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Interaction of Hic-5 with different steroid receptors and its selective coregulator activity

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Interaction of Hic-5 with different steroid receptors and its selective coregulator activity

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INTERACTION OF Hic-5 WITH DIFFERENT STEROID
RECEPTORS AND ITS SELECTIVE COREGULATOR
ACTIVITY

by
Kiran Sriram

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2015

Copyright 2015 Kiran Sriram
i

DEDICATION
To my parents Sumathy and Sriram, and brother Krishna
For the unconditional love and support…

ii

ACKNOWLEDGEMENTS
Firstly, I wish to thank Dr. Michael Stallcup for his constant guidance and support
throughout my master’s program. He has been a wonderful advisor, mentor and professor.
His invaluable suggestions, feedback and encouragement through my ups and downs have
helped me mature as a researcher. I will always be grateful for the opportunity to work with
him and everyone in his lab. I would like to offer my sincere appreciation to my committee
members Dr. Ansgar Siemer and Dr. Zoltan Tokes for their comments and suggestions.
Secondly I would like to express my gratitude to Dr. Rajas Chodankar, a previous post-doc
in our lab. She introduced me to the field of research and got me comfortable in the
academic setting. I am extremely thankful for all the patience she showed in answering my
innumerable questions during experiments and every single time I messed something up.
She was easily approachable and I must thank her for all the talks and advices she gave me
off lab work. I owe this thesis to her.
I would like to show my appreciation to every other person I have worked with in Stallcup
lab. I should thank Dan for his suggestions with experiments and movies and those late
evening career advices. I thank Coralie for her suggestions with my experiments and Brian
for answering my questions and helping with experiments. It was a pleasure working with
Chen Yin and I thank her for all the life advices she has given me. Also I express my
regards to Yixin with whom I have shared long talks on classes, lab work and food.
Finally, I would like to thank my parents, brother, my roommates for putting up with me
and my tantrums and Vrishika, Suraj and Dinesh who are my family away from home.

iii

TABLE OF CONTENTS
DEDICATION (i)
ACKNOWLEDGEMENTS (ii)
LIST OF FIGURES (v)
ABSTRACT (vi)
CHAPTER 1: INTRODUCTION 1
1.1 Steroid Hormone Receptors 1
1.2 Coregulators 3
1.3 Hydrogen Peroxide Inducible Clone-5 (Hic-5) 4
1.3.1 Structure of Hic-5 5
1.3.2 Localization of Hic-5 and nuclear/cytoplasmic shuttling 6
1.3.3 Hic-5 as a coregulator for steroid hormone receptors 7
CHAPTER 2: RESULTS 12
2.1 Generation of stable cell lines expressing Wild type and Mutant rGRα 12
2.2 Interaction of GRγ with Hic-5 21
2.3 Hic-5 binds weakly to ERα compared to GRα suggesting role in weaker 25
coregulator activity
CHAPTER 3: DISCUSSION 29
CHAPTER 4: MATERIALS AND METHODS 33
4.1 Cell Culture 33
4.2 Generation of Stable cell lines 33
iv

4.2.1 Kill curve analysis 33
4.2.2 Transfection 33
4.2.3 Limiting dilution 34
4.3 Plasmids 34
4.3.1 GRγ plasmids 34
4.3.2 ERα plasmids 35
4.4. Mammalian Two Hybrid Assay 35
4.5 Immunoblot Analysis 36
Table 1: Immunoblot conditions 37
BIBLIOGRAPHY 38

v

LIST OF FIGURES

Figure

Title

Page
number
1 Domain structure of steroid receptors 2
2 Schematic representation of domain structure of Hic-5. 6
3 Hic-5 interacts similarly with GRα and GRγ 10
4 Interaction of Hic-5 with ERα weaker than with GRα 11
5 Expression levels of mixed populations prior to making
clonal cell lines
13
6 Expression levels of the WT clones obtained from two
different limiting dilutions.
16, 17
7 Expression levels of the Mut clones obtained from two
different limiting dilutions.
19, 20
8A Hic-5 interacts similarly with GRα and GRγ and through
the tau-2 domain.
23, 24
8B Expression levels of transfected VP16 plasmids encoding
the various domains of GRα and GRγ.
23, 24
9A Hic-5 interacts weakly with ERα compared with GRα. 27, 28
9B Expression levels of plasmids used for Mammalian Two-
hybrid assay.
27, 28

vi

ABSTRACT
Hic-5 (Hydrogen Peroxide Inducible clone-5) is a paxillin family protein that acts as a
transcription coregulator for various steroid receptors such as Androgen Receptor (AR),
Progesterone Receptor (PR), Glucocorticoid Receptor (GR) and Estrogen Receptor (ER).
Hic-5 supports the hormone regulation of certain glucocorticoid target genes and estrogen
target genes while blocking the hormone regulation of certain other genes. Although Hic-
5 affects regulation of multiple subsets of genes regulated by GRα, GRγ and ERα, it serves
as a selective coregulator for GRα by regulating more GRα target genes than target genes
for the other two steroid receptors in the U2OS cell line.
Most nuclear receptor coregulators have been shown to interact with the AF-2 interaction
region on the Ligand Binding Domain (LDB) of nuclear receptors. However, Hic-5 binds
robustly to the less conserved tau-2 region that overlaps the Hinge domain and LBD of
GRα. Here we explored the importance of Hic-5 interaction with this tau-2 region on GRα
in blocking glucocorticoid regulation of a subset of genes. We also compared the
interaction of Hic-5 with GRγ and ERα to that with GRα to explain its selective coregulator
activity for GRα over the other two nuclear receptors.
In this thesis, we report that the smaller number of genes coregulated by Hic-5 for ERα
compared with GRα could be explained by a weak and possibly an indirect interaction of
Hic-5 with ERα compared with a strong and direct interaction with GRα. However, since
we show that Hic-5 interacts similarly with GRα and GRγ, the mechanism of narrower
coregulation for GRγ target genes should be explained by something else.

1

CHAPTER 1
INTRODUCTION
1.1. Steroid Hormone Receptors
Steroid hormones regulate a variety of events such as metabolism, growth and
development, immune response, development of sexual characteristics and maintenance
of electrolyte balance. These hormones are classified into five groups namely
glucocorticoids, mineralocorticoids, estrogens, progestogens and androgens. They are
synthesized and secreted into the blood stream by endocrine glands such as the adrenal
cortex and gonads. These lipid soluble molecules enter the cell by diffusion through the
plasma membrane and bind steroid hormone receptors localized in the nucleus or
cytoplasm. Upon binding they activate the steroid receptors and regulate transcription of
target genes.
Steroid hormone receptors or steroid receptors (SR) belong to a group of structurally and
functionally related proteins called the nuclear receptor (NR) superfamily. They are
ligand inducible transcription factors (TFs) that regulate transcription of target genes.
They contain an N-terminal regulatory domain that includes a hormone independent
activation function domain (AF-1), a central DNA binding domain (DBD), a hinge region
that allows for protein flexibility and a C-terminal Ligand binding domain (LBD) that
encompasses a hormone dependent activation function domain (AF-2). Further down the
LBD of some steroid receptors there is a C-terminal tail whose function has not been
established yet. The DBD is the most conserved region in all the nuclear receptors and
contains two zinc-finger motifs that target the receptor to specific DNA sequences called
2

the hormone response elements (HRE). The LBD contains a dimerization interface which
is responsible for hormone activated dimerization of receptors before binding the
response elements (Chawla et al. 2001). The N-terminal DBD and the C-terminal LBD
are independently well folded and structurally stable while the Hinge domain and the C-
terminal tail may be conformationally flexible and disordered (Scanlan et al. 1999) (Fig
1).

Fig 1: Domain structure of steroid receptors
Steroid hormone receptors contain a hormone independent N-terminal activation function
domain (AF-1), a conserved DNA binding domain (DBD) that recognizes and binds to
response elements, a flexible Hinge domain, a Ligand binding domain (LBD) that
includes the C-terminal activation function domain (AF-2) and dimerization surface apart
from ligand binding site. (Laperrière D et al. 2012).

Once the hormone binds the steroid receptor, a conformational change is induced
changing the receptor to its active form. On activation, the receptors dissociate from the
chaperone complex which contains Hsp-90. The free receptors either homodimerize or
heterodimerize and bind to the response elements on the promoter or enhancer of target
3

genes. Upon binding, the steroid receptor assembles a host of proteins at the site which
eventually recruit RNA Polymerase II (RNA Pol II) to the promoter of the target gene.
Coregulators are the proteins that are recruited to the site by the steroid receptors to help
facilitate the opening of the chromatin for the transcription complex to be assembled. In
some cases, basal level transcription occurs even in the absence of coregulators. They
serve in enhancing the level of transcription of such genes by interacting with the
enhancer elements (McKenna et al. 1999).
1.2 Coregulators
Transcription coregulators are proteins that interact with transcription factors (TFs) and
are required for efficient transcriptional regulation. Coactivators are proteins that interact
with the TFs and enhance their transactivation while corepressors are proteins that
interact with TFs and lower the transcription rate of their target genes. Many steroid
receptor coregulators have been identified since the landmark identification and cloning
of steroid receptor coactivator -1 (SRC-1). The SRC family of proteins including SRC-1,
SRC-2/GRIP-1 and SRC-3 are the most characterized group of steroid receptor
coregulators. (McKenna et al. 1999).
Coregulators are of two major classes. The first class of coregulators are enzymes that
make covalent modifications on histones and modify the chromatin structure. Another set
of coregulators in the same class induce conformational changes on the chromatin
structure in an ATP dependent manner. The second class of coregulators serve as scaffold
proteins that regulates the recruitment of transcription machinery.
4

Most coregulators identified till date have been shown to interact with either the AF-1 or
AF-2 activation function domains of the steroid receptors. However, a steroid receptor
binding protein, Hydrogen Peroxide Inducible Clone -5 (Hic-5), that can act as a
coactivator for GR was identified in 2000. Hic-5 was found to be binding strongly to the
tau-2 region, the region spanning the C-terminal of Hinge region and N-terminal of LBD
region of GR (Yang et al. 2000). Interestingly, it was also found to be interacting with the
LBD region of AR while serving as a coactivator (Fujimoto et al. 1999).
1.3. Hydrogen Peroxide Inducible Clone-5 (Hic-5)
Hic-5 was characterized as a transforming growth factor – β (TGF-β) inducible clone in
mouse osteoblasts. Initially named TGF-β stimulated clone-5 (TSC-5), it was later
renamed Hydrogen peroxide inducible clone-5 as it was found to be partially mediated by
H2O2 (Shibanuma et al. 1994). Hic-5 is a 55 kDa protein and is the most homologous
member to paxillin in the paxillin family. Like the other members of the family, Hic-5 is
a molecular adaptor protein and is localized at focal adhesions, where the cell adheres to
extra cellular matrix via integrins (Nishiya et al. 2002).
Hic-5 is prominently expressed in smooth muscle layer of tissues such as large intestine
and uterus, and also in high amounts in spleen and lung tissues. In a cell culture system,
Hic-5 is more prominent in cells of mesenchymal origin while it is low in many epithelial
cell lines (Shibanuma et al. 2011). Similar results were observed by Jia et al. when they
studied the distribution of Hic-5 in rat tissues (Jia et al. 2001).
Many studies regarding the physiological relevance of Hic-5 are being carried out since
the last decade. A Hic-5 knock-out mouse model was created in 2011 by
5

Kim-Kaniyama et al. There was no embryonic lethality and no apparent abnormality in
function was observed, although, a mild vascular remodeling was observed (Kim-
Kaniyama et al. 2011).
In another set of studies, Hic-5 was shown to be involved in the pathophysiology of
endometriosis by serving as a transcriptional coactivator for PR. They observed a loss of
menstrual cycle regulated expression of Hic-5 in endometriosis (Aghajanova et al. 2009).
Dorgee et al. in 2012 showed that dysregulation of Hic-5 is associated with prostate
cancer progression. A recent study suggests Hic-5 regulates mesangial cell proliferation
in proliferative glomerulonephritis in mice (Jamba et al. 2015). Hence, the significance of
Hic-5 has been increasingly noted.
1.3.1. Structure of Hic-5
The N-terminal region of the protein contains four LD domains that are rich in Leucine
and Aspartate residues. The C-terminal region of the protein comprises four LIM (Lin-11,
Isl-1, Mec-3) domains each having two zinc fingers. Since both the LD and LIM domains
are protein-protein interaction surfaces, it makes sense that Hic-5 serves as a molecular
adaptor providing an interface for multiple proteins to interact and co-operate
(Shibanuma et al. 2011) (Fig 2).

6

Fig 2: Schematic representation of domain structure of Hic-5.
Hic-5 is a 461 amino acid long protein with a molecular weight of 55 kDa. It has four LD
domains in the N-terminus that are rich in Leucine and Aspartate residues. In the
C-terminus, there are four LIM domains that are double zinc-finger motifs each about 50
amino acids in length. The LIM domains are rich in Cysteine/Histidine residues. Due to
the presence of these interaction surfaces, Hic-5 functions as a platform/molecular
adaptor in assembling and interaction of various signaling proteins (Turner CE, 2000).

1.3.2. Localization of Hic-5 and nuclear/cytoplasmic shuttling
Hic-5 is localized in both the cytoplasm and the nucleus. It performs different functions
in both the places. In the nucleus, unlike the other members of paxillin family Hic-5 was
found to be involved in regulation of transcription by acting as a coactivator for AR
(Fujimoto et al. 1999) and GR (Yang et al. 2000). In another study it was shown that Hic-
5 is involved in regulating expression of c-fos and p21. It promotes the formation of a
transcriptional complex by recruiting Sp1 and Smad3 proteins and Mediator complex
(MED 1) (Shibanuma et al. 2011, Chodankar et al. 2014).

7

1.3.3. Hic-5 as a coregulator for steroid hormone receptors
Earliest study reporting Hic-5 as a coactivator was in 1999 when Fujimoto et al reported
Hic-5 (ARA55) as a coactivator for AR by binding to AR-LBD using a yeast-two hybrid
system. They reported that the C-terminal half of ARA55 was sufficient to interact with
AR (Fujimoto et al. 1999).
Around the same time, Hic-5 was identified as a protein that interacted with and served as
a coactivator for GR. With a yeast-two hybrid system, Hic-5 was shown to bind robustly
to the tau-2 transactivation domain of mouse GR (513-562 a.a). Although Hic-5 bound to
GR-DBD-tau-2 domain just with its C-terminal region, the full length Hic-5 was required
for efficient transactivation of reporter gene in yeast and mammalian systems. Transient
reporter gene assays in CV-1 cells showed that Hic-5 enhanced the activity of GR, AR,
mineralocorticoid receptor (MR) and PR robustly but had little effect on the activity of
ER and thyroid receptor (TR). Interestingly, the tau-2 region is conserved between GR,
MR, PR and AR while there is very little sequence homology with ER (Yang et al. 2000).
These results raise the question of whether the tau-2 region is important for interacting
with Hic-5 and thus important for serving as a coactivator.
To answer the above question, mutations (T548A and E554A) were made on the tau-2
domain of rat GR by site-directed mutagenesis and the interaction of Hic-5 with the
mutated GR was observed by transient reporter gene assays in mammalian cells.
Unpublished data from our lab shows that the binding of Hic-5 to the GR-DBD-tau-2
region was disrupted by the mutations. However, on co-immunoprecipitation after
transient overexpression of Hic-5 and mutant full length GR in U2OS cells and Cos-7
cells, the interaction of Hic-5 with wild-type and mutant GR were found to be similar
8

(Chodankar, unpublished data). Stable cell lines expressing the wild-type and mutant GR
were needed to further study the endogenous interaction and effect on transactivation
activity of Hic-5 for the receptor upon mutation.
In another study, Hic-5 was shown to have gene specific roles in Dex-regulated
transcription. On Hic-5 depletion followed by global gene-expression analysis it was
observed that Hic-5 selectively affected both activation and repression of GRα target
genes. Three different classes of genes for which of Hic-5 is involved in GRα function
were classified; Modulated, Independent and Blocked classes (Chodankar et al. 2014).
For the Modulated genes Hic-5 could act as both a coactivator and as a corepressor. For
the genes where Hic-5 acted as a coactivator, Hic-5 was required for recruitment of
MED1 complex which in turn was required for bringing RNA Pol II to the transcription
complex at the promoter. Upon Hic-5 depletion, GRα was still recruited to the GBR in
response to Dex. Chromatin remodeling was not affected and the general transcription
factors CBP and p300 were also recruited (Chodankar et al. 2014).
The Blocked genes are the genes that were not Dex responsive until Hic-5 was depleted.
For this class of genes it was found that the chromatin remained closed at the GBRs and
the GR binding was inhibited in the presence of Hic-5. Since Hic-5 binds robustly to tau-
2 region close to GRα-DBD, it is proposed that Hic-5 might restrict the robust occupancy
of GR in these sites. This would indicate Hic-5 acts before the Dex induced binding of
GR to the response elements which is new for a coregulator.
For the Independent genes, Hic-5 had little or no coregulator activity, and the depletion of
Hic-5 did not affect the Dex regulation of these genes (Chodankar et al. 2014).
9

In a follow up study conducted later, Hic-5 facilitated glucocorticoid and estrogen
regulation of some genes via GRγ and ERα respectively but blocked hormone regulation
of others. However, it was observed that the Hic-5 was a selective coregulator for GRα
and regulated a larger number of genes in the Modulated class for GRα over GRγ and
ERα. Interestingly, Hic-5 regulated similar number of genes in the Blocked class for the
three steroid receptors (Chodankar et al. 2015).
To understand if the selective coregulator activity can be attributed to a difference in
interaction of Hic-5 with the three steroid receptors, their interaction was studied by
co-immunoprecipitation (Co-IP) experiments.
GRγ is a natural splice variant of GRα and differs from GRα due to a single arginine
insertion in the DBD separating the two zinc fingers (Rivers et al. 1999). Since the tau-2
region is conserved, Hic-5 is expected to interact similarly to GRα and GRγ. Co-IP data
suggests that interaction of endogenous Hic-5 with GRα and GRγ was similar in the same
cell line U2OS with the respective GR isoforms overexpressed (Chodankar et al. 2015)
(Fig 3).

10

Figure 3: Hic-5 interacts similarly with GRα and GRγ
Co-IP experiments preformed to study interaction of endogenous Hic-5 with GRα in
U2OS-GRα cell line (A) and interaction of endogenous Hic-5 with GRγ in U2OS-GRγ
cell line (B) (Chodankar et al. 2015).

In another set of experiments conducted, it was observed that Hic-5 does not bind
efficiently to ERα. ERα could not be detected on an immunoblot after Co-IP with Hic-5
in endogenous levels in U2OS-ERα cell line. Although Hic-5 pulled down ERα when
both the proteins were overexpressed, Hic-3 interaction with GRα was 3 times stronger
than with ERα under similar conditions (Chodankar et al. 2015) (Fig 4). Since ERα does
not contain a conserved tau-2 region as in GR isoforms, this result is understandable.
However further studies were required to elucidate if Hic-5 could interact with any other
part of ERα.

11

Figure 4: Interaction of Hic-5 with ERα weaker than with GRα
Co-IP experiments preformed to study interaction of endogenous Hic-5 with ERα in
U2OS-ERα cell line (A) and interaction of overexpressed HA-Hic-5 with overexpressed
Flag-ERα in Cos-7 cell line (B) (Chodankar et al. 2015).

The above findings led us to create stable cell lines expressing wild-type and mutant GRα
in U2OS-cells to study the importance of tau-2 domain in regulation of endogenous target
genes of GR in the future. Also we further wanted to test if the difference in interaction of
Hic-5 with different steroid receptors could explain the difference in the number of target
genes regulated by Hic-5 for each of them.

12

CHAPTER 2
RESULTS
2.1. Generation of stable cell lines expressing Wild type and Mutant
rGRα
In previous studies, Hic-5 has been shown to interact with the tau-2 domain of mouse
GRα (533-562 a.a) (Yang et al., 2000). Mutations in the tau-2 domain disrupt the
interaction between Hic-5 and rat GRα shown by Mammalian Two Hybrid Assay
(Unpublished data from our lab). To understand the importance of interaction of Hic-5
with GRα in regulating Blocked and Modulated classes of genes, stable U2OS clonal cell
lines expressing flag tagged full length Wild-type and Mutant (T548A and E554A) rGRα
were created.
Post transfection with the plasmids (pcDNA-3X-flag-GR-WT) and (pcDNA-3X-flag-GR-
T2M), transfected cells were selected with antibiotic G418 at a concentration of
600 µg/mL (determined by kill curve analysis) and grown as mixed populations. The GR
expression levels in these mixed populations were checked prior to making clonal cell
lines. An important observation was that the GR-Mut cells took longer to grow and get
confluent compared to the GR-WT cells.

13

Figure 5: Expression levels of mixed populations prior to making clonal cell lines.
U2OS cells plated in 6-well plates were transfected with 5µg of plasmids
(pcDNA-3X-flag-GR-WT) and (pcDNA-3X-flag-GR-Mut) with Lipofectamine and Plus
reagent. Transfected cells were selected with G418 and whole cell extracts were collected
to check protein expression levels. Samples were loaded on to 8% SDS-PAGE and
analyzed by immunoblotting using antibodies against GR, Flag and β-actin. Lanes 1&2
represent expression levels of U2OS- Parental (Par) cells, lanes 3&4 represent expression
levels of cells 3 days after transfection and lanes 5&6 represent expression levels of cells
4 days after transfection.

14

The GR expression levels of the transfected cells 3 days and 4 days post transfection for
both WT and Mut mixed populations were comparatively higher than the GR expression
levels of the U2OS – Parental (Par) cell lines (Fig 5). This shows that the transfection
worked and the cells are over-expressing GR, more than the basal level GR expression in
the un-transfected parental cell line. The strong band around 100 kDa detected with a
Flag antibody further supports the success of transfection, as it is not observed in the first
two lanes containing samples from parental cell line. Thus the mixed populations of both
U2OS-GR-WT and U2OS-GR-Mut cells were used to make clonal populations of cells
expressing WT-GR or Mut-GR respectively.
Limiting Dilution – birth of clonal cell lines:
The mixed population of U2OS-GR-WT cells were plated into 96-well plates at a density
of 1 - 2 cells per well. Clones were picked from the 96-well plates 4 - 5 days post plating
and transferred into 24-well plates. Clones were eventually scaled up into 6-well plates
and whole cell extracts were collected to check the GR expression levels. The cells were
maintained in the selection media containing G418 throughout this stage to effectively
eliminate propagation of un-transfected cells. The expression levels of the clones that
survived until 6-well plate stage are as in (Fig 6A). The relative intensity ratios of GR
expression of the U2OS – GRα cell line and the clones obtained compared to parental
U2OS cell line are as in (Fig 6B). These quantities were normalized internally to their
respective β-actin expression levels.
A faint band in the first lane in the GR blot shows the basal expression of GR in U2OS
parental cell line. There is a clear overexpression of GR in the U2OS – GRα cell line
(lane 2) and in some of the clones obtained (lanes 3 – 9). After quantification and
15

normalization to actin levels, it was determined that the GR expression of clones WT-G3,
WT-B5 and WT-F6 were approximately 62, 27 and 40 times respectively to parental
level expression (Fig 6B). Further, a strong band around 100 kDa detected using antibody
against Flag in the corresponding lanes confirms that the clones overexpress the plasmid.
These clones could be potential candidates to establish U2OS cell lines expressing flag-
tagged wild-type GR. The clones WT-G3, WT-B5 and WT-F6 need to be revived and
scaled up, eventually depriving them of the selective antibiotic G418. Also they need to
be tested regularly to check the expression levels of GR while doing in-vivo experiments
with them.

16

17

Figure 6: Expression levels of the WT clones obtained from two different limiting
dilutions. (A) Mixed population of U2OS cells transfected with (pcDNA-3X-flag-GR-
WT) plasmid and selected with G418 were plated in 96-well plates with 1 – 2 cells per
well after limiting dilution. The cells were maintained in selective media, grown until 16
– 32 cell stage, transferred to 24-well plates and 6-well plates later. WCE from the
surviving clones were collected and samples were resolved in 8% SDS-PAGE.
Immunoblot was performed using antibodies against GR, Flag and β-actin. Lane 1
represents expression levels of U2OS parental cell line. Lane 2 represents expression
levels of U2OS-GRα cell line. Lanes 3-9 represent expression levels of U2OS-GR-WT
clones obtained. (B) Western Blot quantification (ChemiDoc XRS system with Image
Lab Software) of GR expression levels of clones compared to U2OS – Parental,
normalized internally to their respective β-actin levels.

18

Similarly, the mixed population of U2OS-GR-Mut cells were plated into 96-well plates at
a density of 3 – 4 cells per well. However, these cells took longer than the wild-type cells
to form clones. Clones were picked 7-10 days after plating and transferred into 24-well
plates. Very few clones survived after this stage unlike the U2OS-GR-WT clones. The
clones that survived were transferred into 6-well plates and whole cell extracts were
collected once they were confluent. A notable observation was that the GR-mutant
clones took much longer (around 7 days) to get confluent in a 6-well plate compared to
GR-wildtype clones (3-4 days).
The expression levels of the GR-mutant clones obtained are as in (Fig 7A). Only one of
the mutant clones obtained overexpressed the mutated GR plasmid, Mut-A6 (Lane 5) in
levels comparable to the U2OS – GRα (lane 2). The strong band detected in the
corresponding lane with Flag antibody confirms the same. However, all the other clones
expressed GR similar to parental level expression (Lane 1), suggesting there is no
overexpression of the plasmids transfected. The lack of bands in the corresponding lanes
in the Flag blot confirms the same. After quantification of the GR western blot and
normalizing against actin, it was determined that the clone Mut-A6 expresses
approximately 6 times more than parental levels (Fig 7B). The clone Mut-A6 serves as a
potential candidate to be developed into a U2OS cell line expressing flag-tagged mutant
rGRα (T548A and E554A). Like the U2OS-GR-WT clones, this mutant clone needs to
be revived, scaled up, deprived off G418 and checked regularly for GR expression levels
while using for experiments.

19

20

Figure 7: Expression levels of the Mut clones obtained from two different limiting
dilutions. (A) Mixed population of U2OS cells transfected with pcDNA-3X-flag-GR-
Mut plasmid and selected with G418 were plated in 96-well plates with 3 – 4 cells per
well after limiting dilution. The cells were maintained in selective media, grown until 16-
32 cell stage, transferred to 24-well plates and 6-well plates later. WCE from the
surviving clones were collected and samples were resolved in 8% SDS-PAGE.
Immunoblot was performed using antibodies against GR, Flag and β-actin. Lane 1
represents expression levels of U2OS parental (Par) cell line. Lane 2 represents
expression levels of U2OS-GRα cell line. Lanes 3-9 represent expression levels of
U2OS-GR-Mut clones obtained. (B) Western Blot quantification (ChemiDoc XRS
system with Image Lab Software) of GR expression levels of clones compared to U2OS –
Parental, normalized internally to their respective β-actin levels.

21

2.2. Interaction of GRγ with Hic-5
Hic-5 serves as a co-regulator for GRα by affecting Dex regulated expression of three classes
of genes; the Modulated class, the Independent class and the Blocked class. Previous studies
have shown that Hic-5 acts as a selective co-regulator for GRα over GRγ by regulating more
Dex regulated genes in the Modulated class for GRα than for GRγ. However, the number of
genes where the hormone regulated genomic occupancy of GRγ was blocked by Hic-5 was
similar to that with GRα (Chodankar et al. 2014).
To understand if this difference could be explained by difference in interaction of Hic-5 with
the two different isoforms, endogenous and overexpression interaction studies were done.
Previously, a strong hormone dependent interaction of Hic-5 with GRα was observed by
endogenous coimmunoprecipitation in U2OS-GRα cells (Chodankar et al. 2014). Similar
hormone dependent interaction of Hic-5 was observed with GRγ in U2OS – GRγ cells
(Chodankar et al., 2015). To further confirm the in-vivo interaction of Hic-5 with the two GR
isoforms through the tau-2 domain, the activation of luciferase gene due to the interaction of
overexpressed Hic-5 and GR domains was tested in a mammalian two hybrid system.
CV-1 cells were transfected with GK1-Luc plasmids encoding luciferase reporter gene
(controlled by Gal4 response elements), plasmids encoding Gal4-DBD fused to Hic-5 and
plasmids encoding VP16-AD fused to GRα/GRγ DBD or DBD-tau2 domains. VP16-AD
fused to GRα-DBD-tau2 gave a dramatic increase in the reporter gene expression suggesting
that Hic-5 bound strongly to GRα-DBD-tau2. This confirms the results from previous studies
that Hic-5 binds to GRα and acts as a co-regulator by binding to its tau-2 domain. Similarly,
an induction was observed with plasmids encoding VP16-AD fused to GRγ-DBD-tau2
22

(406-571a.a) but not with GRγ-DBD (406-541a.a) (Fig 8A). This suggests that Hic-5 interacts
with GRγ via the same tau-2 domain.
The expression levels of the transfected GR plasmids were checked using an Immunoblot
using VP16 antibody to confirm that the dramatic increase in luciferase activity was not due to
higher amount of GR fragments present Fig 8B shows that the expression levels of the
plasmids were similar. Fainter bands observed in Lanes 3 and 5 (compared with lanes 2 and 4)
suggest Hic-5 binds strongly to tau-2 domain even at lower concentrations but not to
GR-DBD even if present in abundance.

23

24

Figure 8: (A) Hic-5 interacts similarly with GRα and GRγ and through the tau-2
domain. CV-1 cells plated in 24 well plates at a density of 80,000 cells per well were
transfected with 200 ng of GK1-LUC reporter plasmid, 500 ng of plasmid encoding
Gal4-DBD fused to Hic-5 and 500 ng of plasmid encoding VP16-AD fused to GRα or
GRγ fragments. 48 hours post transfection, lysates were collected and assayed for
luciferase activity. The Relative Luciferase units shown here are the mean of 3 technical
replicates with standard deviation. (B) Expression levels of transfected VP16 plasmids
encoding the various domains of GRα and GRγ. CV-1 cells were plated in 6-well
plates at a density of 100,000 cells per well and transfected with 2500 ng of VP16-GRα-
DBD, VP16-GRα-DBD-tau2, VP16-GRγ-DBD and VP16-GRγ-DBD-tau2. Whole cell
extracts were collected 48 hours post transfection with RIPA buffer and samples were
resolved on an 8%SDS-PAGE. Immunoblots were done using antibodies against VP16
and β-actin for internal control.

25

2.3. Hic-5 binds weakly to ERα compared to GRα suggesting role in
weaker coregulator activity.
Although Hic-5 failed to enhance transient reporter gene activation by ERα
(Yang et al., 2000), recent studies have shown that Hic-5 acts as a coregulator for a
subset of endogenous target genes of ERα, shown by genome wide studies after Hic-5
depletion (Chodankar et al., 2015). However, Hic-5 supports hormone regulation by ERα
for fewer genes than by GRα.
To understand if this narrower coregulation observed with ERα could be explained by
difference in interaction of Hic-5 with the two steroid receptors, binding of Hic-5 to ERα
and GRα were studied.
A hormone dependent interaction between endogenous Hic-5 and endogenous ERα could
not be detected by coimmunoprecipitation in U2OS-ERα cells. Also, the interaction of
HA tagged Hic-5 with Flag tagged GRα was found to be three times stronger than with
Flag tagged ERα after overexpression in Cos-7 cells and immunoprecipitating with HA
antibody (Chodankar et al., 2015). To further compare the interaction of Hic-5 with GRα
and ERα in-vivo, their association was studied by a mammalian two-hybrid assay.
Plasmids encoding VP16-AD fused to various domains of hERα namely ER-AF1
(1-166 a.a), ER-DBD-hinge (167-339 a.a), ER-DBD-LBD (167-595 a.a) were prepared.
CV-1 cells were transfected with GK-1 Luc plasmid encoding luciferase gene controlled
by Gal4 response elements, pM-Hic-5 plasmid encoding Gal4-DBD fused to Hic-5 and
VP16 plasmids encoding VP16-AD fused to different domains of GRα or ERα.
26

VP16-AD fused to GRα-DBD-tau2 gave a strong induction of luciferase expression as
expected. However, none of the ER fragments fused to VP16-AD were able to activate
the reporter gene (Fig 9A), indicating that none of the hERα domains showed detectable
interaction with Hic-5 in comparison with GRα-tau2 domain. Thus the interaction of
Hic-5 with ERα observed by co-IP studies (Figure 4B) could be indirect unlike that with
GRα. This could also explain the narrower coregulation activity of Hic-5 for ERα.
To make sure the difference in luciferase induction was not due to difference in amount
of VP16-AD fused plasmids, an immunoblot using VP16 antibody was done to check the
expression levels of the plasmids. All the plasmids expressed at similar levels in Cos-7
cells (Fig 9B).

27

(A)
28

Figure 9: (A) Hic-5 interacts weakly with ERα compared with GRα. CV-1 cells were
transfected with 200 ng of Gk1-Luc reporter plasmid, 500 ng of plasmid encoding Gal4-
DBD fused to Hic-5 and 500 ng of plasmid encoding VP16-AD fused to GRα or ERα
fragments. 24 hours post transfection, Estrogen (E2) treatment was started at 100 nM
concentration as indicated. 48 hours post transfection, lysates were collected and assayed
for luciferase activity. The Relative Luciferase units shown here are the mean of 3
technical replicates with standard deviation. (B) Expression levels of plasmids used for
Mammalian Two-hybrid assay. Cos-7 cells were plated into 6-well plates at density of
100,000 cells per well and transfected with 2500 ng of VP16 plasmids encoding the
different domains of GRα and ERα. 24 hours post transfection, Estrogen (E2) treatment
was started at a concentration of 100 nM. 48 hours post transfection, lysates were
collected and samples were resolved in 8% SDS-PAGE. Western Blotting was performed
with antibodies against VP16 and β-actin as internal control.

29

CHAPTER 3
DISCUSSION
Hic-5 mediates transcriptional regulation by GRα, affecting Dex responsiveness of
multiple subsets of genes. Hic-5 either supports or opposes hormone regulation of a
subset of GR target genes (Modulated class) or completely inhibits Dex responsiveness of
some other genes (Blocked class) (Chodankar et al., 2014). On these Blocked class of
genes, Hic-5 prevents the genomic occupancy of GRα by preventing GRα mediated
chromatin remodeling. In this study, we attempt to elucidate the importance of Hic-5
interaction with GR for regulating the Blocked and Modulated genes.
Previously it was shown that Hic-5 acts as a coregulator for mouse GRα by binding to its
tau-2 region (Yang et al., 2000). Our unpublished results in the lab show that mutations
(T548A and E554A) on the tau-2 region in rat GRα disrupt its interaction with Hic-5, via
transient reporter gene assay. To study the interaction of endogenous mutated tau-2
region and Hic-5, stable U2OS cell lines expressing flag-tagged full length wild-type and
mutant (T548A and E554A) rGRα were created.
Stable U2OS cell lines could be created with good overexpression of wild-type rGR.
However, only one mutant clone could be established as many clones did not survive
after transfer to bigger dishes. This could be explained by inappropriate transfection
methods or growth compromise due to mutation in GR. Further work in establishing
stable cell lines would involve expanding the U2OS-GR-WT cells and checking GR
expression levels. For creating U2OS-GR-Mut cells, different transfection methods such
as nucleofection, virus mediated transfer, etc. needs to be tried.
30

After the establishment of mutant cell lines, the interaction between endogenous mutated
GR and Hic-5 could be studied by co-immunoprecipitation. If the interaction is still
comparable to that in U2OS-GR-WT cells, it is possible that these specific amino acids
mutated (T548A and E554A) are not important for interaction with Hic-5. Other
possibilities are in contrast to the reports earlier, Hic-5 interacts with GR indirectly or
Hic-5 interacts with an additional region of GR like the LBD.
It would be interesting to observe the expression patterns of initially defined Blocked
genes in the mutant cell line. Since Hic-5 binds GR in a region close to the GR-DBD, it
could be possible that the interaction plays a major role in chromatin remodeling at GBR
for GR to bind. It was earlier observed that on Hic-5 depletion FAIRE signals were
robustly induced at GBRs of few Blocked genes suggesting a possible role of Hic-5 in
chromatin remodeling at these sites (Chodankar et al. 2014). Thus, we expect more
chromatin remodelers to be recruited to the GBR in Blocked genes and better binding of
GR on disruption of GR-Hic-5 interaction. This would result in upregulation of the genes
in the Blocked class in the GR mutant cell line compared to the wild-type. With the
Modulated genes we expect no induction of Dex regulated activation or repression in the
mutant cell line if the interaction between Hic-5 and tau-2 region is disrupted.
Hic-5 as a coregulator for GRγ
Similar to GRα, Dex responsiveness via GRγ is mediated by Hic-5 for three subsets of
genes (Mod, Independent and Blocked class). However, Hic-5 serves as a coregulator for
fewer genes in Mod class for GRγ than for GRα and similar number of Blocked genes for
both cases (Chodankar et al., 2015). Our results show that the binding of Hic-5 to the
tau-2 region on GRγ is similar to the binding of GRα-tau-2 and Hic-5. Thus, the narrower
31

coregulation for GRγ for Modulated genes should be explained by some other molecular
mechanisms. It would be interesting to decipher this mechanism as it would explain how
Hic-5 selectively coregulates steroid receptors.
The efficient binding of Hic-5 to GRγ as much as to GRα suggests that Hic-5 possibly
uses the same mechanism in both cases to suppress the Blocked genes. It would be
interesting to see if mutations on the tau-2 region of GRγ upregulates the Blocked genes.
We could also perform interaction studies followed by Mass Spec (MS) analysis to see if
Hic-5 recruits different chromatin remodelers for GRα and GRγ. Chromatin remodelers
might be involved as Hic-5 prevents genomic occupancy of GRγ on its Blocked genes.
Weaker interaction could explain weaker coregulator activity of Hic-5 for ERα
Hic-5 affects estrogen response of ERα target genes in three different classes; Modulated,
Independent, and Blocked. It was observed in previous studies that Hic-5 regulates fewer
genes in Mod class for ERα than for GRα, although similar number of Blocked genes
were identified in both cases (Chodankar et al. 2015). Previous co-immunoprecipitation
results and results from this study suggest that Hic-5 binds more weakly to ERα than to
GRα. Thus we conclude that the coregulator activity of Hic-5 for ERα could be explained
by an indirect interaction between these proteins. Indirect interaction serves as a fulcrum
in bringing more specificity. If more proteins are involved in the interaction, it brings in
more specificity based on the expression of the proteins in different cell types and thus
interaction would vary among cell types. Higher specificity could explain narrower
regulation of genes in Mod class. The proteins that mediate indirect interaction between
ERα and Hic-5 could be identified by doing a Mass spectrometry analysis after pulling
down Hic-5-ERα complex in-vivo.
32

On broader terms, answering the question as to why Hic-5 regulates hormone
responsiveness of some genes while completely inhibiting hormone responsiveness of
another subset of genes would help us understand the importance of information
contained in the genome by itself. It is possible that the signal for activation or repression
comes from the DNA sequence onto which the receptors bind or the chromatin structure
in those sites. We could look for histone marks on the enhancers of the target genes after
hormone treatment to understand if they are pre-programmed to be activated or repressed
before steroid receptor binding and recruitment of coregulators.
Also, since Hic-5 is expressed in a tissue specific manner while the steroid receptors
control various pathways in many cell types, the selective regulation by Hic-5 could play
a major role in cell type specific genomic occupancy of the steroid receptors and thus the
cell type specific gene expression.

33

CHAPTER 4
MATERIALS AND METHODS
4.1. Cell Culture
U2OS cells were cultured in McCoy’s 5A medium supplemented with 10% Fetal Bovine
Serum (FBS). CV-1 and Cos-7 cells were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) supplemented with 10% FBS.
4.2. Generation of Stable cell lines
4.2.1. Kill Curve Analysis:
U20S cells were plated into 6 well plates with 600,000 cells per well. They were treated
with G418 (Geneticin, Santa Cruz Biotechnology) at different concentrations (50, 100,
200, 400, 600, 800, 1000, 2000 µg/mL) to determine the minimal concentration required
to kill cells. The concentration at which 90% of cells were dead after 72 hours of
treatment was used for selection after transfection.
4.2.2. Transfection:
U2OS cells were seeded into 6 well plates at a density of 600,000 cells per well.
Previously prepared plasmids encoding flag-tagged wild-type and mutant (T548A and
E554A) full length rGR (pcDNA-3X-flag-GR-WT) and (pcDNA-3X-flag-GR-T2M)
were transfected into U2OS cells the next day. 5000 ng of the plasmids were transfected
into each well with Lipofectamine along with Plus reagent and Opti-MEM (from
34

Invitrogen, Life Technologies) according to the manufacturer’s protocol. The cells were
allowed to reach 90% confluency in the wells and split in 1:5 ratio.
4 days after transfection, the cells were treated with antibiotic Geneticin (G418) at a
concentration of 600 ng/µL to select cells that have taken up the transfected plasmids.
The transfected cells were scaled up gradually from 6 well culture plates to T-75 culture
flasks (maintained in G418 throughout) before performing limiting dilution to obtain
individual clones. Also, whole cell extracts were collected from the mixed population
after transfection to check the expression levels of the plasmids.
4.2.3. Limiting dilution:
The scaled up mixed populations of U2OS-GR-WT and U2OS-GR-T2M were plated into
96 well plates at a density of 1-2 cells per well for WT and 3-4 cells per well for T2M.
Clones were picked from 96 well plates after 1 week of plating. The clones were
eventually scaled up to 24 well plates, 12 well plates and 6 well plates. Whole cell
extracts were collected from surviving clones, maintained in G418, using RIPA buffer
and anaylsed for GR expression using Immunoblots.
4.3. Plasmids:
4.3.1. GRγ plasmids:
Previously constructed plasmids VP16-GRγ-DBD (406-541a.a) and VP16-GRγ-DBD-
tau2 (406-571 a.a) were transformed into DH5α competent cells. Transformed cells were
picked from agar plates containing selective antibiotic (ampicillin). These cells grown in
LB broth overnight and plasmids were prepared using Qiagen Plasmid maxi-prep kit.
35

4.3.2. ERα plasmids:
hERα fragments namely ER-AF1 (1-166 a.a), ER-DBD-hinge (167-339 a.a) and ER-
DBD-LBD (167-595 a.a) were PCR amplified using appropriate forward and reverse
primer sets from a flag-hERα template. These fragments were cloned into the BamHI and
EcoRI restriction sites of pVP16 plasmid vector using T4 DNA Ligase and 10X Buffer
from Promega. Ligation was verified by a diagnostic digestion with the restriction
enzymes BamHI and EcoRI.
The plasmids were transformed into DH5α competent cells by heat shock method.
Transformed colonies were picked the following day from agar plates containing the
selective antibiotic (ampicillin). These cells were grown in LB broth overnight and
plasmids were prepared the following day using Qiagen Plasmid maxi-prep kit. The
plasmids were sent to Genewiz .Inc for sequencing.
4.4. Mammalian Two Hybrid Assay
CV-1 cells were plated at a density of 80,000 cells per well in 24 well plates. The cells
were transfected the following day with plasmids using Lipofectamine 2000 reagent
along with Opti-MEM (from Invitrogen, Life Technologies) according to manufacturer’s
protocol.
The plasmids transfected were
i. GK-1-Luc (Webb et al. 1998) (containing the Luciferase gene downstream of
Gal-4 Response elements) – 200 ng
ii. pM-Hic-5 (Yang et al. 2000) (encoding fusion protein of Gal-4-DBD and full
lengthHic-5) – 500 ng
36

iii. pVP16-GR/ER fragments (encoding fusion protein of VP16 AD and various
GR/ER domains) – 500 ng
24 hours post transfection, media was changed and Estrogen treatment (100 nM
concentration) was started where required. 48 hours post transfection, lysates were
collected and luciferase assay was performed using Promega Luciferase Assay system
according to the manufacturer’s protocol.
4.5. Immunoblot Analysis
Cells were allowed to grow until they reach 80% confluency in 6-well plates and lysates
were collected using RIPA buffer with 50 mM Tris, 150 mM NaCl, 1% NP-40, 0.25%
Sodium Deoxycholate, 0.1% SDS and 1X Protease Inhibitor Cocktail. Cells were washed
twice with Dulbecco’s PBS. 600 µL of RIPA was added to each well and incubated on
ice for 1µminutes. Cell scraper was used to collect lysates which were then centrifuged at
12000 rpm for 5-10 minutes and supernatant was used for immunoblot analysis. Protein
samples were resolved by SDS-Polyacrylamide Gel Electrophoresis. Proteins were
transferred onto a nitro-cellulose membrane and blocked with 5% non-fat milk for 1 hour
at room temperature. The membrane was then incubated overnight at 4°C, with required
Primary Antibody at the concentrations mentioned below (Table 1). Post incubation and
washes in 1X-TBST, the membranes were incubated with respective anti-rabbit or anti-
mouse secondary antibodies conjugated with Horseraddish peroxidase for 45 minutes at
room temperature. ECL reagent from Thermo Scientific was used for developing and
detection.

37

Table 1: Immunoblot conditions
Primary
Antibody
Company Antibody
dilutions
Secondary
Antibody
Protein
amount loaded
GR (H-300) –
Cat #: sc-8992
Santa Cruz
Biotech
1:1000 Anti-Rabbit 30 µg
Flag (M2) –
Cat # : F3165
Sigma –Aldrich 1:1000 Anti-Mouse 30 µg
β-actin –
Cat #: A5441
Sigma –Aldrich 1:5000 Anti-Mouse 10 µg
VP16 (I-21) –
Cat # : sc-7545
Santa Cruz
Biotech
1:500 Anti-Mouse

30 µg

38

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Abstract (if available)

Abstract Hic-5 (Hydrogen Peroxide Inducible clone-5) is a paxillin family protein that acts as a transcription coregulator for various steroid receptors such as Androgen Receptor (AR), Progesterone Receptor (PR), Glucocorticoid Receptor (GR) and Estrogen Receptor (ER). Hic-5 supports the hormone regulation of certain glucocorticoid target genes and estrogen target genes while blocking the hormone regulation of certain other genes. Although Hic-5 affects regulation of multiple subsets of genes regulated by GRα, GRγ and ERα, it serves as a selective coregulator for GRα by regulating more GRα target genes than target genes for the other two steroid receptors in the U2OS cell line. ❧ Most nuclear receptor coregulators have been shown to interact with the AF-2 interaction region on the Ligand Binding Domain (LDB) of nuclear receptors. However, Hic-5 binds robustly to the less conserved tau-2 region that overlaps the Hinge domain and LBD of GRα. Here we explored the importance of Hic-5 interaction with this tau-2 region on GRα in blocking glucocorticoid regulation of a subset of genes. We also compared the interaction of Hic-5 with GRγ and ERα to that with GRα to explain its selective coregulator activity for GRα over the other two nuclear receptors. ❧ In this thesis, we report that the smaller number of genes coregulated by Hic-5 for ERα compared with GRα could be explained by a weak and possibly an indirect interaction of Hic-5 with ERα compared with a strong and direct interaction with GRα. However, since we show that Hic-5 interacts similarly with GRα and GRγ, the mechanism of narrower coregulation for GRγ target genes should be explained by something else.

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Asset Metadata

Creator Sriram, Kiran (author)

Core Title Interaction of Hic-5 with different steroid receptors and its selective coregulator activity

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 07/07/2015

Defense Date 06/03/2015

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

Tag coregulator,OAI-PMH Harvest,steroid receptors,Transcription

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Stallcup, Michael R. (committee chair), Siemer, Ansgar (committee member), Tokes, Zoltan A. (committee member)

Creator Email ksriram@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-587097

Unique identifier UC11300561

Identifier etd-SriramKira-3555.pdf (filename),usctheses-c3-587097 (legacy record id)

Legacy Identifier etd-SriramKira-3555.pdf

Dmrecord 587097

Document Type Thesis

Format application/pdf (imt)

Rights Sriram, Kiran

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 a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA