturer’s protocol. In each
experiment, the total amount of DNA per dish was held constant by the addition of
pcDNA3.1 (+) vector when appropriate (Invitrogen, Carlsbad, CA). Following
transfection, cells were grown for 24 h (DU-145, HBL-100, and MCF-7) or 48 h (PC-3)
in RPMI medium (without phenol red) that contained 5% charcoal/dextran-stripped FBS
(Gemini Bio Products, Calabasas, CA) and, where indicated, DHT (1 or 10 nM) or 10
nM E2 for the last 24 h of growth. Whole-cell extracts were prepared in 0.25 M Tris-HCl
pH 8.0 by repeated freezing and thawing. CAT assays were performed using the Quan-
T-CAT kit (Amersham Pharmacia Biotech, Piscataway, NJ) (see below) and total cellular
protein was measured using the BioRad (Hercules, CA) Protein Asssay kit. Relative
CAT activities (c.p.m./O.D.^) are reported as the mean ± SE of three independent
dishes.
Chloram phenicol Acetyltransferase (CAT) Assays
Cell extracts were prepared as described above. 40 pi amounts of cell extracts
were transferred into 1.5 ml microcentrifuge tubes. 10 pi of substrate mix was added to
each tube and incubated at 37°C for 30 minutes. After incubation, 1 ml of diluted bead
mix was added to each reaction tube and allowed to stand at room temperature for 5
minutes. Beads were then spun down into tight pellets by centrifugation for 5 minutes
and radioactive supernatant was collected and discarded. Pellet was washed with 1 ml of
42
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
wash buffer and then respun for 3-5 minutes. Supernatants were again discarded and 1
mi of scintillation cocktail was added to each tube. Pellets were mixed by brief vortexing
and reactions were counted and analyzed..
G lutathione-S-T ransferase (GST) Pull-Downs
Glutathione-S-transferase (GST) and GST-fiision proteins were expressed and purified as
described (Hong et al., 1996). Glutathione-Sepharose-bound GST protein, GST-AR (1-
555), or GST-GRIP1 fragments (5-765, 563-1121, or 1121-1462) were incubated with
3 S S-radiolabeled full-length BRCAl or BRCAl fragments transcribed and translated in
vitro from pcDNA3.1 vectors using a TNT-Coupled Reticulocyte Lysate System
(Promega) in the presence of 3 5 -S methionine. Associated BRCAl was eluted, resolved
by SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed by autoradiography.
10% of total labeled BRCAl incubated in each reaction was loaded for comparison.
RESULTS
BRCAl enhances AR signaling
In order to assess the role of BRCAl in AR signaling, we cotransfected PC-3
prostate cancer cells with a wild-type AR expression vector and increasing amounts of a
wild-type BRCAl expression vector. Androgen-dependent activation of AR was
enhanced by coexpression of exogenous BRCAl (Figure 2-1). Substitution of the
BRCAl plasmid with parent vector (pcDNA3.1) failed to generate a coactivation response
indicating that functional BRCAl is required. No coactivation by BRCAl was observed
43
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
in the absence of DHT (Figure 2-1). Furthermore, BRCAl failed to stimulate the reporter
gene in the absence of exogenous AR (data not shown).
Exogenous BRCA l does not increase AR stability
The androgen receptor is highly stabilized by DHT (approximately 2-5 fold). To
test if BRCAl enhancement of AR signaling is biochemical and not due to stabilization,
we transfected CV-1 cells with AR alone (no DHT versus 10 nM DHT) and with AR +
BRCAl (10 nM DHT). As a control baseline, we used untransfected CV-1 cells.
Following transfection, the cells were incubated for 48 hours, lysed in RIPA buffer, and
the extracted lysates were then quantified by BCA protein quantification assay. 7.5 fig of
total lysates were resolved on a 4-20 % gradient gel and transferred onto a PVDF
membrane. Membranes were blotted with anti-AR antibodies and detected by ECL
chemiluminescent reagent. Untransfected cell lysates contained no detectable AR,
whereas transfected cell lysates all demonstrated AR bands. As expected, DHT stabilized
AR approximately 4 fold. However, coexpression of BRCAl in the presence of DHT did
not result in a statistically significant increased AR stability over DHT alone (Figure 2-2).
Hence, the 2-3 fold observed enhancement of AR activity by BRCAl cannot be attributed
to a stabilization phenomenon.
BRCAl enhances AF-1 of the AR
Full-length BRCAl was coexpressed in PC-3 cells with either a constitutively
active AR variant comprising the NTD and the DBD, i.e. AR(NTD-DBD), to assess AR
AF-1 activity, or a variant comprising the DBD and LBD, i.e. AR(DBD-LBD), to assess
44
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
AR AF-2 activity. BRCAl enhanced the activity of AR(NTD-DBD) but was unable to
activate AR(DBD-LBD) in either the presence or absence of ligand (Figure 2-3).
Therefore, BRCAl is able to coactivate the AF-1 function of the AR.
Potentiation of pl60 coactivation by BRCAl
The pl60 coactivators (i.e. SRCl/NcoAl, GRIPl/TIF2/NcoA2, and
AIBl/p/CIP/ACTR) are nuclear proteins that bind to NRs and potentiate ligand-dependent
receptor signaling by recruiting transcriptional regulatory proteins, including histone
acetyltransferases (McKenna et al., 1999; Xu et al., 1999) and methyltransferases (Chen
et al., 1999). They interact with, and coactivate the AR through both AF-1 and AF-2 of
the receptor (Ma et al., 1999). To determine if BRCAl is involved in pl60-mediated
coactivation of AR signaling, we cotransfected mammalian cell lines with expression
vectors for BRCAl and/or GRIP1, SRC-la, or AIB1 along with AR. BRCAl and the
p i60 coactivators individually were able to enhance AR activity in PC-3 cells in a
hormone-dependent fashion (Figure 2-4). Coexpression of BRCAl with each of the
pl60 coactivators resulted in synergistic coactivation of AR signaling (Figure 2-4), results
that were duplicated using GRIP1 and BRCAl in a second prostate cancer cell line (DU-
145), and two breast cell lines (HBL-100 and MCF-7) (Figure 2-5). The relatively small
effects observed in MCF-7 cells may be due to the overexpression of endogenous A1B1
(Anzick et al., 1997).
45
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
BRCAl enhances GRIPl-mediated coactivation of AR LBD
Since BRCAl was able to potentiate pl60-mediated coactivation of wild-type AR,
we argued that it should be able to do the same with the AR LBD. As expected, in
transiently transfected PC-3 cells, hormone-dependent reporter gene activation by the
AR(DBD-LBD) was enhanced by GRIP1 coexpression (Figure 2-6). Furthermore, this
activity was potentiated by coexpression of BRCAl, although BRCAl had little or no
effect on AR(DBD-LBD) signaling in the absence of GRIP1 coexpression. Similar
results were observed when AIB1 was substituted for GRIP1 (data not shown).
Therefore, BRCAl can coactivate AR AF-2, but only in the presence of co-associated
pi 60 coactivators.
BRCAl interacts with the N-terminus of AR and the C-terminus of GRIP1
The ability of BRCAl to coactivate AR and to potentiate pl60-mediated
enhancement of AR signaling suggests physical interactions. To determine if BRCAl
physical associates with the AR and/or GRIP1, in vitro protein binding experiments were
performed using glutathione-S-transferase (GST)-fused fragments of either AR or GRIP1
and radiolabeled full-length BRCAl. Schematic representations of the AR, BRCAl and
GRIP1, including major domains and motifs, are shown (Figure 2-7). In these
experiments, full-length BRCAl was found to interact specifically with both GST-AR (1-
555) (Figure 2-8) and GST-GRIPlc (1121-1462) (Figure 2-9). No interactions were
detected with GST-GRIP1 (5-765, 563-1121) or GST-protein alone.
46
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Both AR and GRIP1 Interact with the N-terminus of BRCAl
In order to map the interactions of both the AR-NTD and GRIPlc onto BRCAl,
3 5 S-radiolabeled, overlapping fragments of BRCAl were incubated with GST-AR (1-
555) orGST-GRIPlc (1121-1462) and analyzed as described above. Pull-down results
localize both AR and GRIP1 interactions to a region in the N-terminus of BRCAl
spanning amino acids 1-404 (Figure 2-10). This region of BRCAl contains a cysteine-
rich zinc-binding domain, or RING finger, which is believed to function in protein-
protein interactions (1). However, it is not determined if this RING finger is important in
mediating AR and/or GRIP1 binding.
BRCAl coimmunoprecipitates with GRIP1, but not with GRIP1AAD2
To test if BRCAl and GRIP1 interacts in mammalian cells, we cotransfected
SV40-transformed COS-7 monkey kidney cells with BRCAl and either HA-GRIP1 or
HA-GRIP1AAD2, which lacks the AD2 interaction domain. As a negative control,
untransfected cells were grown simultaneously. Whole cell lysates (WCL) were
immunoprecipiated with either a BRCAl polyclonal antibody (C-20, Santa Cruz), or a
non-specific rabbit polyclonal antibody (rabbit IgG, Zymed). Immune complexes were
stringently washed and eluted proteins were resolved by SDS-PAGE and transferred onto
a PVDF membrane. Membranes were probed for coimmunoprecipitated HA-GRIP or
HA-GRIPAAD2 using a rat anti-HA antibody (Roche Pharmaceuticals). Full-length HA-
GRIP 1, but not the truncated AAD2 mutant, coimmunoprecipitated with BRCAl (Figure
2-11). This interaction was specific for BRCAl as no coimmunoprecipiated HA-GRIP 1
47
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
was detected when normal rabbit IgG was used. Likewise, no detectable band was seen
in the untransfected negative control. To demonstrate that these results were not due to
differences in HA-GRIP 1 or HA-GRIP 1AAD2 expression, samples of each WCL used
for the coimmunoprecipitation assays were screened by western blotting to detect HA-
tagged proteins in transfected versus untransfected cells (Figure 2-10). Using a rat anti-
HA antibody, appropriate bands of expected molecular weights were detected in each of
the samples derived from transfected cells, but no bands were observed in the
untransfected cells.
BRCAl synergy with GRIP1 is dependent upon an intact AD2 domain
Since BRCAl was unable to interact with the GRIP1AAD2 mutant by GST pull
down assay and coimmunoprecipitation, we wanted to test if this loss of interaction
resulted in a corresponding loss of functional synergy. In CV-1 cells, we transfected AR,
BRCAl and combinations of either HA-GRIP1, HA-GRIPIAADI, or HA-GRIP1AAD2.
Luciferase activities were assayed using the methods described above [Methods]. Both
HA-GRIP 1 and HA-GRIPAAD1 were able to enhance BRCAl activity on AR signaling.
HA-GRIP 1AAD2, however, was unable to function with BRCAl, suggesting that
BRCAl and GRIP1 synergy is dependent upon direct interaction between the two
coactivators (Figure 2-12).
48
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
DISCUSSION
Several independent lines of evidence suggest that BRCAl may play a role in
transcriptional regulation. BRCAl contains a transcriptional activation domain (Chapman
and Verma, 1996; Monteiro et al., 1996), that functions as a coactivator of p53-dependent
gene expression (Ouchi et al., 1998; Somasundaram et al., 1997; Zhang et al., 1998) and
has been shown to be associated with the RNA polymerase II holoenzyme complex
(Anderson et al., 1998; Scully et al., 1997). The recent finding that BRCAl inhibits ER
activity (Fan et al., 1999) suggested, at that time, that BRCAl may protect against breast
cancer risk due to this inhibition. In the present study, we demonstrate that BRCAl
enhances androgen-responsive reporter gene expression by interacting with, and
activating AF-1 of the AR NTD. Furthermore, BRCAl can potentiate the effect of pl60
coactivators on AR signaling in breast and prostate cell lines, possibly by modulating
nuclear receptor-pl60 coactivator interactions. Interestingly, both the AR and the pl60
coactivators appear to interact with the N-terminus of BRCAl. This is also the region of
BRCAl where both p53 (Ouchi et al., 1998; Somasundaram et al., 1997; Zhang et al.,
1998) and c-myc binding occurs (Wang et al., 1998), revealing a novel feature of this
relatively small, but important, region. It is possible that BRCAl potentially modulates
transcription by stabilizing interactions between transcription factors associated with its
N-terminus and the transcriptional initiation complex bound to its C-terminus. Indeed,
deletion of this C-terminal interaction domain results in the loss of BRCAl’s coactivation
function as it can no longer recruit the preinitiation complex to sites of active gene
transcription (Ouchi et al., 1998; Somasundaram et al., 1997; Zhang et al., 1998).
Therefore, mutations which lead to premature truncation of BRCAl likely result in
defective, or impaired, function due to loss of this bridging interaction, though
49
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
presumably, BRCAl’s ability to associate with transcriptional regulatory proteins through
its N-terminus is unaffected. Thus, it is possible that truncated forms of BRCAl may act
as dominant negative inhibitors in heterozygous mutation carriers resulting in significant
dysregulation of transcription by sequestering limiting factors, thereby, diminishing the
effectiveness of the remaining wild-type BRCAl protein.
The results of this study suggest a complex interplay between AR, the pl60
coactivators and BRCAl in modulating cell proliferation, and by implication cancer risk,
in tissues like the prostate and breast. In prostate, loss of BRCAl was initially thought to
be associated with an increased risk for developing cancer, although later studies
considering specific mutations failed to demonstrate any such correlation in mutation
carriers (Hubert et al., 1999; Johannsson et al., 1999; Struewing et al., 1997). In
retrospect, this is not surprising since BRCAl appears to function as a coactivator of AR
signaling. As such, the loss of BRCAl might, in fact, protect against prostate cancer by
decreasing AR activity.
In the case of breast cancer risk, women with only one functional copy of BRCAl
exhibit a significant correlation between AR-CAG size alleles and early-onset disease
(Rebbeck et al., 1999), suggesting that BRCAl acts to maintain AR’s protective effect on
the breast by potentiating AR activity during normal signaling events. According to this
model, when there is partial loss of BRCAl function, AR signaling might be significantly
diminished, potentially leading to greater breast cell proliferation. The fact that both lower
AR activity and AIB1 alterations in BRCAl mutation carriers result in similar cancer
phenotypes and risk profiles supports this hypothesis. Although further studies are
required, these findings may help in our understanding of the role of BRCAl in breast
cancer development, and may be useful in remodeling current treatment protocols based
on the unique biology of BRCAl-associated cancers.
50
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 2-1. W ild-type BRCAl coactivates AR transactivation in PC -3
prostate cancer cells. Transiently transfected cells were assayed for stimulation of
ARR3 tk-CAT reporter activity by dihydrotestosterone (DHT). ARR3 tk-CAT is composed
of a minimal thymidine kinase (TK) promoter under the control of three identical
fragments of the rat probasin promoter (nucleotides -244 to -96) each comprising two
androgen responsive elements (i.e., ARBS-1 and ARBS-2) (Snoek et al., 1998). Cells
were cotransfected with 2.0 |ig ARR3 tk-CAT, 50 ng pCMV-hAR, and increasing
amounts of pcDNA-BRCAl as indicated. Total transfected DNA was held constant by
the addition of pcDNA3.1 vector when appropriate. Chloramphenicol acetyl transferase
(CAT) activities were normalized for total cellular protein and data presented are the mean
+ SE of three independent dishes. Fold is measured relative to DHT-dependent AR
activity with no transfected BRCAl. N.B. Figure courtesy o f Dr. Ryan Irvine.
51
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 2-1
600
I j no hormone
o 400
1 0
s 200
2.5 ug
ARR3TK-CAT + +
pCMV-hAR + -f
pcDNA-BRCA1 - -
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
fold
Figure 2-2. AR is not stabilized by BRCAl coexpression. SxlC^/well PC-3
prostate cancer cells were grown overnight on 24-well tissue culture plates. Next day,
cells were transfected with the following amounts of plasmids, as described above: 50 ng
CMV-AR, 250 ng pcDNA3.1-BRCAl. Transfections were performed in triplicates using
Superfect reagent (Gibco-BRL) as per Superfect protocol. Cells were then incubated for
48 hours either in the absence or precence of 10 nM DHT. After the incubation time,
lysates were harvested in RIPA buffer and quantified by BCA Protein Quantification
Assay (Pierce). 7.5 ng of total lysates/sample were loaded onto a 4-20% gradient gel
(Bio-Rad), resolved by SDS-PAGE, and then transferred onto a PVDF membrane.
Western immunoblot analysis was performed using a rabbit-anti(AR) polyclonal antibody
(lmg/ml) (Santa Cruz). Gel Quantification and analysis was done using a GS-710
calibrated imaging densitometer (Bio-Rad). Bar graphs are shown as relative
transmission O.D. (relative to AR, 10 nM DHT). There is no significant increase in AR
band density observed in the presence of BRCAl.
53
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 2-2
2
1.75
1.5
S
| 1.25
d
o 1
U
« 0.75
o >
a
0.5 -
0.25 -
0
10nM DHT
CMV-AR
pcDNA-BRCAl
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1.30X
Figure 2-3. BRCA l works through AR AF-1 in PC-3 cells. Cells were
cotransfected with 50 ng pCMV-hAR, 10 ng pcDNA-AR (NTD-DBD), or 0.5 n g
pcDNA-AR (DBD-LBD), 2.0 ng ARR3 tk-CAT, and 2.5 ng pcDNA-BRCAl as
indicated. Mammalian expression vectors pcDNA-AR (NTD-DBD) and pcDNA-AR
(DBD-LBD) encode AR amino acids 1-647 and 538-919, respectively. AR (NTD-DBD)
is a constitutive activator of ARR3 tk-CAT and thus, potentiation of its activity by BRCAl
is ligand independent. N.B. Figure courtesy o f Dr. Ryan Irvine.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 2-3
500-
| | no hormone PC-3
£ 400
$ 200
£ 100
ARR3TK-CAT
AR(wt)
AR (NTD-DBD)
AR (DBD-LBD)
BRCA1
+ + + +
+ +
+ + + +
+ +
+ + +
+
+
+
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 2-4. Synergistic coactivation of AR signaling by BRCA l and
members of the pl60 family of nuclear receptor coactivators. PC-3 cells
were cotransfected with 2.0 pg pSG5-GRIPl, pcDNA3.1-AIBl, or pSG5-SRC-la, 2.0
pg ARR3 tk-CAT, 25 ng pCMV-hAR, and 2.5 pg pcDNA-BRCAl as indicated. In each
case, AR transactivation activity in the presence of transfected BRCAl and pl60
coactivator was greater than the additive effects of BRCAl and p i60 coactivator assayed
separately. N.B. Figure courtesy of Dr. Ryan Irvine.
57
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 2-4
500
S 200-
ARR3TK-CAT
SRC-1 a
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 2*5. Potentiation of AR signaling by BRCA l occurs in both
prostate* and breast-derived cell lines. Prostate cell line DU-14S and breast cell
lines HBL-100 and MCF-7 were cotransfected with 2.0 pg ARR3 tk-CAT, 25 ng pCMV-
hAR, 2.0 pg pSG5-GRIPl, and 2.5 pg pcDNA-BRCAl as indicated. The data
presented are fold + SE relative to DHT-dependent AR activity with no transfected
BRCAl or GRIP1. As in PC-3, a synergistic coactivation of AR signaling by BRCAl
and GRIP1 occurred in these cell lines. The relatively small effects observed in MCF-7
may be due to the overexpression of endogenous AIB1 in this cell line (Anzick et al.,
1997). N.B. Figure courtesy o f Dr. Ryan Irvine.
59
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 2-5
D H '
GRIP1
BRCA1
DU-145 HBL-100 MCF-7
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
+ +
Figure 2-6. BRCA l potentiates G R IPl-m ediated coactivation of AR AF-2
on the MMTV promoter. PC-3 cells were transfected with 2.0 pg MMTV-CAT, 1.0
pg pcDNA-AR (DBD-LBD), 2.0 pg pSG5-GRIPl, and 2.5 pg pcDNA-BRCAl as
indicated. BRCAl failed to coactivate AR AF-2 in the absence of exogenously expressed
GRIP1 suggesting that it cannot make a functional contact with AR LBD. Since BRCAl
potentiates GRIPl-mediated coactivation of AR AF-2, it is likely that GRIP1 recruits
BRCAl through direct contacts. N.B. Figure courtesy o f Dr. Ryan Irvine.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 2-6
200
> 150-
< 100-
•S 50-
2
DHT + + + +
MMTV-CAT + + + +
AR (DBD-LBD) + + + +
GRIP1 + +
BRCA1 + - +
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 2-7. Schematic diagram s of AR, GRIP1, and BRCA1 showing the
locations of various functional domains. Domains of AR: AF-l/AF-2,
autonomous activation functions 1 and 2; NTD, N-terminal domain; DBD, DNA-binding
domain; LBD, ligand-binding domain; Q/P/G, glutamine/proline/glycine poly-amino acid
stretches. Domains of GRIP 1: bHLH, basic helix-loop-helix sequence; PAS, Per-Amt-
Sim domain; NR boxes, nuclear receptor binding domains (LXXLL motifs); CID, CBP
interaction domain; AD 1/AD2, activation domains. Domains of BRCA1: RING, zinc-
finger domain; NLS, nuclear localization signals; BRCT, BRCA1 carboxy terminus.
Numbers represent relative amino acid positions.
63
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 2-7
A. A ndrogen Receptor
AM A M
E
1 KID
J U
D 60 LBD 919
B. GRIP1
bHLHIPAS
1
NR Box
563 765
C K V A D 1 A02
1121 1462
C. BRCA1
RUG
r u ~
1
NLS
560
BRCT
■
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 2-8. Full-length BRCA1 interacts with the N-term inal domain of
the androgen receptor. Glutathione-S-transferase (GST) and GST-fusion proteins
were expressed and purified as described (Chen et al., 1999). Glutathione-Sepharose-
bound GST protein and GST-AR (1-555) were incubated with 3 S S-radiolabeled BRCA1
transcribed and translated in vitro from pcDNA3.1 vector encoding full-length BRCA1.
Associated BRCA1 was eluted, resolved by SDS-polyacrylamide gel electrophoresis
(PAGE) and analyzed by autoradiography. 10% of total labeled BRCA1 incubated in
each reaction was loaded for comparison.
Figure 2-9. Full-length BRCA1 interacts with the C-term inus of GRIP1.
Glutathione-S-transferase (GST) and GST-fusion proteins were expressed and purified as
described (Chen et al., 1999). Glutathione-Sepharose-bound GST protein or GST-
GRIP1 fragments (5-765,563-1121, or 1121-1462) were incubated with 3 S S-radiolabeled
BRCA1 transcribed and translated in vitro from pcDNA3.1 vector encoding full-length
BRCA1. Associated BRCA1 was eluted, resolved by SDS-polyacrylamide gel
electrophoresis (PAGE) and analyzed by autoradiography. 10% of total labeled BRCA1
incubated in each reaction was loaded for comparison
65
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURES 2-8 & 2-9
AF-1 AF-2
a r r ■ ■ i z r: ~ i 1
1 NTD OBO LBO 919
bHLH/PAS NR Box CID/AD1 AD2
Q R 'P I I M W 1 1 1 T ------ —
1 963 765 1121 1462
RING NLS BRCT
BRCA1 I 1 " II ' — — 1
1 500 1863
a
£ <[|A
i !o i s 5 ?
° O o c
BRCA1
■
AR
FIGURE 2-8
< 3
BRCA1
GRIP1
FIGURE 2-9
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 2-10. Autoradiographs showing the localization of the AR NTD
and G R IPlc interactions on BRCA1. Plasmids containing fragments of BRCA1
were generated as described. Diagrams of the functional domains corresponding to amino
acids are provided for reference. Unpurified in vitro translated BRCA1 fragments were
incubated with GST, GST-AR (1-555) or GST-GRIP1 (1121-1462). Both AR and
GRIP1 binding is shown to localize to the N-terminus of BRCA1.
67
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 2-10
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
GST-GRIP1
(1121-1462)
Figure 2-11. BRCA1 coim m unoprecipitates with GRIP1, but not w ith
GRIP1AAD2. COS-7 monkey kidney cells were transfected with HA-GRIP1 or HA-
GRIP1AAD2. Untransfected cell were grown simultaneously as a control. All samples
were grown and treated in triplicates. Whole cell lysates were extracted,
immunoprecipitated with either 1 mg/mL of specific rabbit anti-BRCAl polyclonal
antibodies (C20, Santa Cruz) or 1 mg/mL of non-specific rabbit polyclonal antibodies
(Zymed), and washed with R1PA buffer (plus complete protease inhibitors, Roche
Pharmaceuticals). Immune complexes were eluted in sample buffer and resolved by
SDS-PAGE. Proteins were transferred onto PVDF membranes and probed for HA-
GRIP1 or the AAD2 mutant using a rat anti-HA polyclonal antibody (Roche
Pharmaceuticals).
69
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 2-11
IP (anti-) C-20 IgG C-20 C-20
1 2 3 4
I 1 I ------------------------------ 1
HA-GRIP1
HA-GRIP1AAD2
Coimmunoprecipitation Assay
HA-GRIP1
HA-GRIP1AAD2
Western Immunoblot Analysis
1 - Untransfected
2 - BRCA1/HA-GRIP1
3-BRCA1/HA-GRIP1
4 - BRCA1/HA-GRIP1AAD2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 2*12. Synergy with GRIP1 is dependent upon an intact AD2
dom ain. CV-1 ceils were transfected with AR, BRCA1 and/or HA-GRIP1 plasmids.
H A-GRIP1AAD1 and HA-GRIP1A AD 2 mutants were substituted for HA-GRIP1 where
indicated. GRIP1 and GRIP1 mutant effects on BRCA1 synergy were assessed by
luciferase assay and plotted with respect to AR and AR+BRCA1 activities.
71
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 2-12
f
I
12
10 J
8
6
4 ■
2 ■
IGRIP1
□GRIP1AAD1 mmMm
□QHIP1AA02 in u n
n n n
10 nM DHT
m
+ + + - + + + ■ + + +
AR + + + + + + + + + + + +
BRCA1
- - + + - -
+ +
-
+ +
GRIP1
- - - + - - ■ • m • m
GRIP1AAD1
- - - - - ■
+
m • •
GRIP1AAD2 - - - - - - m m •
+
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
REFERENCES
Anderson, S. F., Schlegel, B. P., Nakajima, T., Wolpin, E. S., and Parvin, J. D.
(1998). BRCA1 protein is linked to the RNA polymerase n holoenzyme complex via
RNA helicase A. Nature genet 19,254-256.
Anzick, S. L., Kononen, J., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X.-
Y., Sauter, G., Kallioniemi, O.-P., Trent, J. M., and Meltzer, P. S. (1997). AIBl, a
steroid receptor coactivator amplified in breast and ovarian cancer. Science 277,965-967.
Chamberlain, N. L., Driver, E. D., and Miesfeld, R. L. (1994). The length and location
of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect
transactivation function. Nucleic Acids Res 22, 3181-6.
Chapman, M. S., and Verma, I. M. (1996). Transcriptional activation by BRCA1.
Nature 382, 678-679.
Chen, D., Ma, H., Hong, H., Koh, S. S., Huang, S.-M., Schurter, B. T., Aswad, D.
W., and Stallcup, M. R. (1999). Regulation of transcription by a protein
methyltransferase. Science 284,2174-2177.
Chen, J.-J., Silver, D., Cantor, S., Livingston, D. M., and Scully, R. (1999). BRCA1,
BRCA2, and Rad51 operate in a common DNA damage response pathway. Cancer Res
59, 1752s-1756s.
73
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Chen, Y., Lee, W. H., and Chew, H. K. (1999). Emerging roles of BRCA1 in
transcriptional regulation and DNA repair [In Process Citation], J Cell Physiol 181, 385-
92.
Duncan, J. A., Reeves, J. R., and Cooke, T. G. (1998). BRCA1 and BRCA2 proteins:
roles in health and disease. Mol Pathol 51, 237-247.
Edwards, A., Hammond, H. A., Lin, J., Caskey, C. T„ and Chakraborty, R. (1992).
Genetic variation at trimeric and tetrameric tandem repeat loci in four human population
groups. Genomics 12, 241-253.
Fan, S., Wang, J., Yuan, R., Ma, Y., Meng, Q., Erdos, M. R., Pestell, R. G., Yuan,
F., Aubom, K. J., Goldberg, I. D., and Rosen, E. M. (1999). BRCA1 inhibition of
estrogen receptor signaling in transfected cells. Science 284, 1354-6.
Giguere, B., Hollenberg, S. M., Rosenfeld, M. G., and Evans, R. M. (1986).
Functional domains of the human glucocorticoid receptor. Cell 46,645-652.
Giovannucci, E., Stampfer, M. J., Krithivas, K., Brown, M., Brufsky, A., Talcott, J.,
Hennekens, C. H., and Kantoff, P. W. (1997). The CAG repeat within the androgen
rceptor gene and its relationship to prostate cancer. Proc Natl Acad Sci USA 94, 3320-
3323.
Green, S., Issemann, I., and Sheer, E. (1988). A versatile in vivo and in vitro eukaryotic
expression vector for protein engineering. Nucl Acids Res 16.
74
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Hackenberg, R., Hawighorst, T., Filmer, A., Nia, A. H., and Schulz, K. D. (1993a).
Medroxyprogesterone acetate inhibits the proliferation of estrogen- and progesterone-
receptor negative MFM-223 human mammary cancer cells via the androgen receptor.
Breast Cancer Res Treat 25,217-224.
Hardy, D. O., Scher, H. I., Bogenreider, T„ Sabbatini, P., Zhang, Z. F., Nanus, D.
M., and Catterall, J. F. (1996). Androgen receptor CAG repeat lengths in prostate cancer:
correlation with age of onset. J Clin Endocrinol Metab 81,4400-5.
Hong, J., Kohli, K., Trivedi, A., Johnson, D. L., and Stallcup, M. R. (1996). GRIP1,
a novel mouse protein that serves as a transcriptional co-activator in yeast for the hormone
binding domains of steroid receptors. Proc Natl Acad Sci USA 93,4948-4952.
Hubert, A., Peretz, T., Manor, O., Kaduri, L., Wienberg, N., Lerer, I., Sagi, M., and
Abeliovich, D. (1999). The Jewish Ashkenazi founder mutations in the BRCA1/BRCA2
genes are not found at an increased frequency in Ashkenazi patients with prostate cancer.
Am J Hum Genet 65,921-924.
Irminger-Finger, I., Siegel, B. D., and Leung, W. C. (1999). The functions of breast
cancer susceptibility gene 1 (BRCA1) product and its associated proteins. Biol Chem
380, 117-128.
75
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Irvine, R. A., Ma, H., Yu, M. C., Ross, R. K., Stallcup, M. R., and Coetzee, G. A.
(2000). Inhibition of pl60-mediated coactivation with increasing androgen receptor
polyglutamine length. Hum Mol Genet 9,267-274.
Irvine, R. A., Yu, M. C., Ross, R. K., and Coetzee, G. A. (1995). The CAG and GGC
microsatellites of androgen receptor gene are in linkage disequilibrium in men with
prostate cancer. Cancer Res 55, 1937-1940.
Johannsson, O., Loman, N., Moller, T., U., K., Borg, A., and Olsson, H. (1999).
Incidence of malignant tumours in relatives of BRCA1 and BRCA2 germline mutation
carriers. Eur J Cancer 35, 1248-1257.
Lobaccaro, J.-M., Lumbroso, S., Belon, C., Galtier-Dereure, F., Bringer, J., Lesimple,
T., Heron, J.-F., Pujol, H., and Sultan, C. (1993). Male breast cancer and the androgen
receptor gene. Nature Genet 5, 109-110.
Ma, H., Hong, H., Huang, S. M., Irvine, R. A., Webb, P., Kushner, P. J., Coetzee,
G. A., and Stallcup, M. R. (1999). Multiple signal input and output domains of the 160-
kilodalton nuclear receptor coactivator proteins. Mol Cell Biol 19,6164-6173.
Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K.,
Blumberg, B., Kastner, P., Mark, M., and Chambon, P. e. a. (1995). The nuclear
receptor superfamily: the second decade. Cell 583, 835-839.
76
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
McKenna, N. J., Xu, J., Nawaz, Z., Tsai, S. Y., Tsai, M.-J., and O'Malley, B. W.
(1999). Nuclear receptor coactivators: multiple enzymes, multiple complexes, multiple
functions. J Ster Biochem Mol Biol 69, 3-12.
Monteiro, A. N. A., August, A., and Hanafusa, H. (1996). Evidence for a transcriptional
activation function of BRCA1 C-terminal region. Proc Natl Acad Sci USA 93, 13595-
13599.
Munoz de Toro, M., Maffini, M. V., Kass, L., and Luque, E. H. (1998). Proliferative
activity and steroid hormone receptor status in male breast carcinoma. J Steroid Biochem
Mol Biol 67, 333-339.
Ouchi, T., Monteiro, A. N., August, A., Aaronson, S. A., and Hanafusa, H. (1998).
BRCA1 regulates p53-dependent gene expression. Proc Natl Acad Sci U S A 95,2302-6.
Rahman, N., and M.R., S. (1998). The genetics of breast cancer susceptibility. Annu
Rev Genet 32, 95-121.
Rebbeck, T. R., Kantoff, P. W., Krithivas, K., Neuhausen, S., Blackwood, M. A.,
Godwin, A. K., Daly, M. B., Narod, S. A., Garber, J. E., Lynch, H. T., Weber, B. L.,
and Brown, M. (1999). Modification of BRCA1-associated breast cancer risk by the
polymorphic androgen-receptor CAG repeat. Am J Hum Genet 64, 1371-7.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Ross, R. K., Coetzee, G. A., Reichardt, J. K., Yu, M. C., Feigelson, H., Stanczyk, F.
Z., Kolonel, L. N., and Henderson, B. E. (1998). Androgen metabolism and prostate
cancer: establishing a model of genetic susceptibility. Cancer Res 58,4497-4504.
Scully, R., Anderson, S. F., Chao, D. M., Wei, W., Ye, L., Young, R. A., Livingston,
D. M., and Parvin, J. D. (1997). BRCA1 is a component of the RNA polymerase II
holoenzyme. Proc Natl Acad Sci U S A 94,5605-10.
Snoek, R., Bruchovsky, N., Kasper, S., Matusik, R. J., Gleave, M., Sato, N., Mawji,
N. R., and Rennie, P. S. (1998). Differential transactivation by the androgen receptor
cancer cells. Prostate 36,256-263.
Somasundaram, K., Zhang, H., Zeng, Y. X., Houvras, Y., Peng, Y., Wu, G. S.,
Licht, J. D., Weber, B. L., and El-Deiry, W. S. (1997). Arrest of the cell cycle by the
tumour-suppressor BRCA1 requires the CDK-inhibitor p21WAFl/CiPl. Nature 389,
187-90.
Spurdle, A. B., Dite, G. S., Chen, X., Mayne, C. J., Southey, M. C„ Batten, L. E.,
Chy, H., Trute, L., McCredie, M. R., Giles, G. G., Armes, J., Venter, D. J., Hopper,
J. L., and Chenevix-Trench, G. (1999). Androgen receptor exon 1 CAG repeat length
and breast cancer in women before age forty years. J Natl Cancer Inst 91,961-6.
Struewing, J. P., Hartge, P., Wacholder, S., Baker, S. M., Berlin, M., McAdams, M.,
Timmerman, M. M., Brody, L. C., and Tucker, M. A. (1997). The risk of cancer
78
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews. N
Engl J Med 20, 1401-1408.
Szelie, J., Jimenez, J., Soto, A. M., Luizzi, M. F., and Sonnenshein, C. (1997).
Androgen-induced inhibition of proliferation in human breast cancer MCF7 cells
transfected with androgen receptor. Endocrinology 138, 1406-1412.
Tilley, W. D., Marcelli, M., J.D., W„ and M.J., M. (1989). Characterization and
expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci USA
86, 327-331.
Wang, Q., Zhang, H., Kajino, K., and Greene, M. I. (1998). BRCA1 binds c-Myc and
inhibits its transcriptional and transforming activity in cells. Oncogene 17, 1939-48.
Webb, P., Lopez, G. N., Uht, R. M., and Kushner, P. J. (1995). Tamoxifen activation
of the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like
effects of antiestrogens. Mol Endocrinol 9,443-456.
Wooster, R., Mangion, J., Eeles, R., Smith, S., Dowsett, M., Averill, D., Barrett-Lee,
P., Easton, D. F., Ponder, B. A. J., and Stratton, M. R. (1992). A germline mutation in
the androgen receptor gene in two brothers with breast cancer and Reifenstein syndrome.
Nature Genet 2, 132-134.
Xu, L., Glass, C. K., and Rosenfeld, M. G. (1999). Coactivator and corepressor
complexes in nuclear receptor function. Curr Op Gen Dev 9.
79
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Zhang, H., Somasundaram, K., Peng, Y., Tian, H., Bi, D., Weber, B. L., and El-
Deiry, W. S. (1998). BRCA1 physically associates with p53 and stimulates its
transcriptional activity. Oncogene 16, 1713-21.
Zhu, X., Daffada, A. A. I., Chan, C. M. W., and Dowsett, M. (1997). Identification of
an exon 3 deletion splice variant androgen receptor mRNA in human breast cancer. Int J
Cancer 72, 574-580.
80
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
CHAPTER 3. An Emerging Role for BRCA1 in Estrogen Receptor
and Progesterone Receptor Regulation: Clearing up the Controversy
ABSTRACT \ > ’ i
!
BRCA1 is a tumor suppressor with roles in DNA repair, cell-cycle control, and
transcriptional regulation. We have previously demonstrated that BRCA1 functions in
androgen receptor signaling by enhancing receptor-mediated transcription activation,
both alone and in the presence of the p i60 nuclear receptor coactivators, GRIP1,
SRC la, and AIB1. Furthermore, we have shown that this functional coactivation is
mediated by direct physical interactions between BRCA1 and AR, as well as, BRCA1
and the pl60 coactivators. In the current study, we now describe an emerging role for
BRCA1 in the positive regulation of estrogen receptor (ER) and progesterone receptor
(PR) signaling, supporting a general role for BRCA1 as a coactivator of the steroid
receptors. This enhancement of both ER and PR signaling is potentiated in the presence
of GRIP1, suggesting a functional coordination between the two coactivators during
hormone signaling. Although these results contradict a previous report describing
BRCA1 as an inhibitor of ER signaling, several lines of evidence lead us to conclude
that BRCA1, under physiologic conditions, is indeed a coactivator of ER: (1) dose-
response curves demonstrate that BRCA1 functions as a coactivator under conditions of
low exogenous ER, and exhibits non-specific inhibition (squelching artifact) at higher
81
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
doses of exogenous ER; (2) synergy between BRCA1 and GRIP1 depends upon
receptor dose, showing enhancement at low-dose ER, and inhibition at high-dose ER;
and (3) previous studies demonstrate that forced overexpression of BRCA1 results in
general growth arrest and cellular toxicity, which may be mistaken for inhibition under
transient transfection assays. Therefore, these results imply inherent differences in
coactivator function on ER activity, as assessed by transient transfection assays, that
may depend upon ER dose rather than actual physiological function. Indeed, ER dose
curves reveal differential transcription activation kinetics throughout a broad dose range
characterized by three distinct phases, a linear, Dose-Dependent Phase at low doses, an
Inhibitory (Squelching) Phase at intermediate to moderately-high doses, and a
Refractory (Coactivator-Independent) Phase at high doses of transfected ER. Together,
these results support the role for BRCA1 as a coactivator of ER, and may explain the
putative inhibition observed by the previous study using high doses of ER and BRCA1.
Hence, when studying coactivator function in nuclear receptor signaling, it is important
to utilize nuclear receptor conditions that closely mirrors physiological conditions and
will not result in artifacts due to improper methodology. Finally, we demonstrate that
BRCA1 is also a coactivator of the progesterone receptors (PR), both alone and in the
presence of GRIP1.
82
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
INTRODUCTION
Breast cancer is the most common malignancy among women in the United
States with approximately 178,700 new cases diagnosed each year (Landis S et al.,
1998). It is the second most common cause of cancer-related death. In spite of the
importance of this disease, relatively little is known about the control mechanisms of
cell proliferation in breast epithelium, although proliferative activity is considered to be
a critically important determinant of carcinogenesis (Cohen SM and Ellwein LB, 1990).
Development of malignant phenotypes appear to involve multiple stages with the
accumulation of mutational events occurring within the DNA of cells. Cell division
increases the risk of genetic errors and serves to propagate the errors to daughter cells.
Since single-stranded DNA errors can be repaired, the rate of DNA repair and the rate
of cell division are both important in establishing a mutation in the genome. Single
stranded DNA damage may be converted through mitotic activity to gaps or mutations,
and through nondisjunction into more substantial changes. Activation or alteration of
proto-oncogene expression and the loss or inactivation of tumor-suppressor genes,
which control normal cellular activity, is considered to be particularly important. Many
of the genes which are altered in human cancers are growth factors, growth factor
receptors, signal transducers or transcription factors. The progressive accumulation of
genetic alterations is expected to lead to the development of carcinoma in situ and,
83
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
subsequently, with accumulation of additional genetic alterations, to invasive
carcinoma.
Circumstantial Evidence for the Importance of Proliferative Activity in Breast
Cancer Development and the Role of Ovarian Hormones
There is considerable evidence that ovarian hormones have an important effect
on breast cancer risk (Henderson BE et al., 1982). Late menarche and early menopause
(or ovariectomy) are associated with a lower risk of developing breast cancer (Kelsey
JL et al., 1993; Moore DH et al., 1983). The protective effect of early menopause
(Kelsey JL et al., 1993; MacMahon B et al., 1973; Trichopolous D et al., 1972) is an
important risk factor that can be used to illustrate the potential role of ovarian hormones
in the etiology of breast cancer. For almost all non-hormone dependent cancers, when
the logarithm of incidence is plotted against age, the resultant "curve" approximates a
single straight line, but for breast cancer (and endometrial cancer) it can be
approximated by two straight lines, one with a very steep slope before age 50 years and
another with a diminished slope thereafter. The increase in incidence with age is much
steeper during the premenopausal period than after the menopause (Pike MC, 1987).
Women who stop menstruating before age 40, either naturally or through surgical
intervention, have half the risk of breast cancer of women who continue to menstruate
to age 50. This strongly suggests that the hormonal pattern of premenopausal women,
i.e. cyclic production of relatively large amounts of estradiol and progesterone, causes a
84
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
greater rate of increase in the risk of breast cancer than the hormonal pattern of
postmenopausal women (constant low estrogen and very low progesterone). However,
the protective effect of early menopause gives no information on the relative importance
of these two ovarian hormones in determining risk.
Studies of endometrial cancer have led to the "unopposed-estrogen hypothesis"
for this cancer (Henderson BE et al., 1982; Siiteri PK, 1978). This hypothesis maintains
that estrogen “unopposed” by a progestin increases the risk of endometrial cancer by
stimulating normal endometrial-cell division; high-dose progestins block the action of
estrogen on the endometrium. This hypothesis provides a very satisfactory explanation
of the major risk factors for endometrial cancer: a reduced risk from early menopause,
high parity and combination-type oral contraceptive (COC) use, in contrast with an
increased risk from obesity and postmenopausal estrogen-replacement therapy (ERT)
(Ferenczy A et al., 1979). Endometrial-cell division in premenopausal women is
effectively confined to the pre-ovulatory follicular phase and immediate postovulation
phase of the menstrual cycle, when serum estradiol levels are relatively low, but
unopposed (Ferenczy A et al., 1979; Key TJA and Pike MC, 1988). Even so, it is
during this phase of the cycle that endometrial proliferation has been shown to be
maximal. This is in contrast to the postovulatory luteal phase, when serum progestrone
levels begin to rise and endometrial cell proliferation ceases, despite elevating levels of
estradiol. Early menopause reduces endometrial cancer risk by reducing the
unopposed-estradiol concentration, and the associated cell division rate, from the
relatively high level during the premenopausal follicular phase to the low level during
85
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
the postmenopausal period. This reduction in estradiol concentration is more than
sufficient to compensate for the fact that all estrogen exposure in the postmenopausal
period is not opposed by a progestin. Parity reduces risk because pregnancy is
associated with high progesterone levels. COC use reduces the risk because COCs
reduce the period of endometrial exposure to unopposed estrogen from the 14 days of
the normal follicular phase to the 7 days per 28-day cycle during which the COC is not
used, and because endogenous estrogen concentrations are very low during these 7
days. Postmenopausal obesity is associated with significantly increased endogenous
estrogen levels, which are also more bioavailable due to the decreased levels of sex-
hormone binding globulin (SHBG) and increased peripheral conversion of androgens to
estrones in obese women (Folsom AR et al., 1990; Longcope C, 1971; MacDonald PC
et al., 1978). Premenopausal obesity is also associated with a greatly increased
endometrial cancer risk. Premenopausal obesity is associated with more anovular (no
progesterone) cycles, where the endogenous estrogen level is similar to follicular-phase
levels, and the endometrium is thus exposed to unopposed estrogens for a longer period
of time. ERT increases the bioavailable estradiol concentration of a postmenopausal
woman to approximately two-thirds of the level found in the early follicular phase
(Ferenczy A et al., 1979), greatly increasing the endometrial-cell division rate.
The success of the unopposed-estrogen hypothesis in explaining the
epidemiology of endometrial cancer has stimulated an effort to develop a hypothesis
along similar lines for breast cancer. However, it appears clear that an “unopposed-
estrogen” hypothesis is not tenable for breast cancer. Although postmenopausal obesity
86
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
is associated with a small increase in breast cancer risk, premenopausal obesity is
actually associated with a decrease in relative risk. Moreover, COCs with an estrogen-
progestin mix in each pill do not protect against breast cancer and even high-dose ERT
[conjugated equine estrogen (1.25 mg daily)] produces, at most, only a modest increase
in breast-cancer risk even after 20-years use (Colditz GA et al., 1995; Key TJA and
Pike MC, 1988). In contrast to the situation with endometrial cancer, there is evidence
that use of a progestin as part of the estrogen-replacement therapy for postmenopausal
women is associated with an increased, not a decreased, risk of breast cancer (Bergkvist
L et al., 1989). COC use by women of reproductive age may actually increase breast
cancer risk (Collaborative Group on Hormonal Factors in Breast Cancer, 1996;
Collaborative Group on Hormonal Factors in Breast Cancer, 1996; Pike MC et al.,
1993), in stark contrast to the clear protective effect against endometrial cancer. In light
of these disparate results, characterization of control mechanisms for proliferative
activity in normal breast-epithelial cells would clearly be useful in understanding breast
carcinogenesis.
The Estrogen Receptor and Progesterone Receptor
Steroid hormones play important roles in promoting proliferation and cell
differentiation in normal breast epithelium and breast cancer cells. Since only cells with
steroid-hormone receptors will respond to the steroids, these biological effects are
87
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
thought to be mediated through transcriptional activation of specific sets of genes
recognized by particular receptor proteins.
Estrogen Receptor: Two Subtypes, ER-a and ER-f3
Substantial progress has been made in the last decade in understanding the
mechanisms of action for both the estrogen receptor (ER) and progesterone receptor
(PR), members of the steroid-thyroid-retinoid nuclear receptor superfamily (Evans RM,
1988; Tsai MJ and O’Malley BW, 1994). The first of the two subtypes of estrogen-
receptor genes (ER-a) has been localized to the long arm of chromosome 6 (band q24-
27) (Kumar V et al., 1987) and is termed the “classical” estrogen receptor. It is a
relatively large gene, spanning at least 140 kilobases, and contains eight exons which
encode the ER-a protein product. These exons, which are, respectively, 684, 191, 117,
336, 139, 134, 184, and 4537 base pairs in size (Figure 3.1), have been well
characterized and have various functions as described below. The transactivational
amino-terminal hypervariable region (AF-1) is predominantly coded for by exon 1.
Exons 2 and 3 each code for one zinc finger of the DNA binding domain (DBD). The
hinge region (H) is coded for by exon 4. The large hydrophobic hormone binding
domain (HBD/AF-2) is encoded by five different exons including part of exon 4, exons
5, 6 and 7, and part of exon 8 (Ponglikitmongkol M et al., 1988). Combined, these 4
domains make up the 595 amino acid estrogen receptor a (Figure 3.1 A).
88
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
The second estrogen receptor gene (ER-P), which was initially cloned from a rat
prostate cDNA library (Kuiper GG et al., 1996), and subsequently from humans
(Mosselman S et al., 1996) and mice (Tremblay GB et al., 1997), has been shown to
map to chromosome 14 (band q22-24) (Enmark E et al., 1997). The ER-P gene spans a
region of 30-40 kilobases, and, like ER-a, is also comprised of eight exons which code
for a 530 amino acid protein product (Figure 3.IB). These exons have been shown by
various groups to share significant homology to their ER-a counterparts, especially in
the areas of the DBD (97%) and the HBD (59.1%) (Enmark E et al., 1997; Kuiper GG
et al., 1996; Mosselman S et al., 1996; Ogawa S et al., 1998; Tremblay GB et al., 1997).
It was this high degree of sequence conservation between the two subtypes that initially
clued researchers into suspecting that ER-P was actually a second type of estrogen
receptor. Subsequent studies have since proven that ER-P is indeed a novel estrogen
receptor subtype, suggesting that ER-a is not the only mechanism by which estrogens
may have their influence on different cell types. In fact, because of the heterogeneous
tissue distribution patterns of the two estrogen receptors (Enmark E et al., 1997), it is
now suspected that there may exist heretofore unrecognized mechanisms of estrogen
signaling in tissues that either exclusively express just the ER-a or ER-P subtype, or
express both (Kuiper GG and Gustafsson J, 1997).
89
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Progesterone Receptors: PR-A, PR-B, and PR-C
Unlike the estrogen receptors, there is only one type of progesterone receptor
(PR), though multiple isoforms have been shown to exist. The two major isoforms of
PR are PR-A (94 kDa) and PR-B (114 kDa), both of which are encoded by the same
gene, which is located on the long arm of chromosome 11 (band q22-23) and spans over
90 kilobases (Misrahi M et al., 1993). Whereas it was once thought that the two
isoforms arose from the same mRNA transcript through different in frame translational
initiation sites, it is now known that the two isoforms are actually the products of two
different transcripts generated by alternative estrogen-responsive promoters (Kastner P
et al., 1990). These promoters, however, lack the consensus palindromic estrogen
responsive element (GGTCAnnnTGACC) typical for other estrogen-responsive genes
(Klein-Hitpass et al., 1987; Klock G et al., 1987), suggesting that alternative estrogen-
responsive regulatory elements may exist, such as the recently described imperfect
palindromic elements that mediate estrogen’s induction of TGF-a (El-Ashry D et al.,
1996). Progesterone receptor is encoded by eight exons of 2380, 152, 117, 306, 145,
131, 158, and 153 base pairs (Misrahi M et al., 1993). These exons code for similar
functional domains as their GR counterparts, demonstrating the high degree of
functional and structural conservation among the different steroid receptors (the
androgen receptor also shares this basic structural identity) (Figure 3.2). Recently, a
third isoform of PR (60 kDa PR-C) has been described in literature, but it remains to be
seen if this form actually plays a major functional role in progestin-mediated signaling
90
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
(Wei LL et al., 1997). PR-C is smaller than the other two PR isoforms as it is truncated
in its N-terminus.
The Structure o f ER and PR
The estrogen and progesterone receptors appear to have little in common
functionally, but they share a remarkable amount of structural similarities. Through the
use of in vitro mutagenesis assays, deletion mutation studies and domain-swapping
experiments, four major regions have been demonstrated for both ER and PR. These
domains are, in order from the carboxy-terminal end: a hormone-binding domain
(HBD), a hinge region (H), a DNA-binding domain (DBD), and a variable or regulatory
domain (AF-1) (Figure 3.3) (Green S and Chambon P, 1987; Green S et al., 1986;
Greene GL et al., 1986; Kumar V et al., 1986; Misrahi M et al., 1987).
The hormone-binding domains of ER and PR are critical for both hormone
recognition and receptor regulation. In the absence of ligand, this region appears to be
inhibitory, preventing the receptor (ER or PR) from binding to its DNA response
element in the promoter region of a target gene. This inhibition is caused by the
interaction between the HBD and a heat shock-90 protein (HSP-90), which leads to the
formation of a transcriptionally inactivated complex of proteins consisting of
monomeric receptor, HSP-90, p59, and possibly HSP-70 (Rehberger P et al., 1992;
Segnitz B and Ghering U, 1995). It is believed that the formation of this complex
prevents the receptor from binding DNA, possibly by disrupting key areas of the DNA-
91
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
binding domain. Receptors lacking the hormone-binding domain no longer have this
regulatory element and results in an altered receptor that is constituitively active (Tora
L et al., 1989).
Binding of ligand to receptor is thought to result in an allosteric change that
allows the receptor-hormone complex to bind to its DNA response element. In the
presence of a hormone agonist, the receptor undergoes a conformational change
resulting in the dissociation of the monomeric receptor from the heat shock complex.
This change in conformation then results in the spontaneous dimerization of receptors,
followed by DNA binding via their specific hormone response elements (HRE) and
activation of a second, ligand-dependent transcriptional activation domain (AF-2)
contained within the HBD (Giangrande P et al., 1997; Wahli W and Martinez E, 1991).
This ligand-mediated process is highly regulated and hormone specific. Only an
agonist can induce the proper allosteric effects to enable both DNA-binding and
transcriptional activation. Antiagonists that bind to the HBD have been shown to elicit
DNA-binding, but cannot induce the correct conformational changes necessary for
mz/if-activation (Xu J et al., 1996). Because of all of these important functions, it is
easy to understand why this domain is highly conserved within each class of steroid
receptors.
The other highly conserved domain is the DNA binding domain, which lies
immediately downstream of the highly variable, ligand-independent transcriptional
activation domain (AF-1). This region enables each receptor to recognize and bind its
own special hormone response element once ligand-binding and dimerization has
92
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
occurred. However, in addition to this DNA binding function, this domain has also
been demonstrated to be important in dimerization, nuclear localization of receptor, and
HSP-90 binding (Levenson A and Jordan C, 1994).
The amino acids between the hormone-binding and DNA-binding domains have
been referred to as the hinge region, since it is thought to be important in establishing
the allosteric association of the hormone-binding and the regulatory domains. This
region also contains sequences that are critical in directing the receptor protein to the
nucleus after it is synthesized in the cytoplasm (Fuller PJ, 1991; Picard D et al., 1990).
These “nuclear-localization signals” are sufficient to direct these proteins to the nucleus.
In addition to the primary nuclear localization signals in the hinge region, a second
signal is present in the hormone-binding domain that specifies nuclear localization in
the presence of hormone (Guiochon-Mantel A et al., 1991). The nuclear localization of
the steroid-unoccupied form of ER and PR requires continuous metabolic activity.
Various inhibitors of energy synthesis in cultured cells expressing ER or PR
demonstrate that the nuclear residency of the receptor reflects a dynamic state. In the
presence of energy inhibitors, receptor diffuses from the nucleus to the cytoplasm.
When the inhibitors are removed and glucose is returned to the culture medium, ER and
PR are transported back to the nucleus (Guiochon-Mantel A et al., 1991). Both the
steroid-occupied and unoccupied forms of receptor reside in the nucleus, not the
cytoplasm, of the intact cell. ER and PR can be thought of, primarily, as regulators of
transcriptional activity and their location in the nucleus is most appropriate for this
function. Although the DNA sequences of both estrogen- and progesterone-response
93
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
elements in promoter regions have been characterized, only a limited number of specific
ER/PR-inducible genes are identified. The genes responsible for the proliferative
activity of hormone-responsive cells are not known.
Ovarian Hormones and BRCA1 Regulation
Early studies of BRCA1 expression in mice and on cell lines have suggested a
role for ovarian hormones in regulating BRCAi expression. Marquis et al. (Marquis et
al., 1995) showed that BRCAI mRNA expression is upregulated in the mammary
glands of mice during puberty, pregnancy, and, in the case of ovariectomized mice,
following 17B-estradiol and progesterone treatment. These results were supported by a
separate study which also showed an increase in BRCAI expression in mammary
glands during pregnancy (Lane et al., 1995), supporting a role, whether direct or
indirect, for hormones in BRCAI regulation. The finding of a new subclass of Alu
DNA repeats with estrogen-responsiveness in the 5’ region of the BRCAi locus seems
to support a direct role for estrogens in the upregulation of BRCAI transcriptions
(Norris et al., 1995). However, despite the presence of these novel estrogen response
elements (ERE), other studies have shown that hormone-mediated regulation of BRCAI
transcription is indirect, resulting from a general alteration in the proliferative status of
the hormone-responsive cells rather than a direct interaction with specific DNA
elements on the BRCAI gene itself (Gudas et al., 1995; Spillman and Bowcock, 1996).
These findings were further supported by Marks et al. (Marks et al., 1997) using
94
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
estrogen-responsive breast cell lines. To determine whether estrogen induction of
BRCAI expression is direct or the result of the mitogenic activity of the hormone,
various experiments were performed and several lines of evidence emerged supporting
the conclusion that E2 induces BRCAI through a general increase in DNA synthesis
(proliferation) (Marks et al., 1997). Firstly, the kinetics and magnitude of BRCAI
induction are different from that of pS2, an estrogen-responsive gene; secondly,
cycloheximide treatment of cells blocked BRCAI induction, but not pS2, suggesting
that de novo protein synthesis is required in BRCAI upregulation; thirdly, other
hormonal and growth factor treatments of cells result in similar induction of BRCAI,
indicating a general increase in DNA synthesis is sufficient for BRCAI upregulation;
and lastly, reporter constructs controlled by the putative BRCAI 5’ ERE genomic
sequences failed to demonstrate estrogen responsiveness in transfected cells. Hence,
together these results indicate that the upregulation of BRCAI expression observed in
hormonally-regulated tissues and cells in response to ovarian hormone stimulation is
indirectly related to the increased mitogenic potential caused by the hormones on
general cell proliferation, and not through specific induction of the gene itself.
BRCAI Regulation of Hormone Function
Not much is known about BRCAI’s role in hormone regulation, and what is
known is through indirect observation in studies looking at BRCAI’s effect in the
development of hormone-responsive organs. Typically, B rcal/' knockout mice are
95
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
embryonic lethal, with developing embryos suffering a proliferative block early during
embryogenesis (Hakem et al., 1996). However, if Breal7' mice are somehow allowed
to bypass this proliferative block, it is possible to study the role of BRCAI in adult mice
development. To overcome the early lethality of the nullizygous Brcal phenotype, and
to study B rcal’s role in mammary gland development, Xu et al. (Xu et al., 1999)
created a Cre-loxP Brcal mouse model, which utilizes tissue-specific and temporally-
expressed factors to target knockouts in desired tissues, allowing developmentally
important genes to be studied in adult mice as knockouts. Using this approach, this
group was able to knock out Brcal in developing mammary glands and observed that
loss of Brcal resulted in blunted ductal morphogenesis, including increased tumor
formation. Abnormal duct development in these mice were also observed during
pregnancy. Specifically, mice that contained the knockouts failed to develop extensive
mammary glands, indicating that Brcal may function in this hormone-mediated process.
In a separate study utlitizing a different approach to bypass the early lethality of Brcal
knockouts, Cressman et al. (Cressman et al., 1999) generated Brcal '/pSS7* double
knockout mice, some of which survived into adulthood. Of these mice, the most
notable differences observed, as compared to p537' controls, were defects in
spermatogenesis in male mice, and underdeveloped mammary glands in a female
mouse. Interestingly, both of these processes are hormone-regulated events, supporting
a role for Brcal in steroid hormone signaling pathways. Moreover, since loss of Brcal
function results in defects that are phenotypically consistent with those observed during
low or insufficient hormone signaling, it is likely that Brcal functions as an enhancer,
96
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
or coactivator, in steroid receptor signaling, such that loss of its function results in
diminished hormone responsiveness. The results discussed in Chapter 2 certainly
support this conclusion.
In contrast, a recent study by Fan et al. (Fan et al., 1999) showed BRCAI to be
an inhibitor of ER signaling in transfected mammalian cells. In this study, coexpression
of high dose BRCAI with exogenous ER was found to inhibit hormone-dependent ER
signaling, possibly through disruption/dysregulation of the AF-2 region of ER. These
findings provide a possible mechanism whereby loss of BRCAI function may result in
the development of breast cancer, presumably due to unopposed ER signaling.
However, although the data demonstrated in this study clearly suggests a role for
inhibition, it is interesting that these results could only be achieved under extreme, non-
physiological conditions, such as high doses of nuclear receptor (0.5 pg) and BRCAI
(0.5 pg) and high hormone concentration (1 pM). Since overexpression of nuclear
receptors can result in self-inhibition due to squelching (personal communication, Steve
Koh, Laboratory of Michael Stallcup), it is imperative to perform dose-studies to
determine which conditions are suitable for characterizing coactivator/corepressor
function. Moreover, since expression of exogenous BRCAI is known to inhibit cell
growth and proliferation (Holt et al., 1996) and even cause cytotoxicity (Wilson et al.,
1997), further studies are required to determine if BRCAI inhibition is truly
physiologic, or merely an artifact of the conditions used in that study. If BRCAI is a
true inhibitor of ER function, then it should be able to inhibit ER signaling under all
conditions, including low dose receptor. In the current study, we propose that BRCAI
97
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
is indeed a coactivator of ER and not an inhibitor as previously described. Moreover,
since pl60 coactivators are also expressed in breast tissues, it is possible that BRCAI
may function in ER signaling by potentiating pl60 coactivator function, just as in AR
signaling. Lastly, to further expand upon BRCAI’s role in steroid hormone regulation,
we propose to determine if BRCAI functions in progesterone signaling, both alone and
in the presence of GRIP1.
MATERIALS AND METHODS
Transient transfection assays were performed using a chloramphenicol-acetyl
transferase (CAT) assay system. Mammalian cells were transfected with plasmids
encoding various transcription factors and tested for reporter gene activation. In some
cases, protein expression from transfected plasmids were checked by
immunofluorescence. Protein binding assays were performed using glutathione-S-
transferase (GST) fusion proteins and in vitro transcribed and translated protein targets.
Mammalian Expression Vectors and Plasmid Construction
Plasmids pCMV-hAR (Tilley et al., 1989), pSG5-ERa (Green et al., 1988),
pSG5-GRIPl and pSG5-SRC-la (Ma et al., 1999), pcDNA3.1-AIBl (Anzick et al.,
1997), ARRjtk-CAT (Snoek et al., 1998), ERE-C0II6O-CAT (Webb et al., 1995), and
98
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
MMTV-CAT (Giguere et al., 1986) were described previously. To construct the vector
pcDNA-AR (NTD-DBD), an Nhel-BamHI fragment was PCR amplified from pcDNA-
hAR (Irvine et al., 2000) plasmid DNA using AR (NTD-DBD) primer pairs SI and AS1
(Table 2.1) and inserted into the reciprocal restriction sites of pcDNA3.1 (+)• Vector
pcDNA-AR (DBD-LBD) was constructed in sequential cloning steps. First, an Nhel-
Kpnl PCR fragment containing the AR Kozak sequence was amplified using primers SI
and AS2 and inserted into the corresponding sites of pcDNA3.1 (+). Second, a Kpnl-
EcoRI PCR fragment was amplified using primers S2 and AS3 (Table 1) and inserted
into the restored Kpnl site and the downstream EcoRI site of the pcDNA3.1 (+)
multiple cloning site. BRCAI mammalian expression plasmid pcDNA-BRCAl was
constructed by inserting a 5’ Notl-Xhol 3’-treated BRCAI insert derived from pBSK-
lhFL plasmid (Chen et al., 1999) into corresponding endonuclease restriction sites of a
pcDNA3.1/mycHisC(-) vector (Invitrogen).
Proteins with N-terminal hemagglutinin A (HA) epitope tags were expressed in
transient mammalian cell transfections and in vitro from pSG5.HA, which has SV40
and T7 promoters (Chen et al., 1999). the following proteins were expressed from
previously described pSG5.HA derivatives: GRIP1AAD1 (full-length GRIP1 with
amino acids 1057-1109 deleted) (Ma et al., 1999); GRIP1, GRIP15 .7 6 5 , G R IP l?^ ^,
GRIP 1,i2 i-i4 6 2 > and CARM1 (Chen et al., 1999). A polymerase chain reaction (PCR)-
amplified cDNA fragment encoding full-length PRMT1 was cloned into the EcoRI-
BamHI sites of pSG5.HA.
99
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Other previously described mammalian expression plasmids include: pSVARo
(transient transfection assays) (Brinkmann et al., 1989) and pcDNA-hAR (in vitro
expression) (Irvine et al., 2000) both encode androgen receptor, pSG5-ERa encoding
estrogen receptor (Green et al., 1988), pPR (progesterone receptor), and pSG5-GRIPl.
Luciferase reporter gene constructs were previously described: MMTV-LUC for AR
and PR, MMTV(ERE)-LUC for ER (Umesono and Evans, 1989). BRCAI mammalian
expression plasmid pcDNA-BRCAl and overlapping BRCAI fragments were
previously described (Chapter 2).
Bacterial Expression Plasmids
Bacterial expression plasmids encoding GST, GST-AR and GST-GRIP1
fragments were previously described (Chen et al., 1999; Ma et al., 1999).
Tissue culture and transfections
Cells obtained from the American Type Culture Collection (Manassas, VA)
were maintained in RPMI (PC-3, DU-145, and HBL-100) or DMEM (MCF-7) medium
that contained 10% fetal bovine serum (FBS). CV-1 and COS-7 cells were maintained
in DMEM, 10% FBS. 24-48 hours post-transfections, cells were washed with ice-cold
PBS and then lysed with lysis buffer (Promega).
100
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
CV-1 cells were transfected in triplicates (or quadruplicates where indicated)
and cell extracts were assayed for luciferase activity as described below. When
hormone was required, transfections were grown in medium containing charcoal-treated
serum (Gemini Bio Products) and lOnM concentration of appropriate hormone for each
NR: dihydrotestosterone for AR, progesterone for PR, and estradiol for ER.
Chloramphenicol Acetyltransferase (CAT) Assays
(Refer to Chapter 2, Methods)
Luciferase (LUC) Assays
Following tranfections of 24-well plates, cell extracts for luciferase assays were
prepared by adding 125 pi of IX Lysis Buffer (Promega) per well and freezing at
-80°C. Frozen lysates were thawed at room temperature and then shaken on a rotary
shaker at 200 rpm for 15 minutes. 20 pi of lysates were removed and placed onto
opaque 96-well luciferase assay plates for luminometer readings. 40 pi of luciferase
reagent (Promega) were injected into each well and triplicate (quadruplicate) samples
were read on a plate-reading luminometer.
101
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
GIutathione-S-Transferase (GST) Pull-Downs
Glutathione-S-transferase (GST) and GST-fiision proteins were expressed and
purified as described (Hong et al., 1996). Glutathione-Sepharose-bound GST protein,
GST-AR (1-555), or GST-GRIP1 fragments (5-765, 563-1121, or 1121-1462) were
incubated with 3 S S-radiolabeled full-length BRCAI or BRCAI fragments transcribed
and translated in vitro from pcDNA3.1 vectors using a TNT-Coupled Reticulocyte
Lysate System (Promega) in the presence of 3 5 -S methionine. Associated BRCAI was
eluted, resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed by
autoradiography. 10% of total labeled BRCAI incubated in each reaction was loaded
for comparison.
Preparation of Whole Cell Extracts
Culture cells were washed with cold PBS and treated with RIPA Lysis Buffer
(50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, pH 8.0) containing complete
protease inhibitors (Roche). Cells were lysed by incubating on ice for 10 minutes and
then passaging through a 23 gauge needle. Lysates were separated from cellular debris
by centrifugation and quantified by BCA protein quantitation assay (Pierce).
102
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Co-Immunoprecipitation and Western Inununoblotting
Whole cell lysates were obtained as described above. Protein lysates were
precleared with Protein-G-Sepharose beads for 1 hour. Protein G was removed and 2-3
pg of appropriate antibodies were added to each lysate to incubate overnight at 4°C.
The next morning Protein G was added to immunocomplexes and allowed to bind for 2
hours. Beads were washed 3-4 times in RIPA lysis buffer and then boiled in SDS
sample buffer. Eluted proteins were resolved by SDS/PAGE and transferred onto
PVDF membranes for western blot analysis.
Membranes were blocked overnight in 5% milk solution and then incubated for
1-2 hours with primary antibodies in blocking solution. Membranes were then washed
with PBST and incubated with appropriate dilutions of secondary antibodies (blocking
solution) for 1 hour. Antibody complexes were detected with the ECL
chemiluminescent system (Amersham).
RESULTS
BRCAI enhances ER signaling through potentiation of GRIP1
Although BRCAI was demonstrated here to enhance AR activity, it was
previously shown to inhibit ER signaling in transfected cells (Fan et al., 1999). This
103
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
suggests that it may play a unique role in the differential regulation of steroid receptor
signaling pathways. As BRCAI is apparently able to work synergistically with pl60
coactivators (see above), we reasoned that BRCAI should in fact potentiate, rather than
inhibit, ER signaling in the presence of pl60 coactivators. To address this discrepancy,
we investigated whether BRCAI could indeed inhibit ER activity. Transfection of
increasing amounts of BRCAI expression vector did not result in suppression of ER
signaling in PC-3 cells (Figure 3.4). Furthermore, BRCAI had no effect on ER
transactivation in either HBL-100 or MCF-7 breast cell lines (Figure 3.5). In contrast, a
modest, though reproducible, inhibition of ER function was observed in DU-145 cells
transfected with high doses of BRCAI plasmid, corroborating part of the results of Fan
et al. (Fan et al., 1999). When BRCAI was coexpressed with GRIP1, however,
potentiation of GRIP 1-mediated coactivation of ER signaling on the estrogen-
responsive reporter was observed in each one of the transfected cell lines (Figure 3.5).
BRCAI stimulation of GRIP1 coactivation was hormone-dependent and BRCA1-
mediated, as stimulation was not observed in the absence of estrogens or when the
BRCAI plasmid was substituted with the parent vector (data not shown). Therefore, in
the presence of exogenous pl60 coactivators, BRCAI functions in enhancing ER
signaling.
104
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
BRCAI Function in ER Signaling is Dependent Upon Nuclear Receptor Dose
Although we demonstrate that BRCAI functions as a coactivator of ER in the
presence of exogenous pl60 coactivators, it is unclear why our results with BRCAI
alone failed to show inhibition. Upon careful examination of the our methodology and
the methods underlined in the study by Fan et al., we noticed vast differences in the
amounts of nuclear receptor plasmids used, as well as amounts of BRCAI plasmids that
were required to demonstrate inhibition. Specifically, in the study by Fan et al., very
high doses of ER and BRCAI plasmids were transfected into cells, 0.5pg versus
nanograms in our study. Furthermore, surprisingly high, non-physiologic
concentrations of estradiol (luM E2) were used to obtain inhibition. Besides the
possibility of squelching, a non-specific inhibition that occurs due the sequestration of
limiting transcription factors by excessive amounts of transfected plasmids, previous
studies have demonstrated that high levels of BRCAI expression results in cell-cycle
inhibition, as well as cell death (Holt et al., 1996; Wilson et al., 1997). Hence, at the
non-physiological levels of ER and BRCAI expressed by Fan et al., it is possible that
the inhibition they report is due to reasons other than a specific functional relationship
between BRCAI and ER. To test under which conditions BRCAI can mediate
inhibition, we transfected CV-1 mammalian cells with increasing amounts of ER
plasmids, and coexpressed BRCAI (0, 150, 500 nanograms). At low doses of ER (1
ng), BRCAI functions as a coactivator, though the level of enhancement is modest
(Figure 3.6). Since BRCAI is not known to interact with ER directly, this slight
105
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
enhancement is most likely due to interactions with endogenous p i60 coactivators. In
contrast, at increasing doses of ER, BRCAI’s “inhibition” effect becomes evident, with
its inhibition becoming more pronounced as ER doses is increased from 10 to 100
nanograms. At 500 nanograms of transfected ER plasmid, we observe coactivator-
independent signaling as the relatively lower amount of exogenous BRCAI has little
effect.
BRCAI Coactivation Depends on ER Levels
Since it is unlikely that BRCAI could function as both a coactivator and a
repressor of ER, and since BRCAI only appears to exhibit inhibition at increasing
amounts of coexpressed ER, we proposed that the inhibition observed in this study and
in the study by Fan et al., is the result of squelching due to overexpression of ER, rather
than a BRCAI-mediated inhibition. Interestingly, when ER is transfected at higher
doses, it is able to squelch its own activity, suggesting the possibility of a limiting
endogenous factor which is used up more rapidly at higher levels of ER (Figure 3.7). In
contrast, at low levels of ER, the transcriptional activation kinetics is linear and dose-
dependent. Hence, studies using large amounts of exogenous ER should be performed
at various other doses to insure that results are specific for physiologic function, and not
due to artifacts of the transient transfection system’s own limitations. To demonstrate
this ER dose-dependent effect, we transfected CV-1 cells with either low dose (5 ng) or
high dose (500 ng) ER plasmid and increasing amounts of BRCAI plasmid. At low
106
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
amounts of ER, BRCAI demonstrates a steady, dose-dependent increase in
enhancement of ER activity (Figure 3.8). In contrast, at 500 ng of ER plasmid, BRCAI
appears to inhibit ER signaling in a dose-dependent fashion (Figure 3.9).
BRCAI, GRIP1, and BRCA1-GRIP1 Enhancement of ER Also Depends on NR
Levels
Since BRCAI function in ER regulation appears to depend upon ER levels, we
wanted to test if this dependence on NR levels was specific for BRCAI, or generic for
other coactivators. We cotransfected GRIP1 with increasing doses of ER and observed
an initial enhancement of ER signaling by GRIP1 at low levels of ER, but at higher
levels of ER, less enhancement was observed (Figure 3.10). Although GRIP1 did not
appear to “inhibit” ER at higher ER levels like BRCAI does, its own activity was
“inhibited” by the relatively higher doses of ER, suggesting that even GRIPl’s ability to
enhance ER signaling depends on receptor levels. Lastly, we have already
demonstrated that BRCAI can potentiate GRIP 1-mediated enhancement of ER
signaling in various cell lines. We now show that even this enhancement is dependent
upon ER levels, supporting the importance of ER dose in modulating coactivator
function.
107
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
BRCAI and Progesterone Receptor Regulation
We have previously demonstrated that BRCAI functions as a coactivator of
androgen receptor signaling, both alone and in the presence of the pl60 nuclear receptor
coactivators, such as GRIP1, SRCla, and AIB1. Additionally, we have shown that
BRCAI is also a coactivator of ER signaling in the presence of pl60 coactivators,
though it’s direct role remains unclear. However, since BRCAI appears to function in
both AR and ER signaling pathways, we then wanted to test if BRCAI plays a role in
progesterone receptor regulation. To address this question, CV-1 cells were transfected
with PR plasmid and increasing amounts of BRCAI plasmid. Additional experiments
using low-dose AR and ER were simultaneously performed. There was a 3-7 fold
enhancement of nuclear receptor activity from no BRCAI transfection to the highest
dose of BRCAI transfected, which was linear and dose-dependent for each of the
receptors tested (Figure 3.11). Therefore, BRCAI appears to be a novel coactivator of
the steroid receptors, and may play a general role in nuclear receptor regulation, though
further studies are necessary to confirm this function. To confirm if BRCAI and the
pl60 coactivators also cooperate in PR signaling, we cotransfected CV-1 cells with
plasmids encoding BRCAI and GRIP1 (Figure 3.12). BRCAI and GRIP1 alone each
enhanced ligand-dependent PR activity 2.15 and 3.76 fold, but coexpression of the two
coactivators resulted in a 10.00 fold increased in PR activity. Hence, BRCAI and
GRIP1 are able to function synergistically in PR signaling, in addition to both AR and
108
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
ER signaling, suggesting a physiologic role in steroid receptor regulation. Table 3.1 is a
summary of the role of BRCAI in steroid hormone signaling.65
DISCUSSION
There is much evidence to support a functional role for BRCAI in the regulation
of steroid hormone signaling. Through mouse knockout models, BRCAI has been
shown to play a pivotal role in breast ductal morphogenesis and pregnancy related
mammary epithelial cell proliferation (Cressman et al., 1999; Xu et al., 1999), two
events that are known to be regulated by circulating ovarian hormones. In addition,
BRCAI also appears to play a critical role during spermatogenesis, another hormone-
regulated process, as male mice nullizygous for BRCAI fail to undergo
spermatogenesis. Interestingly, other than the steroid hormone-specific defects during
development, and slightly smaller adult sizes, BRCAI-deficient mice do no appear to
exhibit any other somatic abnormalities. Hence, given the specific natures of the
defects observed in these mice, BRCAI appears to function in enhancing steroid
hormone signaling pathways, both in developing and adult mice, such that loss of this
function results in phenotypes that are consistent with classic hormone insufficiency.
Recently, BRCAI was shown to inhibit ER signaling in transfected mammalian
cells (Fan et al., 1999). Given its opposite role in the regulation of AR (Chapter 2), this
suggests that BRCAI may play a unique role in the differential regulation of steroid
109
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
receptor signaling pathways. As BRCAI is apparently able to work synergistically with
pl60 coactivators, we reasoned that BRCAI should in fact potentiate, rather than
inhibit, ER signaling in the presence of the pl60 coactivators. To address this
discrepancy, we investigated whether BRCAI could indeed inhibit ER activity.
Transfection of increasing amounts of BRCAI expression vector did not result in
suppression of ER signaling in PC-3 cells. Furthermore, BRCAI had no effect on ER
transactivation in either HBL-100 or MCF-7 breast cell lines. In contrast, a modest,
though reproducible, inhibition of ER function was observed in DU-145 cells
transfected with high doses of BRCAI plasmid, corroborating part of the results of Fan
et al. (Fan et al., 1999). When BRCAI was coexpressed with GRIP1, however,
potentiation of GRIP 1-mediated coactivation of ER signaling on the estrogen-
responsive reporter was observed in each one of the transfected cell lines. BRCAI
stimulation of GRIP1 coactivation was hormone-dependent and BRCAI-mediated, as
stimulation was not observed in the absence of estrogens or when the BRCAI plasmid
was substituted with the parent vector. Therefore, although BRCAI may inhibit ER
signaling under certain, but not all, conditions (see below), BRCAl’s ability to interact
coordinately with the p i60 coactivators during estrogen receptor signaling most likely
supercedes any inhibition that may occur with BRCAI alone.
Unlike most other steroid receptor inhibitors, which demonstrate inhibition
across a broad spectrum of conditions, BRCAI appears to inhibit ER only under
extreme, non-physiologic conditions, such as high levels of exogenous ER and BRCAI,
as well as high concentrations of hormone. However, under conditions of moderate to
110
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
low ER and BRCAI levels, we show that BRCAI enhances ER signaling. Since
BRCAI is known to cause growth arrest at high doses (Holt et al., 1996) and can also
result in cellular toxicity (Wilson et al., 1997), it is possible that the inhibition
associated with overexpression of high-dose BRCAI is related to these two non-ER
effects. However, this does not explain why BRCAI, at low levels, but under high
levels of ER, still exhibit inhibition. In order to rectify this discrepancy, we performed
dose-dependence experiments studying the role of ER level on BRCAI function. From
these dose-response curves, we have developed a new model for studying nuclear
receptor-coactivator function using the transient transfection system. In this model, we
have identified specific parameters along the ER dose curve that may explain the
disparate BRCAI results obtained under varying doses of exogenous ER.
Rather than maintaining a positively-sloped linear curve throughout a range of
ER levels, dose kinetics of ER under transient transfection conditions reveal three
distinct phases. At low to intermediate levels of ER, there is a linear, dose-dependent
increase in reporter gene activation with increasing amounts of transfected ER (Dose-
Dependent Phase). At intermediate to high levels, ER begins to exhibit self-inhibition,
or squelching, due to the excess of plasmids being transfected into the cells (Inhibitory
Phase). Under these conditions, ER activity is no longer dose-dependent, and increased
transfection of ER plasmid, or cotransfection of other plasmids, may result in non
specific inhibition of reporter gene activity due to further sequestration of limiting
factors. At very high levels of ER, the dose curve begins to exhibit a coactivator-
independent phase (Refractory Phase) during which ER begins to function through a
111
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
mechanism that is no longer responsive to exogenous coactivators. Therefore, dose-
response curves must be performed for each transient transfection system used (i.e. cell
type and density and transfection protocols) to insure that nuclear receptor levels fall
within the linear, dose-dependent phase of the curve. If this is not done and “random”
levels of receptor is used, the results may be misleading. Hence, based upon these dose
curve kinetics, it is now clear how BRCAI appears to function as a coactivator of ER
under one condition, and as an “inhibitor” under another. Indeed, although the results
of Fan et al. clearly demonstrate that BRCAI is an inhibitor under the extreme
conditions used in that study, we now show that BRCAI actually enhances ER
signaling at low dose ER, possibly through interactions with endogenous pl60
coactivators, and inhibits signaling only at high dose ER. These results support the
importance and validity of determining receptor kinetics in order to insure all studies
are performed in the dose-responsive phase of NR levels. Since it is unlikely that
BRCAI or ER is endogenously expressed at the levels described by Fan et al, the results
of that study should be construed with caution until physiologic evidence is generated to
support this role for ER inhibition in vivo.
In addition to being a coactivator of AR and ER, we also demonstrate that
BRCAI is a coactivator of PR in transfected mammalian cells. Furthermore, both
BRCAI and GRIP1 are able to function synergistically in enhancing PR signaling,
consistent with previous results for both AR and ER. In both cases, either alone or in
the presence of GRIP!, this enhancement was found to be dose-dependent throughout a
broad range of BRCAI levels. Although no PR dose curves were performed to
112
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
determine if appropriate PR levels were used in these experiments, our use of very low
amounts of PR makes us confident that the results obtained for BRCA1 in PR signaling
are real and not due to artifacts of the system. Therefore, given the results discussed in
Chapter 2 and this current chapter, we are conclude that BRCA1 is a novel coactivator
of the steroid receptors AR, ER, and PR, and hypothesize that BRCA1 may even play a
broader role in general nuclear receptor regulation. However, until other receptors are
similarly studied, we can only speculate the nature of BRCAl’s role in these other
nuclear receptor pathways.
113
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3-1. Schematic illustration of the estrogen receptor (ER) genome and
messenger RNA with protein structure. A, ER-a genomic DNA. Exons I-VIII,
depicted as boxes separated by variable intronic sequences, are shown with their
respective sizes. The mRNA structure of the ER-a transcript and its protein product are
also shown. ER-fi is the product of a different gene and is located on a separate
chromosome than ER-a. Var, amino terminal hypervariable domain; Zing, zinc finger
DNA-binding domains; H, hinge region; DBD, DNA-binding domain; HBD, hormone-
binding domain; AF-l, ligand-independent transactivation domain; AF-2, ligand-
dependent transactivation domain. B, The protein structure of ER-(J is shown with the
percent identity between the functional domains of the two ER subtypes. The genomic
structure and mRNA transcript for ER-(3 have not been included.
114
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Reproduced with permission o f th e copyright owner. Further reproduction prohibited without permission.
A
Genomic DNA
FIGURE 3-1
Domains
5‘ Exon: I II I I I IV V V I V II V III 3
- -| Var |— |Zinc I |—|Zinc II |— | H | hbd|-^IBd|-^B d[^ B d H HBD | |-
684 bp 191 117 336 139 134 184 4537
B
A/B
mRNA
5'
Estrogen Receptor Proteins
Amino acid: 185
ER-a N H 2|
149
ER-P N H 2' ™
Percent Identity: 17.5%
551 595 250 311
214 248
H
97.0% 3c T o% 59.1%
530
COOH
17.9%
U l
3'
COOH
Figure 3-2. Schematic illustration of the progesterone receptor (PR) genome and
messenger RNA with protein structure. A, PR genomic DNA includes exons and
functional domains. B, The mRNA structure of PR has two distinct ATG sites. PR-B
is encoded by a full-length transcript and initiates from the first of the two ATG
initiation sites, whereas PR-A initiates from a downstream in-frame ATG site. Whether
the two isoforms of PR are translated from the same transcript of from two distinct
transcripts is not clear. C, Protein structures of PR-B and PR-A. AF-1, ligand-
independent transctivation domain; DBD, DNA-binding domain; H, hinge region;
HBD, hormone-binding domain.
116
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 3-2
> X
§j
I n
m
§ '
C O
C M
L U
C M
O C M
— C O
C O
C O
§
C O
<
z
O C
E
a
o I
O C
CL
O C
(L
117
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3-3. Functional domains of the steroid hormone receptors. AF-l, ligand-
independent transactivation domain; DBD, DNA-binding domain; H, hinge region;
HBD, hormone-binding domain; AF-2, ligand-dependent transactivation domain; NLS,
nuclear localization domain; HSP-90, heat shock protein-90 region.
118
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGU RE 3-3
ui
(0
c
I M
(D
E
o
o
Q.
ffl
o
ffl
cc
ffl
c
o
E
h a
0
1
T3
o
ha
0 5
0)
c
o
O
c
o
in
f f l <
u i E
(0
C T O
■ = u . f f l
| < Q
O
o
0 ^ 0)
f f l u . j
z < z
1 «
2 CL
1 C D
Q Z
119
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3-4. BRCA1 does not coactivate or repress ER-a transactivation in PC-3
cells. Transiently transfected cells were assayed for stimulation of ERE-C0II6O-CAT
reporter activity by 17(3-estradiol (E2). Cells were cotransfected with 1.0 pg ERE-
C0II6O-CAT, 100 ng pSG5-ERa, and increasing amounts of pcDNA-BRCAl as
indicated. CAT activities were normalized for total cellular protein and data presented
are the mean ± SE of three independent dishes.
120
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 3-4
300
| 1 no hormone
£• H e2
o 2 0 0 -
(0
~ 100-
C O
1
ERE-Coll60-CAT + +
pSG5-ER-a + +
pcDNA-BRCA1 - -
0.5 ng
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3-5. BRCA1 potentiates pl60-mediated E R -a signaling in both prostate
and breast cell lines. Cells were cotransfected with 1.0 |ig ERE-C0II6O-CAT, 25 ng
pSG5-ERa, 2.0 jxg pSG5-GRIPl, and 2.5 |A g pcDNA-BRCAl as indicated. Data
presented are fold + SE relative to E2-dependent ER-a activity with no transfected
GRIP1 or BRCA1. ER-a was not coactivated by BRCA1 in any of the cell lines.
Indeed, in DU-145 cells, ER-a activity was repressed more than 50% by BRCA1
consistent with the observations of Fan et al. (Fan et al., 1999). In PC-3, DU-145, and
HBL-100 cell lines, BRCA1 enhanced GRIP 1-mediated coactivation of ER-a signaling.
BRCA1 had modest effects on receptor signaling in MCF-7 cells.
122
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
BRCA1 Coactivates Estrogen Receptor
Signaling in the Presence of GRIP1
i
E2
ERE-C0I I 6O-CAT + + + + + + + + + + + + + +
ER-a + + + + + + + + + + + + + +
GRIP1
- - + - - + + - - +
-
m
+ +
BRCA1 + - + - + + - +
PC-3
DU-145 HBL-100 MCF-7
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3-6. BRCA1 regulation of ER depends upon receptor levels. CV-1 monkey
kidney cells were transfected with 1, 10, 100, or 500 nanograms of pSG5-ERa plasmids
and cotransfected with 0, 150, or 500 nanograms of pcDNA3.1-BRCAl plasmids. All
triplicate transfections were performed in a 24-well plate format and 10 nM
concentrations of E2 were used where appropriate. BRCA1 dose curves are represented
as percentages of ER signaling alone (10 nM E2) for each of the receptor levels tested.
124
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Luciferase Acitivity
( % o f E R Signaling)
FIGURE 3-6
180
160
ER, 1 ng
140
120
ER, 500 ng
100
80
ER, 10 ng
60
ER, 100 ng
40
20
E2 = 10 nM
0
500 0 150
BRCA1 Dose (ng)
125
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3-7. Dose-dependent kinetics of ER activity. CV-1 cells were transfected
with increasing amounts (1, 10, 100, 500 ng) of ER plasmid. Transfected cells were
grown in the presence of 20 nM E2 or no exogenous E2. ER plasmid levels (X-axis)
are plotted using a logarithmic scale and luciferase activity is measured as relative light
units (RLU). ER dose kinetics are represented by three distinct phases: 1, coactivator-
dependent; 2, squelching (inhibitory); and 3, coactivator-independent.
126
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 3-7
- • — 20 n M E 2
>
<
8 1
< 0
o
3
50
40
30
20
£ 10
3-Coactivator Independent
Phasfl“
T T
0.001 0.01 0.1
ER Dose (tag)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3-8. BRCA1 is a coactivator at low-dose ER. CV-1 cells were transfected
with an equivalent of 5 nanograms of ER plasmid (24-well plate format), and increasing
amounts of BRCA1 plasmid. Bar graphs are plotted as measured luciferase activity
(RLUxlOOO) and folds are shown above each bar as relative to ER activity.
128
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 3-8
ER (20nM E2)
BRCA1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3*9. BRCA1 inhibits ER signaling at high receptor levels. CV-1 cells were
transfected with 500 nanograms of ER plasmid and increasing amounts of BRCA1
plasmid. Bar graphs are plotted as measured luciferase activity (RLUxlOOO) and folds
are shown above each bar as relative to ER activity.
130
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 3-9
E2(20n) +
BRCA1 -
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3-10. BRCA1-GRIP1 synergy depends on transfected ER levels. CV-l cells
were transfected with increasing amounts of ER plasmid (1, 10, 100, or 500 ng). 150
nanograms of BRCAl plasmid and/or 75 nanograms of HA-GRIP1 were cotransfected
where indicated. Results were plotted as linear graphs of measured luciferase activities
(RLU).
132
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 3-10
3
_ l
O C
2?
>
u
<
0 )
1 0
s
£
o
3
500 — • — no E2
— * — 20nM E2
--X ---B R C A 1
- -GHIP1
— BRCA1 ♦ GRIP1
450
400
350
300
250
200
150
100
0.1 0.01 0.001
Estrogen Receptor (pg)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3-11. BRCA1 is a general coactivator of the steroid receptors. Comparison
of BRCA1-mediated coactivation of AR, ER, and PR by transient transfection assay.
CV-1 cells were transfected with plasmids encoding AR (5 ng), ER (2.5 ng), or PR (1
ng) and increasing amounts of BRCA1 plasmid. Plots are given as luciferase activity
versus BRCA1 amount in micrograms.
134
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 3-11 ER+BRCA1 Dose Curve
lO
ER = 2.5 ng
O C
if
>
g
<
s s
s
£
o
^ 4 0 0
| 3 5 0
3 3 0 0
2 5 0
200
1 5 0
1 0 0
5 0
O
1 4 0
120
1 0 0
8 0
6 0
4 0
1 3 2 0
-1
o
BRCA1 (ng)
K
>
<
s
s
0 )
PR+BRCA1 Dose Curve
■ l-l
PR = 1 ng
AR+BRCA1 Dose Curve
/
AR = 5 ng
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 3-12. BRCA1 potentiates GRIP1 enhancem ent of PR signaling.
Mammalian CV-1 cells were grown on 12-well culture plates and cotransfected with
plasmids encoding PR, BRCA1, and/or HA-GRIP1. 1 nanogram of PR plasmid, 250
nanograms of BRCA1 plasmid, and 100 nanograms of HA-GRIPl plasmid were used as
indicated. Graphs were plotted as relative fold over PR alone. Relative folds are
indicated above each bar graph.
136
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 3-12
BRCA1
GRIP1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
10.00X
REFERENCES
Anzick, S. L., Kononen, J., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X.-Y.,
Sauter, G., Kallioniemi, O.-P., Trent, J. M., and Meltzer, P. S. (1997). AIB1, a steroid
receptor coactivator amplified in breast and ovarian cancer. Science 277, 965-967.
Bergkvist L, Adami H-O, Persson I, Hoover R, and Schairer C (1989). The risk of
breast cancer after estrogen and estrogen-progestin replacement. N Eng J Med 321, 293-
297.
Brinkmann, A. O., Faber, P. W., van Rooij, H. C., Kuiper, G. G., Ris, C., Klaassen, P.,
van der Korput, J. A., Voorhorst, M. M., van Laar, J. H., Mulder, E., and et al. (1989).
The human androgen receptor: domain structure, genomic organization and regulation
of expression. J Steroid Biochem 34, 307-10.
Chen, D., Ma, H., Hong, H., Koh, S. S., Huang, S.-M., Schurter, B. T., Aswad, D. W.,
and Stallcup, M. R. (1999). Regulation of transcription by a protein methyltransferase.
Science 284, 2174-2177.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Chen, Y., Lee, W. H., and Chew, H. K. (1999). Emerging roles of BRCA1 in
transcriptional regulation and DNA repair [In Process Citation]. J Cell Physiol 181,
385-92.
Cohen SM, and Ellwein LB (1990). Cell proliferation in carcinogenesis. Science 249,
1007-1011.
Colditz GA, Hankinson SE, Hunter DJ, Willett WC, Manson JE, Stampfer MJ,
Hennekens C, Rosner B, and Speizer FE (1995). The use of estrogens and progestins
and the risk of breast cancer in postmenopausal women. N Engl J Med 332, 1589-1593.
Collaborative Group on Hormonal Factors in Breast Cancer (1996). Breast cancer and
hormonal contracepdves. Lancet 347, 1713-1727.
Collaborative Group on Hormonal Factors in Breast Cancer (1996). Breast cancer and
hormonal contraceptives: further results. Contraception 54, 1S-106S.
Cressman, V. L., Backlund, D. C., Avrutskaya, A. V., Leadon, S. A., Godfrey, V., and
Roller, B. H. (1999). Growth Retardation, DNA Repair Defects, and Lack of
Spermatogenesis in BRCA1-Deficient Mice. Mol Cell Biol 19,7061-7075.
139
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
El-Ashry D, Chrysogelos S, Lippman M, and Kern F (1996). Estrogen induction of
TGF-a is mediated by an estrogen response element composed of two imperfect
palindromes. J Steroid Biochem Mol Biol 59, 261-269.
Enmark E, Pelto-Huikko M, Grandien K, Lagercrantz S, Lagercrantz J, Fried G,
Nordenskjold M, and Gustafsson J (1997). Human estrogen receptor ( 3 gene structure,
chromosomal localization, and expression pattern. Clin Endocrinol Metab 82, 4258-
4265.
Evans RM (1988). The steroid and thyroid hormone receptor superfamily. Science 240,
889-895.
Fan, S., Wang, J., Yuan, R., Ma, Y., Meng, Q., Erdos, M. R., Pestell, R. G., Yuan, F.,
Aubom, K. J., Goldberg, I. D., and Rosen, E. M. (1999). BRCA1 inhibition of estrogen
receptor signaling in transfected cells. Science 284, 1354-6.
Ferenczy A, Bertrand G, and Gelfand MM (1979). Proliferation kinetics of human
endometrium during the normal menstrual cycle. Am J Obstet Gynecol 133, 859-867.
Folsom AR, Kaye SA, Prineas RJ, Potter JD, Gapstur SM, and Wallance RB (1990).
Increased incidence of carcinoma of the breast associated with abdominal adiposity in
postmenopausal women. Am J Epidemiol 131, 794-803.
140
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Fuller PJ (1991). The steroid receptor superfamily: Mechanisms of diversity. FASEB J
5, 3092-3099.
Giangrande P, Pollio G, and McDonnell D (1997). Mapping and characterization of the
functional domains responsible for the differential activity of the A and B isoforms of
the human progesterone receptor. Jour Biol Chem 272, :32889-32900.
Giguere, B., Hollenberg, S. M., Rosenfeld, M. G., and Evans, R. M. (1986). Functional
domains of the human glucocorticoid receptor. Cell 46, 645-652.
Green S, and Chambon P (1987). Oestradiol induction of a glucocorticoid-responsive
gene by a chimaeric receptor. Nature 325, 75-78.
Green S, Walter P, Kumar V, Krust A, B. J.-M., Argos P, and Chambon P (1986).
Human oestrogen receptor cDNA: Sequence, expression and homology to v-erb-A.
Nature 320, 134-139.
Green, S., Issemann, I., and Sheer, E. (1988). A versatile in vivo and in vitro eukaryotic
expression vector for protein engineering. Nucl Acids Res 16.
141
permission of the copyright owner. Further reproduction prohibited without permission.
Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, and J, S. (1986). Sequence and
expression of human estrogen receptor complementary DNA. Science 231, 1150-1154.
Gudas, J. M., Nguyen, H., Li, T., and Cowan, K. H. (1995). Hormone-dependent
regulation of BRCA1 in human breast cancer cells. Cancer Res 55,4561-5.
Guiochon-Mantel A, Lescop P, Chgristin-Maitre S, Losfelt H, Perrot-Applanat M, and
Milgrom E (1991). Nucleocytoplasmic shuttling of the progesterone receptor. EMBO J
10, 3851-3859.
Hakem, R., de la Pompa, J. L., Sirard, C., Mo, R., Woo, M., Hakem, A., Wakeham, A.,
Potter, J., Reitmair, A., Billia, F., Firpo, E., Hui, C. C., Roberts, J., Rossant, J., and
Mak, T. W. (1996). The tumor suppressor gene Brcal is required for embryonic cellular
proliferation in the mouse. Cell 8 5 ,1009-23.
Henderson BE, Ross RK, Pike MC, and Casagrande JT (1982). Endogenous hormones
as a major factor in human cancer. Cancer Res 42, 3232-3239.
Holt, J. T., Thompson, M. E., Szabo, C., Robinson-Benion, C., Arteaga, C. L., King, M.
C., and Jensen, R. A. (1996). Growth retardation and tumour inhibition by BRCA1 [see
comments] [published erratum appears in Nat Genet 1998 May;19(l):102]. Nat Genet
12, 298-302.
142
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Hong, J., Kohli, K., Trivedi, A., Johnson, D. L., and Stallcup, M. R. (1996). GRIP1, a
novel mouse protein that serves as a transcriptional co-activator in yeast for the
hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93,4948-4952.
Irvine, R. A., Ma, H., Yu, M. C., Ross, R. K., Stallcup, M. R., and Coetzee, G. A.
(2000). Inhibition of pl60-mediated coactivation with increasing androgen receptor
polyglutamine length. Hum Mol Genet 9, 267-274.
Kastner P, Krust A, Turcotte B, Stropp U, Tora L, Gronemeyer H, and Chambon P
(1990). Two distinct estrogen-regulated promoters generate transcripts encoding the two
functionally different human progesterome receptor forms A and B. The EMBO Journal
9, 1603-1614.
Kelsey JL, Gammon MD, and John EM (1993). Reproductive factors and breast cancer.
Epidemiol Rev 15, 36-47.
Key TJA, and Pike MC (1988). The dose-effect relationship between "unopposed"
oestrogens and endometrial mitotic rate: Its central role in explaining and predicting
endometrial cancer risk. Br J Cancer 57,205-212.
143
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Key TJA, and Pike MC (1988). The role of oestrogens and progestagens in the
epidemiology and prevention of breast cancer. Eur J Cancer Clin Oncol 24, 29-34.
Klein-Hitpass, Ryffel G, Heitlinger E, and Cato A (1987). A 13 bp palindrome is a
functional estrogen response element and interacts specifically with estrogen receptor.
Nucleic Acids Res 16, 647-663.
Klock G, Strahle U, and Schutz G (1987). Oestrogen and glucocorticoid responsive
elements are closely related but distinct. Nature 329,734-736.
Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, and Gustafsson J (1996). Cloning of
a novel estrogen receptor expressed in rat prostate and ovary. Proc Natil Acad Sci USA
93, 5925-5930.
Kuiper GG, and Gustafsson J (1997). The novel estrogen receptor-{3 subtype: potential
role in the cell- and promoter-specific actions of estrogens and anti-estrogens. FEBS
Lett 410, 87-90.
Kumar V, Green S, Stack G, Berry M, Jin JR, and Chambon P (1987). Functional
domains of the human estrogen receptor. Cell 51, 941-951.
permission of the copyright owner. Further reproduction prohibited without permission.
Kumar V, Green S, Staub A, and Chambon P (1986). Localisation of the oestradiol-
binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J
5, 2231-2236.
Landis S, Murray T, Bolden S, and Wingo P (1998). Cancer Statistics, 1998. CA Cancer
J Clin 48, 6-30.
Lane, T. F., Deng, C., Elson, A., Lyu, M. S., Kozak, C. A., and Leder, P. (1995).
Expression of Brcal is associated with terminal differentiation of ectodermally and
mesodermally derived tissues in mice [published erratum appears in Genes Dev 1996
Feb 1;10(3):365]. Genes Dev 9, 2712-22.
Levenson A, and Jordan C (1994). Transfection of human estrogen receptor (ER) cDNA
into ER-negative mammalian cell lines. J Ster Biochem Mol Biol 54, 229-239.
Longcope C (1971). Metabolic clearance and blood production rates of estrogens in
postmenopausal women. Am J Obstet Gynecol 111, 778-781.
Ma, H., Hong, H., Huang, S. M., Irvine, R. A., Webb, P., Kushner, P. J., Coetzee, G. A.,
and Stallcup, M. R. (1999). Multiple signal input and output domains of the 160-
kilodalton nuclear receptor coactivator proteins. Mol Cell Biol 19, 6164-6173.
145
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
MacDonald PC, Edman CD, Hemsell DL, Porter JC, and Siiteri PK (1978). Effect of
obesity on conversion of plasma androstenedione to estrone in postmenopausal women
with and without endometrial cancer. Am J Obstet Gynecol 130, 448-455.
MacMahon B, Cole P, and Brown J (1973). Etiology of human breast cancer: A review.
J Natl Cancer Inst 50, 21-42.
Marks, J. R., Huper, G., Vaughn, J. P., Davis, P. L., Norris, J., McDonnell, D. P.,
Wiseman, R. W., Futreal, P. A., and Iglehart, J. D. (1997). BRCA1 expression is not
directly responsive to estrogen. Oncogene 14, 115-21.
Marquis, S. T., Rajan, J. V., Wynshaw-Boris, A., Xu, J., Yin, G. Y., Abel, K. J., Weber,
B. L., and Chodosh, L. A. (1995). The developmental pattern of Brcal expression
implies a role in differentiation of the breast and other tissues. Nat Genet 11, 17-26.
Misrahi M, Atger M, d’Auriol L, Loosfelt H, Meriel C, Fridlansky F, Guiochon-Mantel
A, Galibert F, and Milgrom E (1987). Complete amino acid sequence of the human
progesterone receptor deduced from cloned cDNA. Biochem Biophys Res Comm 143,
740-748.
146
permission of the copyright owner. Further reproduction prohibited without permission.
Misrahi M, Venenci PY, Saugier-Veber P, Sar S, Dessen P, and Milgrom E (1993).
Structure of the human progesterone receptor gene. Biochem et Biophys Acta 1216,
289-292.
Moore DH, Moore DH, and Moore CT (1983). Breast carcinoma etiological factors.
Adv Cancer Res 40, 189-253.
Mosselman S, Polman J, and Dijkema R (1996). ER-P: identification and
characterization of a novel human estrogen receptor. FEBS Lett 392, 49-53.
Norris, J., Fan, D., Aleman, C., Marks, J. R., Futreal, P. A., Wiseman, R. W., Iglehart, J.
D., Deininger, P. L., and McDonnell, D. P. (1995). Identification of a new subclass of
Alu DNA repeats which can function as estrogen receptor-dependent transcriptional
enhancers. J Biol Chem 270,22777-82.
Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, Ouchi Y, and Muramatsu
M (1998). The complete primary structure of human estrogen receptor P (hERP) and its
heterodimerization with ER a in Vivo and in Vitro. Biochem Biophy Res Comm 243,
122-126.
147
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Picard D, Kumar V, Chambon P, and Yamamoto K (1990). Signal transduction by
steroid hormones: Nuclear localization is differentially regulated in estrogen and
glucocorticoid receptors. Cell Regul 1, 291-299.
Pike MC (1987). Age-related factors in cancers of the breast, ovary, and endometrium. J
Chron.Dis 40 (Suppl II), 59S-69S.
Pike MC, Bernstein L, and Spicer DV. (1993). Exogenous hormones and breast cancer
risk. In Current Therapy in Oncology, Niederhuber JE, ed. (St. Louis: Mosby Year
Book, Inc.).
Ponglikitmongkol M, Green S, and Chambon P (1988). Genomic organization of the
human oestrogen receptor gene. EMBO J 7, 3385-3388.
Rehberger P, Rexin M, and Gehring U (1992). Heterotetrameric structure of the human
progesterone receptor. Proc Natil Acad Sci USA 89, 8001-8005.
Segnitz B, and Ghering U (1995). Subunit structure of the nonactivated human estrogen
receptor. Proc Natl Acad Sci USA 92,2179-2183.
Siiteri PK (1978). Steroid hormones and endometrial cancer. Cancer Res 38,4360.
148
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Snoek, R., Bruchovsky, N., Kasper, S., Matusik, R. J., Gleave, M., Sato, N., Mawji, N.
R., and Rennie, P. S. (1998). Differential transactivation by the androgen receptor
cancer cells. Prostate 36, 256-263.
Spillman, M. A., and Bowcock, A. M. (1996). BRCA1 and BRCA2 mRNA levels are
coordinately elevated in human breast cancer cells in response to estrogen. Oncogene
13, 1639-45.
Tilley, W. D., Marcelli, M., J.D., W., and M.J., M. (1989). Characterization and
expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci USA
86, 327-331.
Tora L, Mullick A, Metzger D, Ponglikitmongkol M, Park I, and Chambon P (1989).
The cloned human oestrogen receptor contains a mutation which alters its hormone
binding properties. EMBO J 8 ,1981-1986.
Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, and
Giguere V (1997). Cloning, chromosomal localization, and functional analysis of the
murine estrogen receptor p. Mol Endocrinol 11, 353-365.
Trichopolous D, MacMahon B, and Cole P (1972). The menopause and breast cancer
risk. J Natl Cancer Inst 48,605-613.
149
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Tsai MJ, and O’Malley BW (1994). Molecular mechanisms of action of steroid/thyroid
receptor superfamily. Annu Rev Biochem 63,451-486.
Umesono, K., and Evans, R. M. (1989). Determinants of Target Gene Specificity For
Steroid/thyroid Hormone Receptors. Cell 57, 1139-1146.
Wahli W, and Martinez E (1991). Superfamily of steroid nuclear receptors: Positive
and negative regulators of gene expression. FASEB J 5, 2243-2249.
Webb, P., Lopez, G. N., Uht, R. M„ and Kushner, P. J. (1995). Tamoxifen activation of
the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like
effects of antiestrogens. Mol Endocrinol 9 ,443-456.
Wei LL, Norris BM, and Baker CJ (1997). An N-terminally truncated third
progesterone receptor protein, PRC , forms heterodimers with PRg but interferes in
PRb-DNA binding. J Steroid Biochem Molec Biol 62, 287-297.
Wilson, C. A., Payton, M. N., Elliott, G. S., Buaas, F. W., Cajulis, E. E., Grosshans, D.,
Ramos, L., Reese, D. M., Slamon, D. J., and Calzone, F. J. (1997). Differential
150
permission of the copyright owner. Further reproduction prohibited without permission.
subcellular localization, expression and biological toxicity of BRCA1 and the splice
variant BRCAl-deltal lb. Oncogene 14, 1-16.
Xu J, Nawaz Z, Tsai S, Tsai M-J, and O’Malley B (1996). The extreme C terminus of
progesterone contains a transcriptional repressor domain that functions through a
putative corepressor. Proc Natl Acad Sci USA 93, 12195-12199.
Xu, X., Wagner, K. U., Larson, D., Weaver, Z., Li, C., Ried, T., Hennighausen, L.,
Wynshaw-Boris, A., and Deng, C. X. (1999). Conditional mutation of Brcal in
mammary epithelial cells results in blunted ductal morphogenesis and tumour formation
[see comments]. Nat Genet 22, 37-43.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission
151
CHAPTER 4. Recruitment of Protein Methyltransferases, CARM1
and PRMT1, by BRCA1 in Steroid Receptor Signaling
INTRODUCTION
There is a growing body of evidence supporting a role for BRCA1 in the
regulation of steroid hormone signaling. In addition to our own studies, other labs have
demonstrated that BRCA1 not only interacts with the steroid receptors, but also
functions in either enhancing, or inhibiting in the study by Fan et al, receptor-mediated
transcriptional activation. We have further shown that BRCA1 also interacts with
members of the pl60 family of coactivators, such as GRIPl, SRCla, and AIB1,
resulting in cooperative, and depending upon the levels of NR, sometimes synergistic
enhancement of steroid hormone signaling. As previously mentioned in Chapter 2, the
pl60 coactivators are nuclear receptor regulatory proteins that function in ligand-
dependent receptor signaling through their recruitment of various other transcriptional
regulatory proteins, such as histone acetyltransferases (HATs) and protein
methyltransferases (PMTs). These secondary coactivators function in remodeling
chromatin structure through covalent modification of key residues located on DNA-
bound histones. This modification results in the relaxation of local chromatin structure,
presumably to facilitate transcription initiation by increasing promoter access to the
transcriptional complex.
152
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
That BRCA1 is involved in chromatin remodeling is not a novel observation (Hu et al.,
1999). Recently, BRCA1 has been shown to interact with the histone-
acetyltransferases, p300/CBP, which function as transcriptional coactivators of BRCA1
(Pao et al., 2000). In addition, BRCA1 is also associated with the ATP-dependent
chromatin remodeling complex, SWI/SNF, which was shown to play a critical role in
BRCAl’s ability to coactivate p53-dependent transcription of a p21-reporter gene
(Bochar et al., 2000). Interestingly, BRCA1 has also been demonstrated to interact with
components of the histone deacetylase complex (HDAC), though the physiologic
relevance of this association remains to be determined (Yarden and Brody, 1999). One
possible explanation is that BRCA1 merely functions in physically sequestering the
HDACs away from the promoter regions of DNA undergoing active gene transcription.
This mechanism has been demonstrated for the El A carboxy-terminal domain, which is
hypothesized to promote transcription by disrupting promoter-bound CtBP (E1A
carboxy-terminal binding protein) and HDAC1 (Sundqvist et al., 1998). Alternatively,
BRCA1 may have a dual role in transcription regulation, recruiting chromatin
destabilizing proteins under certain conditions, such as during coactivation
(Somasundaram et al., 1997); and recruiting chromatin stabilizing proteins under others,
i.e. inhibition (Fan et al., 1999; Wang et al., 1998). In any event, it is clear that, in
addition to a putative role in recruiting and stabilizing the transcription initiation
complex to the promoters of activated genes, BRCA1 may also function in the
remodeling of local chromatin structures, thereby, altering the accessibility of DNA
during transcription.
154
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
In the current study, we propose that BRCA1, like the pl60 coactivators, can
function in recruiting protein methyltransferases during hormone-dependent
enhancement of steroid receptor signaling. These results suggest that BRCA1 may
function by coordinating the recruitment and stabilization of several different
coactivators involved in steroid hormone regulation, such as the pl60 coactivators and
the chromatin remodeling PMTs. This study is novel in that no other coactivators, other
than the previously described GRIP1 and SRC la, have yet been observed to interact
with the PMTs during transcriptional regulation, much less during a specific,
physiological process, such as during steroid hormone signaling. Hence, not only do we
show that BRCA1 interacts with the PMTs, but this is the first study to suggest a
potential physiologic role for BRCA1 and chromatin remodeling.
MATERIALS AND METHODS
Transient transfection assays were performed using a firefly luciferase (LUC)
assay system. Mammalian cells were transfected with plasmids encoding various
transcription factors and tested for reporter gene activation. In some cases, protein
expression from transfected plasmids were checked by immunofluorescence. Protein
binding assays were performed using glutathione-S-transferase (GST) fusion proteins
and in vitro transcribed and translated protein targets. Coimmunoprecipitation assays
were performed using exogenously expressed proteins.
155
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Mammalian Expression Vectors and Plasmid Construction
Proteins with N-terminal hemagglutinin A (HA) epitope tags were expressed in
transient mammalian cell transfections and in vitro from pSG5.HA, which has SV40
and T7 promoters (Chen et al., 1999). the following proteins were expressed from
previously described pSG5.HA derivatives: GRIP1AAD1 (full-length GRIP1 with
amino acids 1057-1109 deleted) (Ma et al., 1999); GRIP1, GRIP15 _ 7 6 5 , GRIPl7 3 0 _ I 1 2 1 ,
GRIP11 I2 I.1 4 6 2 , and CARM1 (Chen et al., 1999). A polymerase chain reaction (PCR)-
amplified cDNA fragment encoding full-length PRMT1 was cloned into the EcoRI-
BamHI sites of pSG5.HA.
Other previously described mammalian expression plasmids include: pSVARo
(transient transfection assays) (Brinkmann et al., 1989) and pcDNA-hAR (in vitro
expression) (Irvine et al., 2000) both encode androgen receptor, pSG5-ERa encoding
estrogen receptor (Green et al., 1988), pPR (progesterone receptor), pSG5-GRIPl.
Luciferase reporter gene constructs were previously described: MMTV-LUC for AR
and PR, MMTV(ERE)-LUC for ER (Umesono and Evans, 1989). BRCA1 mammalian
expression plasmid pcDNA-BRCAl and overlapping BRCA1 fragments were
previously described (Chapter 2).
156
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Bacterial Expression Plasmids
Bacterial expression plasmids encoding GST, GST-PRMT1 (Lin et al., 1996)
and GST-CARM1 (Chen et al., 1999) were also previously described.
Tissue culture and transfections
CV-1 cells were transfected and cell extracts were assayed for luciferase activity
as described previously. When hormone was required, transfections were grown in
medium containing charcoal-treated serum (Gemini Bio Products) and lOnM
concentration of appropriate hormone for each NR: dihydrotestosterone for AR,
progesterone for PR, and estradiol for ER.
Luciferase (LUC) Assays
Following tranfections of 24-well plates, cell extracts for luciferase assays were
prepared by adding 125 pi of IX Lysis Buffer (Promega) per well and freezing at
-80°C. Frozen lysates were thawed at room temperature and then shaken on a rotary
shaker at 200 rpm for 15 minutes. 20 pi of lysates were removed and placed onto
opaque 96-well luciferase assay plates for luminometer readings. 40 pi of luciferase
157
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
reagent (Promega) were injected into each well and triplicate (quadruplicate) samples
were read on a plate-reading luminometer.
Glutathione-S-Transferase (GST) Pull-Downs
Glutathione-S-transferase (GST) and GST-fusion proteins were expressed and
purified as described (Hong et al., 1996). Glutathione-Sepharose-bound GST protein,
GST-CARM1, or GST-PRMT1 were incubated with 3 5 S-radiolabeled full-length
BRCA1 or BRCA1 fragments transcribed and translated in vitro from pcDNA3.l
vectors using a TNT-Coupled Reticulocyte Lysate System (Promega) in the presence of
3 5 -S methionine. Associated BRCAl was eluted, resolved by SDS-polyacrylamide gel
electrophoresis (PAGE) and analyzed by autoradiography. 10% of total labeled
BRCAl incubated in each reaction was loaded for comparison.
Preparation of Whole Cell Extracts
Culture cells were washed with cold PBS and treated with RIPA Lysis Buffer
(50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, pH 8.0) containing complete
protease inhibitors (Roche). Cells were lysed by incubating on ice for 10 minutes and
then passaging through a 23 gauge needle. Lysates were separated from cellular debris
by centrifugation and quantified by BCA protein quantitation assay (Pierce).
158
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Co-Immunoprecipitation and Western Immunoblotting
Whole cell lysates were obtained as described above. Protein lysates were
precleared with Protein-G-Sepharose beads for 1 hour. Protein G was removed and 2-3
|ig of appropriate antibodies were added to each lysate to incubate overnight at 4°C.
The next morning Protein G was added to immunocomplexes and allowed to bind for 2
hours. Beads were washed 3-4 times in RIPA lysis buffer and then boiled in SDS
sample buffer. Eluted proteins were resolved by SDS/PAGE and transferred onto
PVDF membranes for western blot analysis.
Membranes were blocked overnight in 5% milk solution and then incubated for
1-2 hours with primary antibodies in blocking solution. Membranes were then washed
with PBST and incubated with appropriate dilutions of secondary antibodies (blocking
solution) for 1 hour. Antibody complexes were detected with the ECL
chemiluminescent system (Amersham).
RESULTS
BRCAl Recruits Protein Methyltransferases In The Enhancement Of Androgen
Receptor Signaling
Previously we have demonstrated that BRCAl interacts with the C-terminus of
the pl60 nuclear receptor coactivator, GRIP1 (Chapter 2). This is also the interaction
domain for another coactivator protein, CARM1 (Hong et al., 1996). CARM1 is a
159
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
member of the protein methyltransferase family, which includes PRMT1, and is an
Nc,NG -dimethylarginine transferase (Aletta et al., 1998). It has previously been shown
to enhance nuclear receptor (NR) signaling through its recruitment by NR-bound p i60
coactivators, GRIP1 and SRCla. However, unlike the pl60 coactivators and BRCAl,
CARM1 is unable to enhance NR signaling in the absence of another coactivator,
making it a secondary coactivator in NR signaling pathways. Preliminary evidence
suggests that PRMTl, which shares significant homology with CARM1, also behaves
in a similar manner (Koh et al., unpublished results). Since BRCAl is a direct
coactivator of the androgen receptor, and both BRCAl and CARM1 interact on the
same domain of GRIP1, we tested to see if BRCAl and CARM1 could interact to result
in functional enhancement of AR signaling.
Expression of either protein methyltransferase (PMT), CARM1 or PRMTl,
resulted in enhanced BRCAl coactivator function of the androgen receptor (Figure 4.1).
In transiently-transfected CV-l monkey kidney cells, both CARM1 and PRMTl was
able to mediate further enhancement of BRCAl coactivation of the AR in a hormone-
dependent manner. This enhancement was dose-dependent as increasing the dose of
either PMT resulted in further increase in enhancement. In addition, recruitment of
PMT coactivator function was BRCAl-dependent, as both CARM1 and PRMTl failed
to enhance AR signaling in the absence of exogenous BRCAl.
160
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Recruitment of PMT Activity by BRCAl in Progesterone and Estrogen Receptor
Signaling Pathways
Since BRCAl was also found to be a coactivator of the progesterone receptor,
and the estrogen receptor, through its interaction with the p i60 coactivators, we tested if
BRCAl could recruit protein methyltransferase activity in either PR and/or ER
signaling. In Figure 4.2, we show that both CARM1 and PRMTl are able to mediate
further enhancement of BRCAl coactivator activity in PR signaling, which was again
hormone- and receptor-dependent. This enhancement was dose-dependent as increasing
the dose of exogenous PMT resulted in increased reporter gene activity.
Similarly, CARM1 was demonstrated to enhance BRCAl function in ER
signaling (Figure 4.3), though BRCAl enhancement of the ER by itself was modest.
Nevertheless, taken together, these results suggest that BRCAl, like GRIP1, is able to
interact functionally with protein methyltransferases, providing a possible role of
BRCAl in the recruitment of these chromatin remodeling proteins during NR signaling.
BRCAl and the Protein Methyltransferases Interact in vitro
To investigate a possible, physical interaction between BRCAl and the two
PMTS, we performed in vitro GST pull-down experiments using bacterially-expressed
glutathione-S-transferase-fused CARM1 and PRMTl to pull-down in vitro transcribed
and translated full-length BRCAl. We show that both GST-CARM1 and GST-PRMT1
161
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
bind to full-length BRCAl, though PRMTl appears to have a stronger association
(Figure 4.4). This interaction is specific for the GST-fusion proteins as no interaction
was detected with the GST control
To map the interaction domain of CARM1 and PRMTl on BRCAl, further GST
pull-down experiments were performed using GST, GST-CARM1, or GST-PRMT1 and
various overlapping fragments of in vitro transcribed and translated BRCAl. We show
that CARM1 interacts exclusively with the N-terminus of BRCAl corresponding to
amino acids 1-404. In contrast, PRMTl appears to bind to a large region spanning
amino acids 1-1142, with the strongest interaction spanning amino acids 1-404. Based
upon coomassie blue staining of the GST-fusion proteins, it is likely that this difference
is due to the use of a larger amount of GST-PRMT1.
Interaction of BRCAl and HA-PRMT1 in Mammalian Cells
To determine if BRCAl interacts with the protein methyltransferases in
mammalian cells, we transfected Cos-7 cells with plasmids encoding both BRCAl and
HA-PRMT1. Whole cell extracts were prepared as described above, split into equal
halves, and immunoprecipiated with a BRCAl rabbit polyclonal antibody (l|ig/ml)
(Santa Cruz), or a non-specific rabbit polyclonal antibody (Zymed). Western blotting
using an anti-HA rat polyclonal antibody (Roche) showed specific interaction between
BRCAl and HA-PRMT1, which was immunoprecipitated by the BRCAl antibody, but
162
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
not the control IgG (Figure 4.5). To show that the HA-PRMT1 » s actually being
expressed, whole cell extract was run and blotted with anti-HA antibodies.
Enhancement of AR Signaling By CARM1 and PRMTl is Not Synergistic with
BRCAl, Unlike with GRIP1
Although both CARM1 and PRMTl are Type I protein methyltransferases, their
histone substrate specificities differ, with CARM predominantly melbylating arginines
on histone 3 (H3) and PRMTl methylating histone 4 (H4). GRIPl-mediated
recruitment of CARM1 or PRMTl coactivator function results in i » modest 3-5 fold
increase in enhancement of NR activity relative to GRIP1 and NR atone (Figure 4.6).
However, when CARM1 and PRMTl is coexpressed with G J*-lp i - synergistic
enhancement of nuclear receptor activity is observed (Koh et al., unpublished data;
Figure 4.7). This synergy is probably the due to the difference in substrate specificities
of the two protein methyltransfereases. In contrast, no synergy is observed when
GRIP1 is substituted with BRCAl (Figure 4.8), suggesting the possibility of different
mechanisms for BRCAl and GRIP1.
Synergistic Enhancement of Androgen Receptor Signaling by BRCAl, GRIP1, and
CARM1 or PRMTl
We have previously shown that BRCAl synergistically enhances GR1P1
function in AR signaling. Here we also show that BRCAl, like G R Ifl > can function in
163
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
recruiting CARM1 or PRMT1 in nuclear receptor signaling pathways. Since BRCA1 is
found to associate with both GRIP1 and the protein methyltransferases, we next tested if
the three coactivators can associate to function together in enhancing AR activity. We
transfected cells with AR and various combinations of BRCA1, GRIP1, CARM1,
and/or PRMT1. From our results (Figure 4.9), we show that BRCA1 and GRIP1 alone
can enhance AR function on reporter gene activity by 3.7 fold and 6.4 fold,
respectively. Furthermore, when GRIP1 was coexpressed with either CARM1 or
PRMT1, there was an additional enhancement of AR activity observed, 22.5 fold and
17.6 fold, respectively. However, when BRCA1 was coexpressed with either GRIP and
CARM1, or GRIP1 and PRMT1, a synergistic enhancement of activity was seen, which
was dependent upon the presence of both GRIP I and BRCA1. For comparison,
coexpression of BRCA1 with GRIP1 only enhanced AR signaling by 25.2 fold. Hence,
coexpression of BRCA1 and GRIP I with either of the two protein methyltransferases
result in synergistic enhancement of androgen receptor function.
Synergy by BRCA1, GRIP1 and CARM1 is Dose-Responsive Throughout a Linear
Range of Exogenous Methyltransferase
To test if the synergistic cooperation among the nuclear receptor coactivators
BRCA1, GRIP1 and CARM1 occurred over a wide dose range of CARM1, we
transfected cells with increasing amounts of plasmid encoding CARM1 alone, or in the
presence of BRCA1, GRIP1, or BRCA1 and GRIP1 (Figure 4.10). In the absence of
164
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
any exogenous coactivator, CARM1 does not enhance AR activity over a broad dose
range (0.05p.g-0.25|ig ). Coexpression of either BRCA1 or GRIP1 results in a linear
increase in AR signaling with increasing amounts of CARM1. However, when BRCA1
and GRIP1 were both coexpressed with CARM1, a synergistic enhancement of AR
activity was observed at all doses of exogenous CARM1 expression. Therefore, it is
possible that BRCA1, GRIP1, and CARM1 play a cooperative role in nuclear receptor
signaling pathways, potentially through the formation of a coactivator complex in vivo.
Synergistic Enhancement of ER and PR Signaling by BRCA1, GRIP1 and the
Protein Methyltransferases
We have previously shown that BRCA1 and GRIP1 can function cooperatively
in activating both ER and PR signaling. In addition, both BRCA1 and GRIP1 can
individually recruit CARM1 and PRMT1 in enhancing NR function. We next wanted
to test if BRCA1 and GRIP1 can synergistically activate ER and PR signaling by
recruiting either CARM1 or PRMT1 (Figure 4.11). When coexpressed with ER, GRIP I
enhances NR-dependent reporter gene activation by 5.31 fold. This enhancement is
increased to 7.3 fold in the presence of CARM1 and 10.34 fold in the presence of
PRMT1. However, when BRCA1 is coexpressed with GRIP1 and CARM1, or GRIP1
and PRMT1, the coactivator effect on ER activity is enhanced to 34.92 fold and 19.96
fold, respectively. Similarly, when GRIP1 was coexpressed with either CARM1 or
PRMT1, PR activity was enhanced 10.37 fold and 10.88 fold, approximately 3 times the
165
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
effect seen by GRIP1 alone on PR activity. However, when BRCA1 was coexpressed
with GRIP1 and CARM1, or GRIP1 and PRMT1, PR activity was enhanced 26.52 fold
and 22.23 fold, respectively. For both ER and PR, neither CARM1 nor PRMT1
enhanced NR activity in the absence of both exogenous BRCA1 and GRIP1. Hence,
these results suggest that the cooperative effect of GRIP1, BRCA1 and the protein
methyltranferases is generic for the steroid hormone receptors.
Synergy Among GRIP1, BRCA1 and CARM1 Requires the AD2 Interaction
Domain of GRIP1
Interaction studies have shown that both CARM1 and BRCA1 binding to GRIP1
requires the C-terminus of GRIP1. Loss of this AD2 domain results in loss of binding
with CARM1 and BRCA1. To test if functional synergy is also dependent upon this
domain, we transfected cells with BRCA1, CARM1 and various deletion mutants of
GRIP1 (Figure 4.12). Both full-length GRIP1 and GRIP1AADI, a mutant with an
internal deletion of the ADI domain, were able to enhance AR activity. The C-terminal
truncation mutant of GRIP1, GRIP1AAD2, however, was unable to function with either
BRCA1 or CARM1 individually. Not surprisingly, no significant enhancement was
observed when this mutant was coexpressed with both BRCA1 and CARM1.
Therefore, an intact AD2 interaction domain is necessary for cooperative synergy with
BRCA1 and CARM1 to occur.
166
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
DISCUSSION
BRCA1 and Chromatin Remodeling
BRCA1 is a coactivator of the steroid receptors AR, ER, and PR. It enhances
hormone-dependent signaling either independently or cooperatively with members of
the pl60 family of nuclear receptor coactivators. These coactivators, which includes
GRIP1, SRCla, and AIB1, are thought to function in nuclear receptor signaling by
recruiting other transcriptional regulatory proteins, such as the chromatin remodeling
histone acetyltransferases (HATs) during steroid hormone signaling. Recently,
coactivator associated arginine methyltransferase 1, or CARM1, was found to interact
with the C-terminus of GRIP1 resulting in enhancement of GRIP 1-mediated NR
coactivation. Interestingly, this is also the interaction domain for BRCA1 (Park et al.,
unpublished data), suggesting a possible association between CARM1 and BRCA1
during transcription regulation.
Given the evidence presented here, we conclude that BRCA1 not only
physically, but also functionally interacts with CARM1. In transiently transfected
mammalian cells, BRCA1 is able to enhance NR signaling in a dose-dependent manner.
Coexpression with either CARM1, or the homologous PRMT1, results in further
enhancement of BRCA1 coactivation function. This enhancement was PMT dose-
dependent and required coexpressed BRCA1 since neither CARM1 nor PRMT1 was
able to enhance NR signaling in the absence of BRCA1. Therefore, BRCA1, much like
167
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
GRIP1, may function in NR signaling through mechanisms involving the recruitment of
the chromatin remodeling proteins, such as the methyltransferases, CARM1 and
PRMT1, in addition to the HATs and SWI/SNF complex. However, it is not currently
known if these proteins associate as a larger multiprotein complex, much less function
together during transcriptional activation.
BRCA1 was found to interact with both CARM1 and PRMT1 by GST pull
down assay. Protein mapping studies using overlapping fragments of BRCA1
demonstrated the interactions to localize to the N-terminus of BRCA1. Furthermore, in
transiently transfected cells, BRCA1 and PRMT1 were shown to interact by
coimmunoprecipitation assay. Taken together, these results support the functional
association observed between BRCA1 and the protein methyltransferases during steroid
hormone signaling. Previously, we have shown that BRCA1 and GRIP I interacts both
in vitro and in live cells (Park et al., unpublished data). Furthermore, GRIP1 is also
known to interact with both CARM1 and PRMT1 (Chen et al., 1999). Since each of
these interactions were demonstrated from transfected cells in the absence of
coexpressed NRs, and in the absence of hormones, it is possible that BRCA1, GRIP1,
and CARM1 exist as a pre-formed complex that is recruited during NR-dependent
hormone signaling, much like the previously described DRIP/TRAP complexes.
Indeed, we show here that BRCA1, GRIPl, and the PMTs are able to function
synergistically in enhancing NR signaling, providing preliminary evidence supporting a
functional role for a BRCA1/GRIP1/CARM1 multi-protein complex, with BRCA1 most
likely serving as a cointegrator, or “docking” protein. However, until protein
168
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
purification and colocalization studies can be performed to corroborate the existence of
such a complex, there is no clear evidence to support that this coactivator complex does
form in vivo.
Physiological Significance of Coactivator Synergy
The physiological significance for the coactivator synergy demonstrated in these
studies remains unclear, though many hypotheses can be proposed. Indeed, the
existence of enormous coactivator complexes, such as the preformed TRAPs, DRIPs,
and SWI/SNF coregulators (Xu et al., 1999), provides much precedence supporting the
existence of additional multiprotein complexes. The p i60 coactivators have already
been demonstrated to associate with CBP/p300, the protein methyltransferases, and
other regulatory proteins. Based upon the results presented here, we conclude that
BRCA1 is also a component of this coactivator complex. With BRCA1 intimately
associated with various aspects of the transcriptional machinery, it is possible that it
may act as a bridge linking DNA-bound receptors and coregulatory proteins with the
basal transcriptional machinery, thereby, facilitating and stabilizing transcription in
response to hormone signaling. Considering the fact that the majority of mutations in
BRCA1 result in premature truncation products, it is likely that these mutations play a
role in phenotypic disease as a direct result of loss of this vital bridging function. This
is supported by the observation that BRCA1 truncation mutants lacking the C-terminus,
which is crucial for RNA polymerase II association, no longer function as coactivators,
169
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
but rather, act as dominant negative inhibitors of transcription (Zhang et al., 1998).
Since these truncation mutants retain intact N-terminal domains, it is thought that they
may function in sequestering limiting transcription factors away from wild-type BRCA1
resulting in diminished BRCAl-related activity. This reduced function may not become
phenotypically apparent for most physiologic processes, but may play a role in more
sensitive pathways, such as AR signaling (Rebbeck et al., 1999). However, why certain
pathways are more affected than others is a complex issue. We demonstrate here that
one possible explanation may involve BRCAl’s apparent ability to coordinate the
formation and function of transcriptional complexes resulting in coactivator synergy, at
least in the regulation of the steroid receptors.
In essence, nuclear receptors play an important role in the recruitment and
stabilization of preinitiation complexes at the promoters of target genes. This function
is accomplished through various direct and indirect interactions between the activated,
DNA-bound nuclear receptors and elements of the basal transcription machinery
(McKenna et al., 1999). At the heart of these interactions, though, are the coactivators
and the multiprotein complexes that they form. These coactivator complexes have
many functions which include stabilization of the transcriptional machinery and
relaxation of chromatin structure (Xu et al., 1999). Among a few of the better known
complexes are the SWI/SNF and P/CAF chromatin remodeling complexes, the TRAP
and DRIP nuclear receptor complexes, and the newly-discovered ARC complex, which
is implicated in the regulation of numerous proteins, such as SREBP, NFkB and VP 16
(Naar et al., 1999; Rachez et al., 1999). With the finding that BRCA1 functions as a
170
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
coactivator in in vitro assays (Bochar et al., 2000; Ouchi et al., 1998; Somasundaram et
al., 1997), and has also been found to be associated with other elements of the
transcriptional machinery, it is highly likely BRCA1 participates in transcription
regulation as part of larger coactivator complexes. It is already known that BRCA1 is
associated with complexes in other pathways, such as the hRAD50-hMREll-p95/nibrin
DNA-repair complex (Zhong et al., 1999) and the SWI/SNF (Bochar et al., 2000)
chromatin remodeling complex, giving precedence for the role we describe here as part
of the pl60 nuclear receptor coactivator complex, which includes CBP/p300 and the
protein methyltransferases. As a component of this, and other, transcriptional
complexes, BRCA1 may play a unique role in facilitating the enormously complex
coordination required for activated transcription.
In the case of the pl60 coactivators and the protein methyltransferases, this
coordination results in significant enhancement of nuclear receptor function as detected
by reporter gene activation. Whether a similar synergy occurs in vivo, however,
remains to be determined. Thusfar, we have been unable to purify a complex
containing BRCA1, the pl60 coactivators, and the protein methyltransferases. It is
possible that these interactions a relatively weak and are broken under the stringent
conditions used to isolate the proteins. Alternatively, these complexes may also be
subject to specific regulation, associating only under specific tissue- and cell-cycle-
dependent conditions. Depending upon its expression throughout the cell-cycle, or its
phosphorylation state, BRCA1 may associate with certain complexes, but not with
others. Therefore, by regulating its expression and/or modifying its structure/function
171
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
by phosphorylation, BRCA1 can play many different roles. It may only be a matter of
time before physical evidence is found to support the putative physiological synergy of
BRCA1 and the NR coactivator complexes. There are many examples of tissue-specific
nuclear receptor coregulators with such temporal/spatial dependent functions, such as
the PPAR-y coactivator, PGC-1, or PPAR-y coactivator-1 (Puigserver et al., 1998), and
the recently described TRF, or TBP related factor, which was found to harbor similar
biochemical functions as TBP, but has a different tissue specificity and cellular
localization pattern than TBP (Hansen et al., 1997). Through this tightly regulated
process, it is possible to control many different pathways using a limited number of
proteins, such as BRCA1.
The nuclear receptors mediate tightly-regulated processes resulting in target
gene expression in response to stimuli signaling many disparate events, including
development, differentiation and reproduction. Although these events are well-
characterized on a physiological level, very little is known about their mechanisms,
especially in relation to other coregulatory factors, on a molecular level. As more
evidence emerges, it is now becoming clear that these transcriptional events are
multistep requiring complex coordination of various elements of the transcriptional
machinery, including the coactivator complexes. These multiprotein complexes are
most likely preformed and behave as modular units during activated receptor signaling.
As a result of this modular characteristic, it is possible to regulate receptor activity by
simply shuffling the various coactivator complexes to and from the promoters of target
genes. Hence, we describe here a situation that supports the formation and possible
172
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
function of BRCA1 in modifying steroid receptor activity through its association with a
large, multiprotein complex which includes the p i60 coactivators and the protein
methyltransferases. We anticipate that with further study, this association will provide
significant insights into BRCAl’s role during steroid hormone signaling, and why loss
if its function results in increased neoplastic disease in specific tissues.
173
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4-1. Recruitment of CARM1 and PRMT1 by BRCA1 in AR signaling. CV-
1 cells were transfected with 10 ng of pCMV-hAR expression vector, 250 ng of
pcDNA3.1-BRCAl plasmid, and either 200 or 400 ng of pSG5.HA-CARMl or
pSG5.HA-PRMTl. 125 ng of MMTV-LUC reporter plasmid was also transfected per
well of the 12-well cultures. pSG5.HA empty vector was used as filler DNA.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-1
&
>
3
5
o
&
>
o
<
0
10nM DHT
AR
BRCA1
0.2 fig PMT
0.4 fig PMT
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4-2. Recruitment of CARM1 and PRMT1 by BRCA1 in PR signaling. CV-
1 cells were transfected with 2.5 ng of PR expression vector, 100 ng of pcDNA3.1-
BRCA1 plasmid, and either 200 or 400 ng of pSG5.HA-CARMl or pSG5.HA-PRMTl.
125 ng of MMTV-LUC reporter plasmid was also transfected per well of the 12-well
cultures. pSG5.HA empty vector was used as filler DNA.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-2
CARM1
p
■
+ + + +
PR + + + + +
BRCA1
- -
+ + +
0.2 |ag PMT - - -
+
-
0.4 |ag PMT
- - - - +
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4
Figure 4-3. Recruitment of CARM1 by BRCA1 in ER signaling. CV-1 cells were
transfected with 2.5 ng of pSG5-ERa expression vector, 300 ng of pcDNA3.1-BRCAl
plasmid, and either 200 or 400 ng of pSG5.HA-CARMl or pSG5.HA-PRMTl. 125 ng
of MMTV-LUC reporter plasmid was also transfected per well of the 12-well cultures.
pSG5.HA empty vector was used as filler DNA.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-3
*
>
1
■ o
o
10nM E2
ER
BRCA1
0.2 ng CARM1
0.4 ng CARM1
+
+
+
+
+
+
+
+
+
+
+
+
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3.5X
Figure 4-4. BRCA1 and the protein methyltransferases interact in vitro. A,
Schematic diagrams of CARM1, PRMT1 and BRCA1. B, Glutathione-S-transferase
(GST) and GST-fusion proteins were expressed and purified as described (Chen et al.,
1999). Glutathione-Sepharose-bound GST protein, GST-PRMT1, or GST-CARM1
were incubated with 3 5 S-radiolabeled BRCA1 transcribed and translated in vitro from
pcDNA3.1 vector encoding full-length BRCA1. Associated BRCA1 was eluted,
resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed by
autoradiography. 10% of total labeled BRCA1 incubated in each reaction was loaded
for comparison. Autoradiographs showing the localization of CARM1 and PRMT1
interactions on BRCA1. Plasmids containing fragments of BRCA1 were generated as
described in Chapter 2. Diagrams of the functional domains corresponding to amino
acids are provided for reference. Unpurified in vitro translated BRCA1 fragments were
incubated with GST, GST-CARM1 or GST-PRMT1. Both CARM1 and PRMT1
binding are shown to localize to the N-terminus of BRCA1.
180
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-4
A.
SAM
CARM1
168 285
(55%) (61%)
SAM
608
(%) homology
PRMT1
RING NLS BRCT
BRCA1
500 1863
181
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4-5. Interaction of BRCA1 and HA-PRMT1 in mammalian cells. T-75
flasks were seeded with lxlO6 Cos-7 monkey kidney cells and transfected with 1.5 (ig
of pSG5.HA-PRMTl and 2.5 jig of pcDNA3.1-BRCAl plasmids. Cells were grown
for 48 hours and lysates were harvested using RIPA buffer (plus complete protease
inhibitors cocktail, Roche Pharmaceuticals). Lysates were either immunoprecipitated
with 1 mg/mL of rabbit anti-BRCAl polyclonal antibodies (C20, Santa Cruz), or non
specific rabbit polyclonal antibodies (Zymed). Proteins were eluted and resolved by
SDS-PAGE and transferred onto PVDF membranes. Coimmunoprecipitated HA-
PRMT1 was detected using a rat anti-HA antibody.
182
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-5
1 2 3 4
, HA-PRMT1/BRCA1
1 Crude Lysate (5^L)
2 HA-PRMT1/BRCA1
3 Untransfected
4 HA-PRMT1/BRCA1
IP (anti-): Lyaata C-20 IgG C-20
WB: ratantt-HA
183
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4-6. GRIP1 recruits CARM1 and PRMT1 during steroid receptor
signaling. CV-1 cells were transfected as follows: AR signaling - I ng of pCMV-hAR,
100 ng of MMTV-LUC reporter plasmid and 75 ng pSG5.HA-GRIPl with either 125
ng pSG5.HA-CARMl or 125 ng pSG5.HA-PRMTl, as indicted; ER signaling - 1 ng of
pSG5-ERa, 100 ng MMTV(ERE)-LUC, and 75 ng pSG5.HA-GRIPl with either 125 ng
pSG5.HA-CARMl or 125 ng pSG5.HA-PRMTl, as indicted; and PR signaling - 1 ng
of PR expression vector, 125 ng of MMTV-LUC, and 100 ng pSG5.HA-GRIPl with
either 250 ng pSG5.HA-CARMl or 250 ng pSG5.HA-PRMTl, as indicted.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-6
>
I
■ o
o
25
20
15
10
5 - I
0
0AR (1 ng)
■ ER (1ng)
□ PR (1ng)
NR
GRIP1
CARM1
PRMT1
+
+
+
+
+
+
+
+
+
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4-7. Synergistic enhancement of NR function by CARM1, PRMT1, and
GRIP1. CV-1 cells were transfected as follows: AR signaling - 1 ng of pCMV-hAR,
100 ng of MMTV-LUC reporter plasmid and 75 ng pSG5.HA-GR!Pl with 125 ng
pSG5.HA-CARMl and/or 125 ng pSG5.HA-PRMTl, as indicted; ER signaling - 1 ng
of pSG5-ERa, 100 ng MMTV(ERE)-LUC, and 75 ng pSG5.HA-GRIPl with 125 ng
pSG5.HA-CARMl and/or 125 ng pSG5.HA-PRMTl, as indicted; and PR signaling - 1
ng of PR expression vector, 125 ng of MMTV-LUC, and 100 ng pSG5.HA-GRIPl with
250 ng pSG5.HA-CARMl and/or 250 ng pSG5.HA-PRMTl, as indicted.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-7
NR
GRIP1
CARM1
PRMT1
+
+
+
+
+
+
+
+
+
+
+
+
+
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4*8. Lack of synergistic enhancement of NR function by CARM1, PRMT1,
and BRCA1. CV-1 cells were transfected as follows: AR signaling - 1 ng of pCMV-
hAR, 100 ng of MMTV-LUC reporter plasmid and 125 ng pcDNA3.l-BRCAl with 125
ng pSG5.HA-CARMl and/or 125 ng pSG5.HA-PRMTl, as indicted; ER signaling - I
ng of pSG5-ERa, 100 ng MMTV(ERE)-LUC, and 125 ng pcDNA3.1-BRCAl with 125
ng pSG5.HA-CARMl and/or 125 ng pSG5.HA-PRMTl, as indicted; and PR signaling -
I ng of PR expression vector, 125 ng of MMTV-LUC, and 250 ng pcDNA3.1-BRCAl
with 250 ng pSG5.HA-CARMl and/or 250 ng pSG5.HA-PRMTl, as indicted. Some
squelching is observed when all the plasmids are cotransfected.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-8
NR
BRCA1
CARM1
PRMT1
+
+
+
+
+
+
+
+
+
+
+
+
+
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4-9. Synergistic enhancment of AR signaling by BRCA1, GRIP1 and
CARM1 or PRM T1. Transfections and luciferase reporter gene assays were
conducted as described in Figures 4-7 and 4-8, with the following exceptions.
pSG5.HA-CARMl and pSG5.HA-PRMTl were not cotransfected. pcDNA3.1-
BRCA1, pSG5.HA-GRIPl and/or pSG5.HA-CARMl or pcDNA3.l-BRCAl,
pSG5.HA-GRIPl and/or pSG5.HA-PRMTl were cotransfected into CV-l cells, along
with pCMV-hAR, and assayed for luciferase activity. Coactivator synergy was
observed whenever BRCA1 and GRIP1 were cotransfected together with either protein
methy 1 transferases.
190
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-9
200
150
100
50
AR
GRIP1
BRCA1
CARM1
PRMT1
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
25.2X
Figure 4-10. CARM1 dose and it role in coactivator synergy. CV-1 cells were
transfected with increasing amounts of pSG5.HA-CARMl and, where indicated, 1 ng of
pCMV-hAR, 125 ng pcDNA3.1-BRCAl and/or 75 ng pSG5.HA-GRIPl. Transiently
transfected cells were cultured and maintained in 10 nM DHT.
192
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-10
>
Z
<
s
< u
400
— — AR+CARM1
- - ♦ - -AR+BRCA1+CARM1
— -A — AR+GRIP1+CARM1
350
-AR*BRCA1 +GRIP1+CARM1
300 -
250
200
150
100
CARM1
(0.05(18) (0.25na>
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4-11. Synergistic enhancement of ER and PR signaling by BRCA1, GRIP1
and the protein methyltranferases, CARM1 and PRMT1. Transfections and
luciferase reporter gene assays were conducted as described in Figure 4-9. pcDNA3.1-
BRCA1, pSG5.HA-GRIPl and/or pSG5.HA-CARMl or pcDNA3.1-BRCAl,
pSG5.HA-GRIPl and/or pSG5.HA-PRMTl were cotransfected into CV-1 cells, along
with either 1 ng of pSG5-ERa or 0.25 ng of PR expression plasmid, and assayed for
luciferase activity.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-11
*
>
<
■ o
o
45
□ER (1ng)
■PR (1ng) 40
35
30
25
20
15
10
5
0
NR + + + + + + + + +
GRIP1
BRCA1
CARM1
PRMT1
+
+
+
+
+
+
+
+
+
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 4-12. Synergy among BRCA1, GRIP1 and CARM1 requires the AD2
interaction domain of GRIP1. CV-1 cells were transfected with 0.5 ng of pCMV-
hAR, 100 ng of MMTV-LUC, and 75 ng of wild-type or mutant pSG5.HA-GRIPl
expression vectors, as indicated. 125 ng of pSG5.HA-CARMl was also cotransfected
whenever indicated.
196
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FIGURE 4-12
X9'9i \
xcia
XS61
+ + •
+ + +
• + +
• + •
■ ■ I
+ + •
+ + +
• + +
' + ■
■ I I
+ + ■
+ + +
• + +
• + •
I ■ i
x ri I +
• ■
+
X l l 1 + + •
■
X9 0 I
■ ■ i •
s s s §
s
c
o
E C oc <
tQ (9 U
197
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Controls G R IP1 G R IP1A A D 1 GRIP1AAD2
REFERENCES
Aletta, J. M., Cimato, T. R., and Ettinger, M. J. (L998). Protein methylation: a signal
event in post-translational modification. Trends Biochem Sci 23, 89-91.
Bochar, D. A., Wang, L., Beniya, H., Kinev, A., Xue, Y., Lane, W. S., Wang, W.,
Kashanchi, F., and Shiekhattar, R. (2000). BRCA1 is associated with a human
SWI/SNF-related complex: linking chromatin remodeling to breast cancer. Cell 102,
257-265.
Brinkmann, A. O., Faber, P. W., van Rooij, H. C., Kuiper, G. G., Ris, C., Klaassen, P.,
van der Korput, J. A., Voorhorst, M. M., van Laar, J. H., Mulder, E., and et al. (1989).
The human androgen receptor: domain structure, genomic organization and regulation
of expression. J Steroid Biochem 34, 307-10.
Chen, D., Ma, H., Hong, H., Koh, S. S., Huang, S.-M., Schurter, B. T., Aswad, D. W.,
and Stallcup, M. R. (1999). Regulation of transcription by a protein methyltransferase.
Science 284, 2174-2177.
Fan, S., Wang, J., Yuan, R., Ma, Y., Meng, Q., Erdos, M. R., Pestell, R. G., Yuan, F.,
Aubom, K. J., Goldberg, I. D., and Rosen, E. M. (1999). BRCA1 inhibition of estrogen
receptor signaling in transfected cells. Science 284,1354-6.
198
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Green, S., Issemann, I., and Sheer, E. (1988). A versatile in vivo and in vitro eukaryotic
expression vector for protein engineering. Nucl Acids Res 16.
Hansen, S. K., Takada, S., Jacobson, R. H., Lis, J. T., and Tjian, R. (1997).
Transcription properties of a cell type-specific TATA-binding protein, TRF [see
comments]. Cell 91, 71-83.
Hong, J., Kohli, K., Trivedi, A., Johnson, D. L., and Stallcup, M. R. (1996). GRIPl, a
novel mouse protein that serves as a transcriptional co-activator in yeast for the
hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93,4948-4952.
Hu, Y. F., Hao, Z. L., and Li, R. (1999). Chromatin remodeling and activation of
chromosomal DNA replication by an acidic transcriptional activation domain from
BRCA1. Genes Dev 13,637-42.
Irvine, R. A., Ma, H., Yu, M. C., Ross, R. K., Stallcup, M. R., and Coetzee, G. A.
(2000). Inhibition of pl60-mediated coactivation with increasing androgen receptor
polyglutamine length. Hum Mol Genet 9, 267-274.
Lin, W. J., Gary, J. D., Yang, M. C., Clarke, S., and Herschman, H. R. (1996). J Biol
Chem 271, 15034-15044.
199
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Ma, H., Hong, H., Huang, S. M., Irvine, R. A., Webb, P., Kushner, P. J., Coetzee, G. A.,
and Stallcup, M. R. (1999). Multiple signal input and output domains of the 160-
kilodalton nuclear receptor coactivator proteins. Mol Cell Biol 19,6164-6173.
McKenna, N. J., Xu, J., Nawaz, Z., Tsai, S. Y., Tsai, M.-J., and O'Malley, B. W. (1999).
Nuclear receptor coactivators: multiple enzymes, multiple complexes, multiple
functions. J Ster Biochem Mol Biol 69, 3-12.
Naar, A. M., Beaurang, P. A., Zhou, S., Abraham, S., Solomon, W., and Tjian, R.
(1999). Composite co-activator ARC mediates chromatin-directed transcriptional
activation. Nature 398, 828-32.
Ouchi, T., Monteiro, A. N., August, A., Aaronson, S. A., and Hanafusa, H. (1998).
BRCA1 regulates p53-dependent gene expression. Proc Natl Acad Sci U S A 95, 2302-
6.
Pao, G. M., Janknecht, R., Ruffner, H., Hunter, T., and Verma, I. M. (2000). CBP/p300
interact with and function as transcriptional coactivators of BRCA1. Proc Natl Acad Sci
U S A 97, 1020-5.
200
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Puigserver, P., Wu, Z., Park, C. W., Graves, R., Wright, M., and Spiegelman, B. M.
(1998). A cold-inducible coactivator of nuclear receptors linked to adaptive
thermogenesis. Cell 92, 829-39.
Rachez, C., Lemon, B. D., Suldan, Z., Bromleigh, V., Gamble, M., Naar, A. M.,
Erdjument-Bromage, H., Tempst, P., and Freedman, L. P. (1999). Ligand-dependent
transcription activation by nuclear receptors requires the DRIP complex. Nature 398,
824-8.
Rebbeck, T. R., Kantoff, P. W., Krithivas, K., Neuhausen, S., Blackwood, M. A.,
Godwin, A. K., Daly, M. B., Narod, S. A., Garber, J. E., Lynch, H. T., Weber, B. L.,
and Brown, M. (1999). Modification of BRCA1-associated breast cancer risk by the
polymorphic androgen-receptor CAG repeat. Am J Hum Genet 64, 1371-7.
Somasundaram, K., Zhang, H., Zeng, Y. X., Houvras, Y., Peng, Y., Wu, G. S., Licht, J.
D., Weber, B. L., and El-Deiry, W. S. (1997). Arrest of the cell cycle by the tumour-
suppressor BRCA1 requires the CDK-inhibitor p21WAFl/CiPl. Nature 389, 187-90.
Sundqvist, A., Sollerbrant, K., and Svensson, C. (1998). The carboxy-terminal region of
adenovirus E l A activates transcription through targeting of a C-terminal binding
protein-histone deacetylase complex. FEBS Lett 429, 183-8.
201
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Umesono, K., and Evans, R. M. (1989). Determinants of Target Gene Specificity For
Steroid/thyroid Hormone Receptors. Cell 57, 1139-1146.
Wang, Q., Zhang, H., Kajino, K., and Greene, M. I. (1998). BRCA1 binds c-Myc and
inhibits its transcriptional and transforming activity in cells. Oncogene 17, 1939-48.
Xu, L., Glass, C. K., and Rosenfeld, M. G. (1999). Coactivator and corepressor
complexes in nuclear receptor function. Curr Op Gen Dev 9.
Yarden, R. I., and Brody, L. C. (1999). BRCA1 interacts with components of the
histone deacetylase complex. Proc Natl Acad Sci U S A 96,4983-8.
Zhang, H., Somasundaram, K., Peng, Y., Tian, H., Bi, D., Weber, B. L., and El-Deiry,
W. S. (1998). BRCA1 physically associates with p53 and stimulates its transcriptional
activity. Oncogene 16, 1713-21.
Zhong, Q., Chen, C. F., Li, S., Chen, Y., Wang, C. C., Xiao, J., Chen, P. L., Sharp, Z.
D., and Lee, W. H. (1999). Association of BRCA1 with the hRad50-hMrell-p95
complex and the DNA damage response. Science 285, 747-50.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
BIBLIOGRAPHY
Abbott, D. W., Thompson, M. E., Robinson-Benion, C., Tomlinson, G., Jensen, R. A.,
and Holt, J. T. (1999). BRCA1 expression restores radiation resistance in BRCA1-
defective cancer cells through enhancement of transcription-coupled DNA repair. J Biol
Chem 274, 18808-12.
Althma, P., Rappaport, R., and Swift, M. (1996). Molecular genotyping shows that
ataxia-telangiectasia heterozygotes are predisposed to breast cancer. Cancer Genet
Cytogenet 92, 130-134.
Altiok, S., Batt, D., Altiok, N., Papautsky, A., Downward, J., Roberts, T. M., and
Avraham, H. (1999). Heregulin induces phosphorylation of BRCA1 through
phosphatidylinositol 3-Kinase/AKT in breast cancer cells. J Biol Chem 274, 32274-8.
Anderson, S. F., Schlegel, B. P., Nakajima, T., Wolpin, E. S., and Parvin, J. D. (1998).
BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA
helicase A. Nature genet 19, 254-256.
Aprelikova, O. N., Fang, B. S., Meissner, E. G., Cotter, S., Campbell, M., Kuthiala, A.,
Bessho, M., Jensen, R. A., and Liu, E. T. (1999). BRCA1 -associated growth arrest is
RB-dependent. Proc Natl Acad Sci U S A 9 6 ,11866-71.
203
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Barlow, A. L., Benson, F. E., West, S. C., and Hulten, M. A. (1997). Distribution of the
Rad51 recombinase in human and mouse spermatocytes. Embo J 16, 5207-15.
Baumann, P., and West, S. C. (1998). Role of the human RAD51 protein in homologous
recombination and double-stranded-break repair. Trends Biochem Sci 23, 247-51.
Berman, D. B., Costalas, J., Schultz, D. C., Grana, G., Daly, M., and Godwin, A. K.
(1996). A common mutation in BRCA2 that predisposes to a variety of cancers is found
in both Jewish Ashkenazi and non-Jewish individuals. Cancer Res 56, 3409-3414.
Borden, K. L. B. (2000). RING domains: master builders of molecular scaffolds? J Mol
Biol 295, 1103-1112.
Broca, P. Traite des Tumeurs 1 ,1886-1889.
Callebaut, I., and Momon, J. P. (1997). From BRCA1 to RAP1: a widespread BRCT
module closely associated with DNA repair. FEBS Lett 400, 25-30.
Chapman, M. S., and Verma, I. M. (1996). Transcriptional activation by BRCA1.
Nature 382, 678-679.
204
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Chen, C.-F., Li, S., Chen, Y., Chen, P.-L., Sharp, Z. D., and Lee, W.-H. (1996). The
nuclear localization sequences of the BRCA1 protein interact with the importin-a
subunit of the nuclear transport signal receptor. J Biol Chem 271, 32863-32868.
Chen, J.-J., Silver, D., Cantor, S., Livingston, D. M., and Scully, R. (1999). BRCA1,
BRCA2, and Rad51 operate in a common DNA damage response pathway. Cancer Res
59, 1752s-1756s.
Chen, Y., Farmer, A. A., Chen, C. F., Jones, D. C., Chen, P. L., and Lee, W. H. (1996).
BRCA1 is a 220-kDa nuclear phosphoprotein that is expressed and phosphorylated in a
cell cycle-dependent manner [published erratum appears in Cancer Res 1996 Sep
1;56(17):4074], Cancer Res 56, 3168-72.
Claus, E. B., Schildkraut, J. M., Thompson, W. D., and Risch, N. J. (1996). The genetic
attributable risk of breast and ovarian cancer. Cancer 77, 2318-2324.
Cortez, D., Wang, Y., Qin, J., and Elledge, S. J. (1999). Requirement of ATM-
dependent phosphorylation of brcal in the DNA damage response to double-strand
breaks [see comments]. Science 286, 1162-6.
Duncan, J. A., Reeves, J. R., and Cooke, T. G. (1998). BRCA1 and BRCA2 proteins:
roles in health and disease. Mol Pathol 51, 237-247.
205
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
FitzGerald, M. G., Bean, J. M., and Hegde, S. R. e. a. (1997). Heterozygous ATM
mutations do not contribute to early onset of breast cancer. Nat Genet 15, 307-310.
Foray, N., Randrianarison, V., Marot, D., Perricaudet, M., Lenoir, G., and Feunteun, J.
(1999). Gamma-rays-induced death of human cells carrying mutations of BRCA1 or
BRCA2. Oncogene 18, 7334-7342.
Ford, D., Easton, D. F., Bishop, D. T., Narod, S. A., and Goldgar, D. E. (1994). Risks of
cancer in BRCA 1 -mutation carriers. Lancet 343, 692-695.
Ford, D., Easton, D. F., Stratton, M., Narod, S., Goldgar, D., and Devilee, P. e. a.
(1998). Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2
genes in BC families. Am J Hum Genet 62, 676-689.
Fomace, A. J. J., Alamo, I. J., and Hollander, M. C. (1988). DNA damage-inducible
transcripts in mammalian cells. Proc Natl Acad Sci USA 85, 8800-8804.
Gowen, L. C., Avrutskaya, A. V., Latour, A. M., Koller, B. H., and Leadon, S. A.
(1998). BRCA1 required for transcription-coupled repair of oxidative DNA damage.
Science 281, 1009-1012.
206
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Gudas, J. M., Li, T., Nguyen, H., Jensen, D., Rauscher, F. J., 3rd, and Cowan, K. H.
(1996). Cell cycle regulation of BRCA1 messenger RNA in human breast epithelial
cells. Cell Growth Differ 7, 717-23.
Haaf, T., Golub, E. I., Reddy, G., Radding, C. M., and Ward, D. C. (1995). Nuclear foci
of mammalian Rad51 recombination protein in somatic cells after DNA damage and its
localization in synaptonemal complexes. Proc Natl Acad Sci U S A 92, 2298-302.
Haber, J. E. (1998). The many interfaces of Mrel 1. Cell 95,583-586.
Hsu, L.-C., and White, R. L. (1998). BRCA1 is associated with the centrosome during
mitosis. Proc Natl Acad Sci 95, 12983-12988.
Irminger-Finger, I., Siegel, B. D., and Leung, W. C. (1999). The functions of breast
cancer susceptibility gene 1 (BRCA1) product and its associated proteins. Biol Chem
380, 117-128.
Ivanov, E. L., and Haber, J. E. (1997). DNA repair: RAD alert. Curr Biol 7, R492-5.
Landis S, Murray T, Bolden S, and Wingo P (1998). Cancer Statistics, 1998. CA Cancer
J Clin 48,6-30.
207
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Lee, J. S., Collins, K. M., Brown, A. L., Lee, C. H., and Chung, J. H. (2000). hCdsl-
mediated phosphorylation of BRCA1 regulates the DNA damage response. Nature 404,
201-4.
Liaw, D„ Marsh, D. J., and Li, J. e. a. (1997). Germline mutations of the PTEN gene in
Cowden disease, and inherited breast and thyroid cancer syndrome. Nat Genet 16, 64-
67.
Maldonado, E., Shiekhattar, R., Sheldon, M., Cho, H., Drapkin, R., Rickert, P., Lees, E.,
Anderson, C. W., Linn, S., and Reinberg, D. (1996). A human RNA polymerase II
complex associated with SRB and DNA-repair proteins [published erratum appears in
Nature 1996 Nov 28;384(6607):384]. Nature 381, 86-9.
Malkin, D., Li, F. P., Strong, L. C., Fraumeni, J. F. J., Nelson, C. E., and Kim, D. H. e.
a. (1990). A cancer family syndrome in twenty-four kindreds. Science 250,1233-1238.
Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P. A., and Harshman, K. e. a.
(1994). A strong candidate for the breast and ovarian cancer susceptibility gene
BRCA1. Science 266,66-71.
208
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Monteiro, A. N. A., August, A., and Hanafusa, H. (1996). Evidence for a transcriptional
activation function of BRCA1 C-terminal region. Proc Natl Acad Sci USA 93, 13595-
13599.
Nelen, M. R., Padberg, G. W., and Peeters, E. A. e. a. (1996). Localization of the gene
for Cowden disease to chromosome 10q22-23. Nat Genet 13, 114-116.
Neuhausen, S. L., Gilewski, T., Norton, L., Tran, T., and McGuire, P. e. a. (1996).
Recurrent BRCA2 6174delT mutations in Ashkenazi Jewish women affected by breast
cancer. Nat Genet 13, 126-128.
Ouchi, T., Monteiro, A. N., August, A., Aaronson, S. A., and Hanafusa, H. (1998).
BRCA1 regulates p53-dependent gene expression. Proc Natl Acad Sci U S A 95, 2302-
6.
Pao, G. M., Janknecht, R., Ruffner, H., Hunter, T., and Verma, I. M. (2000). CBP/p300
interact with and function as transcriptional coactivators of BRCA1. Proc Natl Acad Sci
U S A 97, 1020-5.
Papathanasiou, M. A., Kerr, N. C., Robbins, J. H., McBride, O. W., Alamo, I., Jr.,
Barrett, S. F., Hickson, I. D., and Fomace, A. J., Jr. (1991). Induction by ionizing
209
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
radiation of the gadd45 gene in cultured human cells: lack of mediation by protein
kinase C. Mol CeU Biol 11, 1009-16.
Plug, A. W„ Xu, J., Reddy, G., Golub, E. I., and Ashley, T. (1996). Presynaptic
association of Rad51 protein with selected sites in meiotic chromatin. Proc Natl Acad
Sci U S A 93, 5920-4.
Rahman, N., and M.R., S. (1998). The genetics of breast cancer susceptibility. Annu
Rev Genet 32,95-121.
Rao, B. B„ Boyd, A. A., Volcik, K., and Richards, C. S. (1996). Ashkenazi Jewish
population frequencies for common mutations in BRCA1 and BRCA2. Nat Genet 14.
Rebbeck, T. R. (1999). Inherited genetic predisposition in breast cancer: A population-
based perspective. Cancer Supplement 36, 1673-1681.
Ruffner, H., Jiang, W., Craig, A. G., Hunter, T., and Verma, I. M. (1999). BRCA1 is
phosphorylated at serine 1497 in vivo at a cyclin-dependent kinase 2 phosphorylation
site. Mol Cell Biol 19,4843-54.
210
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Scully, R., Anderson, S. F., Chao, D. M., Wei, W., Ye, L., Young, R. A., Livingston, D.
M., and Parvin, J. D. (1997). BRCA1 is a component of the RNA polymerase II
holoenzyme. Proc Natl Acad Sci U S A 94, 5605-10.
Scully, R., Chen, J., Ochs, R. L., Keegan, K., Hoekstra, M., Feunteun, J., and
Livingstion, D. L. (1997). Dynamic changes of BRCA1 subnuclear location and
phosphorylation state are initiated by DNA damage. Cell 90.
Scully, R., Chen, J., Plug, A., Xiao, Y., and Weaver, D. e. a. (1997). Association of
BRCA1 with Rad51 in Mitotic and Meiotic Cells. Cell 88, 265-275.
Somasundaram, K., Zhang, H., Zeng, Y. X., Houvras, Y., Peng, Y., Wu, G. S., Licht, J.
D., Weber, B. L., and El-Deiry, W. S. (1997). Arrest of the cell cycle by the tumour-
suppressor BRCA1 requires the CDK-inhibitor p21WAFl/CiPl. Nature 389, 187-90.
Struewing, J. P., Hartge, P., Wacholder, S., Baker, S. M„ Berlin, M., McAdams, M.,
Timmerman, M. M., Brody, L. C., and Tucker, M. A. (1997). The risk of cancer
associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews. N
Engl J Med 2 0 ,1401-1408.
Swift, M., Reitnauer, P. J., Morrell, D., and Chase, C. L. (1987). Breast and other
cancers in families with ataxia-telangiectasia. N Engl J Med 316,1289-1294.
211
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Szabo, C., and King, M.-C. (1995). Inherited breast and ovarian cancer. Human
Molecular Genetics 4 , 1811-1817.
Takekawa, M., and Saito, H. (1998). A family of stress-inducible GADD45-like
proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell
95, 521-30.
Tavtigian, S. V., Simard, J., Rommens, J., Couch, F., and Shattuck-Eidens, D. e. a.
(1996). The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds.
Nat Genet 12, 333-337.
Thorlacius, S., Struewing, J. P., Hartge, P., Olafsdottir, G. H., Sigvaldason, H., and
Tryggvadottir, L. e. a. (1998). Population-based study of risk of breast cancer in carriers
of BRCA2 mutation. Lancet 352, 1337-1339.
Vaughn, J. P., Davis, P. L., Jarboe, M. D., Huper, G., Evans, A. C., Wiseman, R. W.,
Berchuck, A., Iglehart, J. D., Futreal, P. A., and Marks, J. R. (1996). BRCA1 expression
is induced before DNA synthesis in both normal and tumor-derived breast cells. Cell
Growth Differ 7,711-5.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Wang, Q., Zhang, H., Kajino, K., and Greene, M. I. (1998). BRCA1 binds c-Myc and
inhibits its transcriptional and transforming activity in cells. Oncogene 1 7 ,1939-48.
Wilson, C. A., Payton, M. N., Elliott, G. S., Buaas, F. W., Cajulis, E. E., Grosshans, D.,
Ramos, L., Reese, D. M., Slamon, D. J., and Calzone, F. J. (1997). Differential
subcellular localization, expression and biological toxicity of BRCAl and the splice
variant BRCAl-deltal lb. Oncogene 14, 1-16.
Wooster, R., Bignell, G., Lancaster, J., Swift, S., and Seal, S. e. a. (1995). Identification
of the breast cancer susceptibility gene BRCA2. Nature 378.
Wooster, R., Neuhasen, S. L., Mangion, J., Quirk, Y., and Ford, D. e. a. (1994).
Localisation of a breast cancer susceptibility gene, BRCA2, to chromosome 13ql2-13.
Science 265, 2088-2090.
Wu, L. C., Wang, Z. W., Tsan, J. T., Spillman, M. A., Phung, A., Xu, X. L., Yang, M.-
C. W., Hwang, L.-Y., Bowcock, A. M., and Baer, R. (1996). Identification of a RING
protein that can interact in vivo with the BRCAl gene product. Nat Genet 14,430-440.
Xu, X., Weaver, Z., Linke, S. P., Li, C., Gotay, J., Wang, X. W„ Harris, C. C., Ried, T.,
and Deng, C. X. (1999b). Centrosome amplification and a defective G2-M cell cycle
213
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. Mol
Cell 3, 389-395.
Yarden, R. I., and Brody, L. C. (1999). BRCA1 interacts with components of the
histone deacetylase complex. Proc Natl Acad Sci U S A 96, 4983-8.
Yu, X., Wu, L. C., Bowcock, A. M., Aronheim, A., and Baer, R. (1998). The C-terminal
(BRCT) domains of BRCA1 interact in vivo with CtIP, a protein implicated in the CtBP
pathway of transcriptional repression. J Biol Chem 273, 25388-92.
Zhang, H., Somasundaram, K., Peng, Y., Tian, H., Bi, D., Weber, B. L., and El-Deiry,
W. S. (1998). BRCA1 physically associates with p53 and stimulates its transcriptional
activity. Oncogene 16, 1713-21.
Zhong, Q., Chen, C. F., Li, S., Chen, Y., Wang, C. C., Xiao, J., Chen, P. L., Sharp, Z.
D., and Lee, W. H. (1999). Association of BRCA1 with the hRad50-hMrel l-p95
complex and the DNA damage response. Science 285, 747-50.
Anderson, S. F., Schlegel, B. P., Nakajima, T., Wolpin, E. S., and Parvin, J. D. (1998).
BRCA1 protein is linked to the RNA polymerase H holoenzyme complex via RNA
helicase A. Nature genet 19, 254-256.
214
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Anzick, S. L., Kononen, J., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X.-Y.,
Sauter, G., Kallioniemi, O.-P., Trent, J. M., and Meltzer, P. S. (1997). AIB1, a steroid
receptor coactivator amplified in breast and ovarian cancer. Science 277,965-967.
Chamberlain, N. L., Driver, E. D., and Miesfeld, R. L. (1994). The length and location
of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect
transactivation function. Nucleic Acids Res 22, 3181-6.
Chapman, M. S., and Verma, I. M. (1996). Transcriptional activation by BRCA1.
Nature 382,678-679.
Chen, D., Ma, H., Hong, H., Koh, S. S., Huang, S.-M., Schurter, B. T., Aswad, D. W.,
and Stallcup, M. R. (1999). Regulation of transcription by a protein methyltransferase.
Science 284, 2174-2177.
Chen, J.-J., Silver, D., Cantor, S., Livingston, D. M., and Scully, R. (1999). BRCA1,
BRCA2, and Rad51 operate in a common DNA damage response pathway. Cancer Res
5 9 ,1752s-1756s.
Chen, Y., Lee, W. H., and Chew, H. K. (1999). Emerging roles of BRCA1 in
transcriptional regulation and DNA repair [In Process Citation]. J Cell Physiol 181,
385-92.
215
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Duncan, J. A., Reeves, J. R„ and Cooke, T. G. (1998). BRCA1 and BRCA2 proteins:
roles in health and disease. Mol Pathol 51, 237-247.
Edwards, A., Hammond, H. A., Lin, J., Caskey, C. T., and Chakraborty, R. (1992).
Genetic variation at trimeric and tetrameric tandem repeat loci in four human population
groups. Genomics 12, 241-253.
Fan, S., Wang, J., Yuan, R., Ma, Y., Meng, Q., Erdos, M. R., Pestell, R. G., Yuan, F.,
Aubom, K. J., Goldberg, I. D., and Rosen, E. M. (1999). BRCA1 inhibition of estrogen
receptor signaling in transfected cells. Science 284, 1354-6.
Giguere, B., Hollenberg, S. M., Rosenfeld, M. G., and Evans, R. M. (1986). Functional
domains of the human glucocorticoid receptor. Cell 46, 645-652.
Giovannucci, E., Stampfer, M. J., Krithivas, K., Brown, M., Brufsky, A., Talcott, J.,
Hennekens, C. H., and Kantoff, P. W. (1997). The CAG repeat within the androgen
rceptor gene and its relationship to prostate cancer. Proc Natl Acad Sci USA 94, 3320-
3323.
Green, S., Issemann, I., and Sheer, E. (1988). A versatile in vivo and in vitro eukaryotic
expression vector for protein engineering. Nucl Acids Res 16.
216
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Hackenberg, R., Hawighorst, T., Filmer, A., Nia, A. H., and Schulz, K. D. (1993a).
Medroxyprogesterone acetate inhibits the proliferation of estrogen- and progesterone-
receptor negative MFM-223 human mammary cancer cells via the androgen receptor.
Breast Cancer Res Treat 25, 217-224.
Hardy, D. O., Scher, H. I., Bogenreider, T., Sabbatini, P., Zhang, Z. F., Nanus, D. M.,
and Catterall, J. F. (1996). Androgen receptor CAG repeat lengths in prostate cancer:
correlation with age of onset. J Clin Endocrinol Metab 81,4400-3.
Hong, J., Kohli, K., Trivedi, A., Johnson, D. L., and Stallcup, M. R. (1996). GRIP1, a
novel mouse protein that serves as a transcriptional co-activator in yeast for the
hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93,4948-4952.
Hubert, A., Peretz, T„ Manor, O., Kaduri, L., Wienberg, N., Lerer, I., Sagi, M., and
Abeliovich, D. (1999). The Jewish Ashkenazi founder mutations in the BRCA1/BRCA2
genes are not found at an increased frequency in Ashkenazi patients with prostate
cancer. Am J Hum Genet 65, 921-924.
Irminger-Finger, I., Siegel, B. D., and Leung, W. C. (1999). The functions of breast
cancer susceptibility gene 1 (BRCA1) product and its associated proteins. Biol Chem
380, 117-128.
217
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Irvine, R. A., Ma, H., Yu, M. C., Ross, R. K., Stallcup, M. R., and Coetzee, G. A.
(2000). Inhibition of pl60-mediated coactivation with increasing androgen receptor
polyglutamine length. Hum Mol Genet 9,267-274.
Irvine, R. A., Yu, M. C., Ross, R. K., and Coetzee, G. A. (1995). The CAG and GGC
microsatellites of androgen receptor gene are in linkage disequilibrium in men with
prostate cancer. Cancer Res 55, 1937-1940.
Johannsson, O., Loman, N., Moller, T., U., K., Borg, A., and Olsson, H. (1999).
Incidence of malignant tumours in relatives of BRCA1 and BRCA2 germline mutation
carriers. Eur J Cancer 35, 1248-1257.
Lobaccaro, J.-M., Lumbroso, S., Belon, C., Galtier-Dereure, F„ Bringer, J., Lesimple,
T., Heron, J.-F., Pujol, H., and Sultan, C. (1993). Male breast cancer and the androgen
receptor gene. Nature Genet 5, 109-110.
Ma, H., Hong, H., Huang, S. M., Irvine, R. A., Webb, P., Kushner, P. J., Coetzee, G. A.,
and Stallcup, M. R. (1999). Multiple signal input and output domains of the 160-
kilodalton nuclear receptor coactivator proteins. Mol Cell Biol 19, 6164-6173.
218
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K.,
Blumberg, B., Kastner, P., Mark, M., and Chambon, P. e. a. (1995). The nuclear
receptor superfamily: the second decade. Cell 583, 835-839.
McKenna, N. J., Xu, J., Nawaz, Z., Tsai, S. Y., Tsai, M.-J., and O'Malley, B. W. (1999).
Nuclear receptor coactivators: multiple enzymes, multiple complexes, multiple
functions. J Ster Biochem Mol Biol 69, 3-12.
Monteiro, A. N. A., August, A., and Hanafusa, H. (1996). Evidence for a transcriptional
activation function of BRCAl C-terminal region. Proc Natl Acad Sci USA 93, 13595-
13599.
Munoz de Toro, M., Maffini, M. V., Kass, L., and Luque, E. H. (1998). Proliferative
activity and steroid hormone receptor status in male breast carcinoma. J Steroid
Biochem Mol Biol 67, 333-339.
Ouchi, T., Monteiro, A. N., August, A., Aaronson, S. A., and Hanafusa, H. (1998).
BRCAl regulates p53-dependent gene expression. Proc Natl Acad Sci U S A 95, 2302-
6.
Rahman, N., and M.R., S. (1998). The genetics of breast cancer susceptibility. Annu
Rev Genet 32,95-121.
219
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Rebbeck, T. R., Kantoff, P. W„ Krithivas, K., Neuhausen, S., Blackwood, M. A.,
Godwin, A. K., Daly, M. B., Narod, S. A., Garber, J. E., Lynch, H. T., Weber, B. L.,
and Brown, M. (1999). Modification of BRCAl-associated breast cancer risk by the
polymorphic androgen-receptor CAG repeat. Am J Hum Genet 64, 1371-7.
Ross, R. K., Coetzee, G. A., Reichardt, J. K., Yu, M. C., Feigelson, H., Stanczyk, F. Z.,
Kolonel, L. N., and Henderson, B. E. (1998). Androgen metabolism and prostate
cancer: establishing a model of genetic susceptibility. Cancer Res 58,4497-4504.
Scully, R., Anderson, S. F., Chao, D. M., Wei, W., Ye, L., Young, R. A., Livingston, D.
M., and Parvin, J. D. (1997). BRCAl is a component of the RNA polymerase II
holoenzyme. Proc Natl Acad Sci U S A 94, 5605-10.
Snoek, R., Bruchovsky, N., Kasper, S., Matusik, R. J., Gleave, M., Sato, N., Mawji, N.
R., and Rennie, P. S. (1998). Differential transactivation by the androgen receptor
cancer cells. Prostate 36,256-263.
Somasundaram, K., Zhang, H., Zeng, Y. X., Houvras, Y., Peng, Y., Wu, G. S., Licht, J.
D., Weber, B. L., and El-Deiry, W. S. (1997). Arrest of the cell cycle by the tumour-
suppressor BRCAl requires the CDK-inhibitor p21WAFl/CiPl. Nature 389, 187-90.
220
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Spurdle, A. B., Dite, G. S., Chen, X., Mayne, C. J., Southey, M. C., Batten, L. E., Chy,
H., Trute, L., McCredie, M. R., Giles, G. G., Armes, J., Venter, D. J., Hopper, J. L., and
Chenevix-Trench, G. (1999). Androgen receptor exon 1 CAG repeat length and breast
cancer in women before age forty years. J Natl Cancer Inst 91, 961-6.
Struewing, J. P., Hartge, P., Wacholder, S., Baker, S. M., Berlin, M., McAdams, M.,
Timmerman, M. M., Brody, L. C., and Tucker, M. A. (1997). The risk of cancer
associated with specific mutations of BRCAl and BRCA2 among Ashkenazi Jews. N
Engl J Med 20, 1401-1408.
Szelie, J., Jimenez, J., Soto, A. M., Luizzi, M. F., and Sonnenshein, C. (1997).
Androgen-induced inhibition of proliferation in human breast cancer MCF7 cells
transfected with androgen receptor. Endocrinology 138, 1406-1412.
Tilley, W. D., Marcelli, M., J.D., W., and M.J., M. (1989). Characterization and
expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci USA
86, 327-331.
Wang, Q., Zhang, H., Kajino, K., and Greene, M. I. (1998). BRCAl binds c-Myc and
inhibits its transcriptional and transforming activity in cells. Oncogene 1 7 ,1939-48.
221
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Webb, P., Lopez, G. N., Uht, R. M., and Kushner, P. J. (1995). Tamoxifen activation of
the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like
effects of antiestrogens. Mol Endocrinol 9,443-456.
Wooster, R., Mangion, J., Eeles, R., Smith, S., Dowsett, M., Averill, D., Barrett-Lee, P.,
Easton, D. F., Ponder, B. A. J., and Stratton, M. R. (1992). A germline mutation in the
androgen receptor gene in two brothers with breast cancer and Reifenstein syndrome.
Nature Genet 2,132-134.
Xu, L., Glass, C. K., and Rosenfeld, M. G. (1999). Coactivator and corepressor
complexes in nuclear receptor function. Curr Op Gen Dev 9.
Zhang, H., Somasundaram, K., Peng, Y., Tian, H., Bi, D., Weber, B. L., and El-Deiry,
W. S. (1998). BRCAl physically associates with p53 and stimulates its transcriptional
activity. Oncogene 16, 1713-21.
Zhu, X., Daffada, A. A. I., Chan, C. M. W., and Dowsett, M. (1997). Identification of
an exon 3 deletion splice variant androgen receptor mRNA in human breast cancer. Int J
Cancer 72, 574-580.
222
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Anzick, S. L., Kononen, J., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X.-Y.,
Sauter, G., Kallioniemi, O.-P., Trent, J. M., and Meltzer, P. S. (1997). AIB1, a steroid
receptor coactivator amplified in breast and ovarian cancer. Science 277, 965-967.
Bergkvist L, Adami H-O, Persson I, Hoover R, and Schairer C (1989). The risk of
breast cancer after estrogen and estrogen-progestin replacement. N Eng J Med 321, 293-
297.
Brinkmann, A. O., Faber, P. W., van Rooij, H. C., Kuiper, G. G., Ris, C., Klaassen, P.,
van der Korput, J. A., Voorhorst, M. M., van Laar, J. H., Mulder, E., and et al. (1989).
The human androgen receptor: domain structure, genomic organization and regulation
of expression. J Steroid Biochem 34, 307-10.
Chen, D., Ma, H., Hong, H., Koh, S. S., Huang, S.-M., Schurter, B. T., Aswad, D. W.,
and Stallcup, M. R. (1999). Regulation of transcription by a protein methyltransferase.
Science 284,2174-2177.
Chen, Y., Lee, W. H., and Chew, H. K. (1999). Emerging roles of BRCAl in
transcriptional regulation and DNA repair [In Process Citation], J Cell Physiol 181,
385-92.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Cohen SM, and Ellwein LB (1990). Cell proliferation in carcinogenesis. Science 249,
1007-1011.
Colditz GA, Hankinson SE, Hunter DJ, Willett WC, Manson JE, Stampfer MJ,
Hennekens C, Rosner B, and Speizer FE (1995). The use of estrogens and progestins
and the risk of breast cancer in postmenopausal women. N Engl J Med 332, 1589-1593.
Collaborative Group on Hormonal Factors in Breast Cancer (1996). Breast cancer and
hormonal contraceptives. Lancet 347,1713-1727.
Collaborative Group on Hormonal Factors in Breast Cancer (1996). Breast cancer and
hormonal contraceptives: further results. Contraception 54, 1S-106S.
Cressman, V. L., Backlund, D. C., Avrutskaya, A. V., Leadon, S. A., Godfrey, V., and
Koller, B. H. (1999). Growth Retardation, DNA Repair Defects, and Lack of
Spermatogenesis in BRCAl-Deficient Mice. Mol Cell Biol 19, 7061-7075.
El-Ashry D, Chrysogelos S, Lippman M, and Kern F (1996). Estrogen induction of
TG F-a is mediated by an estrogen response element composed of two imperfect
palindromes. J Steroid Biochem Mol Biol 59, 261-269.
224
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Enmark E, Pelto-Huikko M, Grandien K, Lagercrantz S, Lagercrantz J, Fried G,
Nordenskjold M, and Gustafsson J (1997). Human estrogen receptor P gene structure,
chromosomal localization, and expression pattern. Clin Endocrinol Metab 82, 4258-
4265.
Evans RM (1988). The steroid and thyroid hormone receptor superfamily. Science 240,
889-895.
Fan, S., Wang, J., Yuan, R., Ma, Y., Meng, Q., Erdos, M. R., Pestell, R. G., Yuan, F.,
Aubom, K. J., Goldberg, I. D., and Rosen, E. M. (1999). BRCAl inhibition of estrogen
receptor signaling in transfected cells. Science 284, 1354-6.
Ferenczy A, Bertrand G, and Gelfand MM (1979). Proliferation kinetics of human
endometrium during the normal menstrual cycle. Am J Obstet Gynecol 133, 859-867.
Folsom AR, Kaye SA, Prineas RJ, Potter JD, Gapstur SM, and Wallance RB (1990).
Increased incidence of carcinoma of the breast associated with abdominal adiposity in
postmenopausal women. Am J Epidemiol 131,794-803.
Fuller PJ (1991). The steroid receptor superfamily: Mechanisms of diversity. FASEB J
5,3092-3099.
225
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Giangrande P, Pollio G, and McDonnell D (1997). Mapping and characterization of the
functional domains responsible for the differential activity of the A and B isoforms of
the human progesterone receptor. Jour Biol Chem 272, :32889-32900.
Giguere, B., Hollenberg, S. M., Rosenfeld, M. G., and Evans, R. M. (1986). Functional
domains of the human glucocorticoid receptor. Cell 46, 645-652.
Green S, and Chambon P (1987). Oestradiol induction of a glucocorticoid-responsive
gene by a chimaeric receptor. Nature 325, 75-78.
Green S, Walter P, Kumar V, Krust A, B. J.-M., Argos P, and Chambon P (1986).
Human oestrogen receptor cDNA: Sequence, expression and homology to v-erb-A.
Nature 320, 134-139.
Green, S., Issemann, I., and Sheer, E. (1988). A versatile in vivo and in vitro eukaryotic
expression vector for protein engineering. Nucl Acids Res 16.
Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, and J, S. (1986). Sequence and
expression of human estrogen receptor complementary DNA. Science 231,1150-1154.
Gudas, J. M., Nguyen, H., Li, T., and Cowan, K. H. (1995). Hormone-dependent
regulation of BRCAl in human breast cancer cells. Cancer Res 55,4561-5.
226
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Guiochon-Mantel A, Lescop P, Chgristin-Maitre S, Losfelt H, Perrot-Applanat M, and
Milgrom E (1991). Nucleocytoplasmic shuttling of the progesterone receptor. EMBO J
10, 3851-3859.
Hakem, R., de la Pompa, J. L., Sirard, C., Mo, R., Woo, M., Hakem, A., Wakeham, A.,
Potter, J., Reitmair, A., Billia, F., Firpo, E., Hui, C. C., Roberts, J., Rossant, J., and
Mak, T. W. (1996). The tumor suppressor gene Brcal is required for embryonic cellular
proliferation in the mouse. Cell 85, 1009-23.
Henderson BE, Ross RK, Pike MC, and Casagrande JT (1982). Endogenous hormones
as a major factor in human cancer. Cancer Res 42, 3232-3239.
Holt, J. T., Thompson, M. E., Szabo, C., Robinson-Benion, C., Arteaga, C. L., King, M.
C., and Jensen, R. A. (1996). Growth retardation and tumour inhibition by BRCA1 [see
comments] [published erratum appears in Nat Genet 1998 May; 19(1): 102]. Nat Genet
12, 298-302.
Hong, J., Kohli, K., Trivedi, A., Johnson, D. L., and Stallcup, M. R. (1996). GRIP1, a
novel mouse protein that serves as a transcriptional co-activator in yeast for the
hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93,4948-4952.
227
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Irvine, R. A., Ma, H., Yu, M. C., Ross, R. K., Stallcup, M. R., and Coetzee, G. A.
(2000). Inhibition of pl60-mediated coactivation with increasing androgen receptor
polyglutamine length. Hum Mol Genet 9, 267-274.
Kastner P, Krust A, Turcotte B, Stropp U, Tora L, Gronemeyer H, and Chambon P
(1990). Two distinct estrogen-regulated promoters generate transcripts encoding the two
functionally different human progesterome receptor forms A and B. The EMBO Journal
9, 1603-1614.
Kelsey JL, Gammon MD, and John EM (1993). Reproductive factors and breast cancer.
Epidemiol Rev 15, 36-47.
Key TJA, and Pike MC (1988). The dose-effect relationship between "unopposed"
oestrogens and endometrial mitotic rate: Its central role in explaining and predicting
endometrial cancer risk. Br J Cancer 57,205-212.
Key TJA, and Pike MC (1988). The role of oestrogens and progestagens in the
epidemiology and prevention of breast cancer. Eur J Cancer Clin Oncol 24, 29-34.
Klein-Hitpass, Ryffel G, Heitlinger E, and Cato A (1987). A 13 bp palindrome is a
functional estrogen response element and interacts specifically with estrogen receptor.
Nucleic Acids Res 16,647-663.
228
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Klock G, Strahle U, and Schutz G (1987). Oestrogen and glucocorticoid responsive
elements are closely related but distinct. Nature 329,734-736.
Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, and Gustafsson J (1996). Cloning of
a novel estrogen receptor expressed in rat prostate and ovary. Proc Natil Acad Sci USA
93, 5925-5930.
Kuiper GG, and Gustafsson J (1997). The novel estrogen receptor-P subtype: potential
role in the cell- and promoter-specific actions of estrogens and anti-estrogens. FEBS
Lett 410, 87-90.
Kumar V, Green S, Stack G, Berry M, Jin JR, and Chambon P (1987). Functional
domains of the human estrogen receptor. Cell 51,941-951.
Kumar V, Green S, Staub A, and Chambon P (1986). Localisation of the oestradiol-
binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J
5 ,2231-2236.
Landis S, Murray T, Bolden S, and Wingo P (1998). Cancer Statistics, 1998. CA Cancer
J Clin 48 ,6-30.
229
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Lane, T. F., Deng, C., Elson, A., Lyu, M. S., Kozak, C. A., and Leder, P. (1995).
Expression of Brcal is associated with terminal differentiation of ectodermally and
mesodermally derived tissues in mice [published erratum appears in Genes Dev 1996
Feb 1;10(3):365]. Genes Dev 9,2712-22.
Levenson A, and Jordan C (1994). Transfection of human estrogen receptor (ER) cDNA
into ER-negative mammalian cell lines. J Ster Biochem Mol Biol 54, 229-239.
Longcope C (1971). Metabolic clearance and blood production rates of estrogens in
postmenopausal women. Am J Obstet Gynecol 111, 778-781.
Ma, H., Hong, H., Huang, S. M., Irvine, R. A., Webb, P., Kushner, P. J., Coetzee, G. A.,
and Stallcup, M. R. (1999). Multiple signal input and output domains of the 160-
kilodalton nuclear receptor coactivator proteins. Mol Cell Biol 19, 6164-6173.
MacDonald PC, Edman CD, Hemsell DL, Porter JC, and Siiteri PK (1978). Effect of
obesity on conversion of plasma androstenedione to estrone in postmenopausal women
with and without endometrial cancer. Am J Obstet Gynecol 130,448-455.
MacMahon B, Cole P, and Brown J (1973). Etiology of human breast cancer: A review.
J Natl Cancer Inst 50, 21-42.
230
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Marks, J. R., Huper, G., Vaughn, J. P., Davis, P. L., Norris, J., McDonnell, D. P.,
Wiseman, R. W., Futreal, P. A., and Iglehart, J. D. (1997). BRCA1 expression is not
directly responsive to estrogen. Oncogene 14, 115-21.
Marquis, S. T., Rajan, J. V., Wynshaw-Boris, A., Xu, J., Yin, G. Y., Abel, K. J., Weber,
B. L., and Chodosh, L. A. (1995). The developmental pattern of Brcal expression
implies a role in differentiation of the breast and other tissues. Nat Genet 11, 17-26.
Misrahi M, Atger M, d’Auriol L, Loosfelt H, Meriel C, Fridlansky F, Guiochon-Mantel
A, Galibert F, and Milgrom E (1987). Complete amino acid sequence of the human
progesterone receptor deduced from cloned cDNA. Biochem Biophys Res Comm 143,
740-748.
Misrahi M, Venenci PY, Saugier-Veber P, Sar S, Dessen P, and Milgrom E (1993).
Structure of the human progesterone receptor gene. Biochem et Biophys Acta 1216,
289-292.
Moore DH, Moore DH, and Moore CT (1983). Breast carcinoma etiological factors.
Adv Cancer Res 4 0 ,189-253.
Mosselman S, Polman J, and Dijkema R (1996). ER-(J: identification and
characterization of a novel human estrogen receptor. FEBS Lett 392,49-53.
231
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Norris, J., Fan, D., Aleman, C., Marks, J. R., Futreal, P. A., Wiseman, R. W., Iglehart, J.
D., Deininger, P. L., and McDonnell, D. P. (1995). Identification of a new subclass of
Alu DNA repeats which can function as estrogen receptor-dependent transcriptional
enhancers. J Biol Chem 270, 22777-82.
Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, Ouchi Y, and Muramatsu
M (1998). The complete primary structure of human estrogen receptor P (hERP) and its
heterodimerization with ER a in Vivo and in Vitro. Biochem Biophy Res Comm 243,
122-126.
Picard D, Kumar V, Chambon P, and Yamamoto K (1990). Signal transduction by
steroid hormones: Nuclear localization is differentially regulated in estrogen and
glucocorticoid receptors. Cell Regul 1 ,291-299.
Pike MC (1987). Age-related factors in cancers of the breast, ovary, and endometrium. J
Chron.Dis 40 (Suppl II), 59S-69S.
Pike MC, Bernstein L, and Spicer DV. (1993). Exogenous hormones and breast cancer
risk. In Current Therapy in Oncology, Niederhuber JE, ed. (St. Louis: Mosby Year
Book, Inc.).
232
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
PonglikitmongkoL M, Green S, and Chambon P (1988). Genomic organization of the
human oestrogen receptor gene. EMBO J 7, 3385-3388.
Rehberger P, Rexin M, and Gehring U (1992). Heterotetrameric structure of the human
progesterone receptor. Proc Natil Acad Sci USA 89, 8001-8005.
Segnitz B, and Ghering U (1995). Subunit structure of the nonactivated human estrogen
receptor. Proc Natl Acad Sci USA 92, 2179-2183.
Siiteri PK (1978). Steroid hormones and endometrial cancer. Cancer Res 38,4360.
Snoek, R., Bruchovsky, N., Kasper, S., Matusik, R. J., Gleave, M., Sato, N., Mawji, N.
R., and Rennie, P. S. (1998). Differential transactivation by the androgen receptor
cancer cells. Prostate 36,256-263.
Spillman, M. A., and Bowcock, A. M. (1996). BRCA1 and BRCA2 mRNA levels are
coordinately elevated in human breast cancer cells in response to estrogen. Oncogene
1 3 ,1639-45.
Tilley, W. D., Marcelli, M., J.D., W., and M.J., M. (1989). Characterization and
expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci USA
86,327-331.
233
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Tora L, Mullick A, Metzger D, Ponglikitmongkol M, Park I, and Chambon P (1989).
The cloned human oestrogen receptor contains a mutation which alters its hormone
binding properties. EMBO J 8, 1981-1986.
Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, and
Giguere V (1997). Cloning, chromosomal localization, and functional analysis of the
murine estrogen receptor (5 . Mol Endocrinol 11, 353-365.
Trichopolous D, MacMahon B, and Cole P (1972). The menopause and breast cancer
risk. J Natl Cancer Inst 48, 605-613.
Tsai MJ, and O’Malley BW (1994). Molecular mechanisms of action of steroid/thyroid
receptor superfamily. Annu Rev Biochem 63,451-486.
Umesono, K., and Evans, R. M. (1989). Determinants of Target Gene Specificity For
Steroid/thyroid Hormone Receptors. Cell 57, 1139-1146.
Wahli W, and Martinez E (1991). Superfamily of steroid nuclear receptors: Positive
and negative regulators of gene expression. FASEB J 5, 2243-2249.
234
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Webb, P., Lopez, G. N„ Uht, R. M., and Kushner, P. J. (1995). Tamoxifen activation of
the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like
effects of antiestrogens. Mol Endocrinol 9,443-456.
Wei LL, Norris BM, and Baker CJ (1997). An N-terminally truncated third
progesterone receptor protein, PRC , forms heterodimers with PRg but interferes in
PRg-DNA binding. J Steroid Biochem Molec Biol 62, 287-297.
Wilson, C. A., Payton, M. N., Elliott, G. S., Buaas, F. W., Cajulis, E. E., Grosshans, D.,
Ramos, L., Reese, D. M., Slamon, D. J., and Calzone, F. J. (1997). Differential
subcellular localization, expression and biological toxicity of BRCA1 and the splice
variant BRCAl-deltal lb. Oncogene 14, 1-16.
Xu J, Nawaz Z, Tsai S, Tsai M-J, and O’Malley B (1996). The extreme C terminus of
progesterone contains a transcriptional repressor domain that functions through a
putative corepressor. Proc Natl Acad Sci USA 93, 12195-12199.
Xu, X., Wagner, K. U., Larson, D., Weaver, Z., Li, C., Ried, T., Hennighausen, L.,
Wynshaw-Boris, A., and Deng, C. X. (1999). Conditional mutation of Brcal in
mammary epithelial cells results in blunted ductal morphogenesis and tumour formation
[see comments]. Nat Genet 22,37-43.
235
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Aletta, J. M., Cimato, T. R., and Ettinger, M. J. (1998). Protein methylation: a signal
event in post-translational modification. Trends Biochem Sci 23, 89-91.
Bochar, D. A., Wang, L., Beniya, H., Kinev, A., Xue, Y., Lane, W. S., Wang, W.,
Kashanchi, F., and Shiekhattar, R. (2000). BRCA1 is associated with a human
SWI/SNF-related complex: linking chromatin remodeling to breast cancer. Cell 102,
257-265.
Brinkmann, A. O., Faber, P. W., van Rooij, H. C., Kuiper, G. G., Ris, C., Klaassen, P.,
van der Korput, J. A., Voorhorst, M. M., van Laar, J. H., Mulder, E., and et al. (1989).
The human androgen receptor: domain structure, genomic organization and regulation
of expression. J Steroid Biochem 34, 307-10.
Chen, D., Ma, H., Hong, H., Koh, S. S., Huang, S.-M., Schurter, B. T., Aswad, D. W„
and Stallcup, M. R. (1999). Regulation of transcription by a protein methyltransferase.
Science 284,2174-2177.
Fan, S., Wang, J., Yuan, R., Ma, Y., Meng, Q., Erdos, M. R., Pestell, R. G., Yuan, F.,
Aubom, K. J., Goldberg, I. D., and Rosen, E. M. (1999). BRCA1 inhibition of estrogen
receptor signaling in transfected cells. Science 284,1354-6.
236
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Green, S., Issemann, I., and Sheer, E. (1988). A versatile in vivo and in vitro eukaryotic
expression vector for protein engineering. Nucl Acids Res 16.
Hansen, S. K., Takada, S., Jacobson, R. H., Lis, J. T., and Tjian, R. (1997).
Transcription properties of a cell type-specific TATA-binding protein, TRF [see
comments]. Cell 91,71-83.
Hong, J., Kohli, K., Trivedi, A., Johnson, D. L., and Stallcup, M. R. (1996). GRIP1, a
novel mouse protein that serves as a transcriptional co-activator in yeast for the
hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93,4948-4952.
Hu, Y. F., Hao, Z. L., and Li, R. (1999). Chromatin remodeling and activation of
chromosomal DNA replication by an acidic transcriptional activation domain from
BRCAl. Genes Dev 13, 637-42.
Irvine, R. A., Ma, H., Yu, M. C., Ross, R. K., Stallcup, M. R., and Coetzee, G. A.
(2000). Inhibition of pl60-mediated coactivation with increasing androgen receptor
polyglutamine length. Hum Mol Genet 9,267-274.
Lin, W. J., Gary, J. D., Yang, M. C., Clarke, S., and Herschman, H. R. (1996). J Biol
Chem 271,15034-15044.
237
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Ma, H., Hong, H., Huang, S. M., Irvine, R. A., Webb, P., Kushner, P. J., Coetzee, G. A.,
and Stallcup, M. R. (1999). Multiple signal input and output domains of the 160-
kilodalton nuclear receptor coactivator proteins. Mol Cell Biol 19, 6164-6173.
McKenna, N. J., Xu, J., Nawaz, Z., Tsai, S. Y., Tsai, M.-J., and O'Malley, B. W. (1999).
Nuclear receptor coactivators: multiple enzymes, multiple complexes, multiple
functions. J Ster Biochem Mol Biol 69, 3-12.
Naar, A. M., Beaurang, P. A., Zhou, S., Abraham, S., Solomon, W., and Tjian, R.
(1999). Composite co-activator ARC mediates chromatin-directed transcriptional
activation. Nature 398, 828-32.
Ouchi, T., Monteiro, A. N., August, A., Aaronson, S. A., and Hanafusa, H. (1998).
BRCA1 regulates p53-dependent gene expression. Proc Natl Acad Sci U S A 95, 2302-
6.
Pao, G. M., Janknecht, R., Ruffner, H., Hunter, T., and Verma, I. M. (2000). CBP/p300
interact with and function as transcriptional coactivators of BRCAl. Proc Natl Acad Sci
U S A 97,1020-5.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Puigserver, P., Wu, Z., Park, C. W., Graves, R., Wright, M., and Spiegelman, B. M.
(1998). A cold-inducible coactivator of nuclear receptors linked to adaptive
thermogenesis. Cell 92, 829-39.
Rachez, C., Lemon, B. D., Suldan, Z., Bromleigh, V., Gamble, M., Naar, A. M.,
Erdjument-Bromage, H., Tempst, P., and Freedman, L. P. (1999). Ligand-dependent
transcription activation by nuclear receptors requires the DRIP complex. Nature 398,
824-8.
Rebbeck, T. R., Kantoff, P. W., Krithivas, K., Neuhausen, S., Blackwood, M. A.,
Godwin, A. K., Daly, M. B., Narod, S. A., Garber, J. E., Lynch, H. T., Weber, B. L.,
and Brown, M. (1999). Modification of BRCA1-associated breast cancer risk by the
polymorphic androgen-receptor CAG repeat. Am J Hum Genet 64, 1371-7.
Somasundaram, K., Zhang, H., Zeng, Y. X., Houvras, Y., Peng, Y., Wu, G. S., Licht, J.
D., Weber, B. L., and El-Deiry, W. S. (1997). Arrest of the cell cycle by the tumour-
suppressor BRCA1 requires the CDK-inhibitor p21WAFl/CiPl. Nature 389, 187-90.
Sundqvist, A., Sollerbrant, K., and Svensson, C. (1998). The carboxy-terminal region of
adenovirus El A activates transcription through targeting of a C-terminal binding
protein-histone deacetylase complex. FEBS Lett 429, 183-8.
239
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Umesono, K., and Evans, R. M. (1989). Determinants of Target Gene Specificity For
Steroid/thyroid Hormone Receptors. Cell 57, 1139-1146.
Wang, Q., Zhang, H., Kajino, K., and Greene, M. I. (1998). BRCA1 binds c-Myc and
inhibits its transcriptional and transforming activity in cells. Oncogene 77, 1939-48.
Xu, L., Glass, C. K., and Rosenfeld, M. G. (1999). Coactivator and corepressor
complexes in nuclear receptor function. Curr Op Gen Dev 9.
Yarden, R. I., and Brody, L. C. (1999). BRCA1 interacts with components of the
histone deacetylase complex. Proc Natl Acad Sci U S A 96,4983-8.
Zhang, H., Somasundaram, K., Peng, Y., Tian, H., Bi, D., Weber, B. L., and El-Deiry,
W. S. (1998). BRCA1 physically associates with p53 and stimulates its transcriptional
activity. Oncogene 16, 1713-21.
Zhong, Q., Chen, C. F., Li, S., Chen, Y., Wang, C. C., Xiao, J., Chen, P. L., Sharp, Z.
D., and Lee, W. H. (1999). Association of BRCA1 with the hRad50-hMrell-p95
complex and the DNA damage response. Science 285, 747-50.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Antibody-cytokine/chemokine fusion proteins in the immunotherapy of solid tumors

PDF

Investigation of the role of epigenetic modification of DNA and chromatin in aberrant gene silencing in cancer cells

PDF

Engineering antibodies and antibody /cytokine fusion proteins for the treatment of human malignancies

PDF

Genetic risk factors in breast cancer susceptibility: The multiethnic cohort

PDF

Imaging and prodrug -activating derivatives of chTNT-3 (tumor necrosis therapy) monoclonal antibody

PDF

B7.1 fusion proteins for cancer immunotherapy

PDF

Breast cancer in the multiethnic cohort study: Genetic (prolactin pathway genes) and environmental (hormone therapy) factors

PDF

Characterization of the EWS -FLI1 fusion protein in the tumorigenesis of Ewing's family of tumors

PDF

Androgen receptor gene and prostate -specific antigen gene in breast cancer

PDF

BRCA1 mutations and polymorphisms in African American women with a family history of breast cancer identified through high throughput sequencing

PDF

A large-scale genetic association study of prostate cancer in a multi-ethnic population

PDF

Androgens and breast cancer

PDF

Biochemical analysis of somatic mutations in steroid 5alpha-reductase type II in prostate cancer

PDF

Downstream targets of the Ewing's sarcoma EWS /FLI -1 fusion gene

PDF

HER-2/neu-mediated cell migration and invasion

PDF

Characterization of zebularine: A novel inhibitor of DNA methylation with clinical potential

PDF

Association of vitamin D receptor gene polymorphisms with colorectal adenoma

PDF

Analysis of the pathological characteristics of breast carcinoma in Hispanic BRCA carriers

PDF

Development of replication -competent retroviral vectors for efficient, targeted gene therapy of cancer

PDF

Gene regulation of type I collagen and its alteration by FGF -2 during corneal endothelial mesenchymal transformation

Asset Metadata

Creator Park, John Jungha (author)

Core Title Breast cancer susceptibility gene 1: A role in transcriptional regulation

School Graduate School

Degree Doctor of Philosophy

Degree Program Pathobiology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,health sciences, oncology,health sciences, pathology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Press, Michael (committee chair), Coetzee, Gerhard (committee member), Epstein, Alan L. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-228056

Unique identifier UC11334745

Identifier 3073832.pdf (filename),usctheses-c16-228056 (legacy record id)

Legacy Identifier 3073832.pdf

Dmrecord 228056

Document Type Dissertation

Rights Park, John Jungha

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA