University of Southern California Dissertations and Theses

Repression of RNA polymerase III-dependent transcription by the tumor suppressors p53 and PTEN

(USC Thesis Other)

Repression of RNA polymerase III-dependent transcription by the tumor suppressors p53 and PTEN

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content REPRESSION OF RNA POLYMERASE III-DEPENDENT TRANSCRIPTION BY
THE TUMOR SUPPRESSORS p53 AND PTEN
by
Annette Woiwode
____________________________________________________________________
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
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
May 2007
Copyright 2007 Annette Woiwode
ii
Dedication
I would like to dedicate my thesis to my parents Karin and Fritz Woiwode. I know
they are proud of me. I would also like to dedicate my thesis to my sister Melanie
Woiwode. I am so thankful she is here to see me graduate.
iii
Acknowledgements
I would like to thank my advisor Debbie Johnson for her patience, advice, and
support. I would also like to thank my committee members Lucio Comai and David
Ann for their time and input. My fellow PhD student Jody Fromm could not have
been more supportive. Lastly, I cannot imagine having gone through Graduate
School without the love and support of my wonderful husband Ernesto Noriega.
iv
Table of Contents
Dedication ii
Acknowledgements iii
List of figures iv
Abstract v
Introduction: RNA polymerase III, Transcription Factor Complexes, and 1
Promoter Structure
RNA polymerase III transcription is regulated by the cell cycle 4
RNA polymerase III transcription is regulated by tumor suppressors 6
and oncogenes
PTEN 10
The tumor suppressors p53 and PTEN enhance each others activity 16
Chapter 1: p53 represses RNA polymerase III transcription by targeting TBP 18
Chapter 2: PTEN represses RNA polymerase III transcription via its lipid 25
phosphatase activity
Chapter 3: The PI-3 kinase signal transduction pathway regulates RNA 34
polymerase III transcription
Chapter 4: PTEN-mediated repression of RNA polymerase III transcription 38
in LN18 and U87 cells is not due to cell cycle affects
Chapter 5: PTEN mediates repression of RNA polymerase III transcription by 42
decreasing the promoter occupancy of TFIIIB
Chapter 6:Discussion 51
Chapter 7: Materials & Methods 62
References 78
v
List of Figures
Figure 1 p53 selectively represses transcription from a modified tRNA
Arg
gene 19
Figure 2 Overexpression of TBP alleviates p53-mediated repression of RNA 22
polymerase III transcription
Figure 3 p53 does not disrupt TBP/Brf1 binding 24
Figure 4 PTEN represses RNA polymerase III transcription via its lipid 27
phosphatase activity and independently of p53
Figure 5 PTEN selectively represses endogenous RNA polymerase III 30
transcription
Figure 6 Decreasing PTEN expression selectively enhances RNA polymerase 33
III transcription
Figure 7 The PI-3 kinase signal transduction pathway regulates RNA 37
polymerase III transcription
Figure 8 Reduction of PTEN levels in LN18 cells does not induce cell cycle 39
Changes
Figure 9 Cyclin D1 & nucleus-persistent cyclin D1 T286A do not alleviate 41
PTEN-mediated repression of RNA polymerase III transcription in
U87 cells
Figure 10 The affect of PTEN on TFIIIB 47
Figure 11 PTEN expression selectively decreases the promoter occupancy of 50
TFIIIB
Figure 12 Model of PTEN-mediated repression of RNA polymerase III 59
transcription
vi
Abstract
For cells to grow and proliferate they must be capable of protein synthesis. As RNA
polymerase (pol) III transcription products are required for protein synthesis, RNA
pol III transcriptional activity is tightly linked to the rate of cell growth. High levels
of RNA pol III transcription products are seen in transformed and tumor cells,
suggesting that aberrant regulation of RNA pol III transcription contributes to
transformation and tumorigenesis. The tumor suppressor p53 represses RNA pol III
transcription by directly targeting the RNA pol III transcription factor complex
TFIIIB which consists of TBP, Brf1, and Bdp1. TBP and Brf1 associate to form a
stable complex while Bdp1 associates reversibly with the complex. Our studies
determined that p53 mediates repression of RNA pol III transcription by directly
targeting TBP which is limiting for RNA pol III transcription in the presence but not
the absence of p53 expression. In contrast, Brf1 is not limiting for RNA pol III
transcription. Although p53 binds to the TBP/Brf1 complex, it does not disrupt the
complex. As p53 mediates repression of RNA pol III transcription, we determined
whether another tumor suppressor, PTEN, represses RNA pol III transcription.
Whereas p53 is a transcription factor that functions in the nucleus, PTEN is a lipid
phosphatase that functions largely in the cytoplasm. The lipid phosphatase activity
of PTEN directly antagonizes the PI-3 kinase (PI3K) signal transduction pathway.
We determined that PTEN mediates repression of RNA pol III transcription via its
lipid phosphatase activity. We also determined that the PI3K pathway regulates
vii
RNA pol III transcription; therefore, we suggest that antagonism of the PI3K
pathway is one mechanism by which PTEN mediates repression of RNA pol III
transcription. We also determined the effect of PTEN on TFIIIB. PTEN was found
to disrupt the TBP/Brf1 complex with this disruption correlating with a PTEN-
mediated decrease in Brf1 serine phosphorylation. PTEN was also found to decrease
the occupancy of TFIIIB on a tRNA gene. We suggest that the PTEN-mediated
decrease in Brf1 serine phosphorylation leads to disruption of the TBP/Brf1 complex
which in turn leads to a decrease in TFIIIB occupancy on a tRNA gene.
1
Introduction: RNA polymerase III, Transcription Factor
Complexes, and Promoter Structure
RNA polymerase III and its transcription factor complexes
Human RNA polymerase (pol) III is the largest RNA pol and consists of 17 subunits,
5 of which (hRPC25, 15, 14, 10, and 8) are shared between the 3 RNA polymerases.
Each shared subunit is transcribed from 1 gene and incorporated into the 3 RNA
polymerases (44). Six subunits are unique to RNA pol III with 3 of them (hRPC62,
39, and 32) forming a subcomplex that can be dissociated from the RNA pol III core
(44, 104). The enzyme lacking these 3 subunits functions in elongation and
termination but does not show promoter-dependent initiation. The hRPC39 subunit
was found to interact with the TATA-binding protein (TBP) and TFIIB-related factor
1 (Brf1), 2 components of the 3 subunit transcription factor IIIB (TFIIIB), suggesting
that the 3 subunit subcomplex directs RNA pol III binding to the TFIIIB-DNA
complex (104).
TFIIIB consists of a stable TBP/Brf1 complex that associates reversibly with Bdp1
(103). TBP and Brf1 associate directly, with the TFIIB-related N-terminus [amino
acids (aa) 1-300] of Brf1 weakly associating with TBP while the C-terminus (aa 281-
675) interacts strongly with TBP (103).
2
Transcription factor IIIC (TFIIIC) was thought to consist of 2 multi-subunit
complexes — TFIIIC1 and TFIIIC2; however, it is now known that TFIIIC1 is Bdp1
(106). TFIIIC is a stable complex of 5 subunits that are 220, 110, 102, 90, and 63
kilo Daltons (kDa) in mass. TFIIIC220, 110, and 90 contain histone
acetyltransferase activity with TFIIIC90 specifically acetylating K14 on histone H3
(50, 41, 40).
Promoter structure and transcription factor complex recruitment
RNA pol III transcription products are small untranslated RNAs that include 5S
rRNA, tRNAs, U6 small nuclear (sn)RNA, and 7SL RNA. RNA pol III genes have
been grouped into 3 main types based on the location and/or type of elements that
make up their promoters (115). There are also a number of RNA pol III genes whose
promoters do not fit into the 3 main categories. These various promoter structures
allow for divergent pathways of transcription factor recruitment; however, the end
result is always the recruitment of TFIIIB and subsequently RNA pol III to the
transcription start site.
A type 1 promoter, of which the 5S rRNA gene is the only example, consists of 3
internal elements that are required for efficient transcription. Together these
elements constitute the internal control region (ICR). Altering the spacing between
the elements greatly reduces transcription (75). The transcription factor IIIA
3
(TFIIIA) binds to the ICR via protein-DNA interactions. TFIIIA, a single
polypeptide that contains 9 zinc fingers, is only required for the transcription of type
1 genes and serves as a specificity factor for the recruitment of TFIIIC to the 5S
rRNA promoter (107, 69). TFIIIC then serves as an assembly factor for TFIIIB.
The tandemly repeated tRNA genes have type 2 promoters that consist of the highly
conserved internal elements, the A and B blocks, with the A block located at
approximately +10 to +20 from the transcription start site (115). In contrast to type 1
promoters, the spacing between the A and B blocks is variable with optimal spacing
of the B block being ~ 30 to 60 base pairs (bp) downstream of the A block (115).
For assembly of the transcription initiation complex, TFIIIC is first recruited to the B
block via protein DNA interactions (3, 19). In the absence of TFIIIC, TFIIIB cannot
stably associate with the tRNA
Arg
gene; therefore, as with type 1 promoters, TFIIIC
serves as an assembly factor for TFIIIB which then recruits RNA pol III to the
promoter (19).
In contrast to type 1 and 2 promoters, type 3 promoters do not contain internal
elements. The best characterized type 3 promoter is that of a human U6 snRNA
gene. There is a TATA box between -25 and -30 to which TBP binds (36, 75). TBP
loosely associates with the alternatively spliced Brf1 variant — Brf2 — and Bdp1 to
make up TFIIIB which is present at the U6 snRNA promoter (82, 67). The
4
transcription factor complex snRNA activating protein complex (SNAP
C
) is also
involved in the transcription of type 3 promoters.
RNA polymerase III transcription is regulated by the cell cycle
Mitosis
All nuclear transcription is repressed during mitosis with RNA synthesis stopping by
mid-prophase in mammalian cells (76). RNA pol III transcription is repressed
during mitosis, remains low in early G1 phase, gradually increases as G1 progresses,
reaches a peak in late G1, and remains high in S and G2 phases (110, 84).
Repression of transcription is not due to chromatin condensation as a topoisomerase
II inhibitor does not relieve repression (34). However, TFIIIB, which is known to be
targeted for regulation, strongly stimulates transcription when added to mitotic
extracts derived from HeLa cells (111).
Although TBP was found to be hyperphosphorylated during mitosis, this
phosphorylation did not correlate with a reduction in RNA pol III transcription and
addition of TBP to mitotic extracts did not increase transcription (111). In contrast,
addition of Brf1 and Bdp1 increased RNA pol III transcription when added to mitotic
extracts (22, 42). Brf1 is hyperphosphorylated during mitosis by one or more
kinases that become active at metaphase and do not include the mitotic Cdc2/cyclin
B complex (22). Bdp1 is phosphorylated by casein kinase 2 (CK2) during mitosis
5
(42). Protein phosphatase 2A (PP2A) antagonizes the inhibitory effect of the mitotic
kinases (22).

Phosphorylation of the TFIIIB components differentially affects their promoter
occupancy. The occupancy of hyperphosphorylated TBP decreases ~ 20% on 5S
rRNA and tRNA genes, while a greater decrease (~ 50%) is seen on the U6 snRNA
gene (22, 42). Phosphorylated Brf1 occupancy does not decrease appreciably
whereas the occupancy of phosphorylated Bdp1 decreases substantially on 5S rRNA,
tRNA, and U6 snRNA promoters and on chromatin in general (22, 42). In
accordance with the decrease in Bdp1 promoter occupancy, the interaction between
Brf1 and Bdp1 was found to decrease during mitosis (22). The occupancy of RNA
pol III mirrors that of phosphorylated Bdp1 and as such is substantially reduced
during mitosis (22).
Interphase
Following mitosis, RNA pol III transcription remains at low levels during early G1
with TFIIIB increasing transcription when added to extracts derived from HeLa cells
synchronized in early G1 phase (110). TFIIIB is not limiting during S and G2
phases when RNA pol III transcription reaches peak levels (110). TBP protein levels
were found to not change appreciably throughout the cell cycle (110) and the
hyperphosphorylation seen in mitosis is no longer present in early G1. Thus,
6
regulation of TBP does not seem to be the cause of differential regulation of RNA
pol III transcription during the cell cycle (110).
The tumor suppressor retinoblastoma protein (Rb), which binds and
regulates/represses a number of transcription factors when hypophosphorylated, was
found to bind Brf1 during G0 and early G1 with binding decreasing with progression
through G1 (84). When bound by Rb, Brf1/TFIIIB is unable to bind to TFIIIC or
RNA pol III and thus there is no recruitment of a transcription competent initiation
complex (91). Rb function is regulated by phosphorylation by the cyclin D/Cdk4,
cyclin D/Cdk6, and cyclin E/Cdk2 complexes that become active in late G1 (118,
57). Rb remains hyperphosphorylated throughout S, G2, and M phases after which it
is dephosphorylated by protein phosphatase 1 (70). Therefore, the rate of RNA pol
III transcription parallels that of Rb phosphorylation (84).

RNA polymerase III transcription is regulated by tumor
suppressors and oncogenes
RNA pol III transcription is tightly regulated by both tumor suppressors and
oncogenes. The importance of limiting RNA pol III transcription products is
evidenced by the fact that 2 fundamental tumor suppressors, Rb and p53, repress
RNA pol III transcription. Rapid growth, a hallmark of tumor cells, requires high
levels of RNA pol III transcription products with the highest levels of RNA pol III
7
transcription products correlating with a tumorigenic phenotype (83, 113). A study
analyzing 80 tumor samples (representing 19 types of cancer) found that 7SL RNA,
a component of the signal recognition particle involved in protein trafficking, was
overexpressed in all instances relative to adjacent normal tissue (9). In another
study, tRNA, 5S rRNA, and 7SL RNA were found to be overexpressed in human
ovarian cancers (116). A mechanism by which overexpression of RNA pol III
transcription products in tumor cells was found to occur is through an increase in
RNA pol III transcription factors. Reverse transcriptase-polymerase chain reaction
(RT-PCR) found that mRNAs encoding all 5 subunits of TFIIIC were elevated in
ovarian carcinomas (116).
Rb
Primary fibroblasts derived from Rb null mice have elevated levels of RNA pol III
transcription (114). Rb contains a pocket domain that is required for its function as a
tumor suppressor. Rb-mediated repression of RNA pol III transcription is alleviated
by mutations in its pocket domain that occur naturally in tumors (114). The pocket
domain binds a number of Rb target proteins including the E2F family of
transcription factors and, as discussed above, Brf1 (84, 91, 14). The pocket domain
of Rb also binds to TFIIIC with addition of TFIIIC to in vitro transcription assays
alleviating Rb-mediated repression of RNA pol III transcription (14). Rb has also
been shown to directly repress RNA pol III transcription of U6 snRNA by
8
associating with SNAP
C
possibly through direct protein-protein interactions (37).
The closely related pocket proteins p107 and p130 also repress RNA pol III
transcription in a cell cycle dependent manner by stably associating with Brf1 (92).
p53
The tumor suppressor p53 is mutated or lost in greater than 50% of all human
cancers and its inactivation is an important step towards tumor formation (38). As
with Rb, primary fibroblasts derived from p53 null mice have elevated levels of
RNA pol III transcription (5). Endogenous p53 was found to associate with TFIIIB,
and p53 was shown to be a general repressor of RNA pol III transcription by
targeting TFIIIB (5).
c-Myc
c-Myc is a well established oncoprotein that stimulates rapid increases in translation
and cell growth which precede DNA replication and cell division (81, 17). c-Myc
directly targets RNA pol II transcribed genes involved in cell growth for repression
or activation by binding to their promoter regions and/or transcription factors present
at these promoter regions (17). c-Myc was also found to stimulate cell growth
through the activation of RNA pol III transcription. Comparison of RNA pol III
transcription in c-Myc knockout and wild-type fibroblasts showed ~ sevenfold lower
transcript production in the knockout cells (30). The transactivation domain of c-
9
Myc fused to GST was found to bind TBP and Brf1, and co-immunoprecipitation
analysis found that endogenous c-Myc associated with Brf1 (30). Chromatin
immunoprecipitation analysis showed that c-Myc is present at the 5S rRNA,
tRNA
Tyr
, tRNA
Leu
, and tRNA
Arg
promoters (30, 26). Therefore, c-Myc directly
activates RNA pol III transcription by binding TFIIIB (30).
CK2 and ERK
CK2, a ubiquitously expressed serine/threonine kinase that is active in the nucleus
and the cytoplasm, both activates and represses RNA pol III transcription in a cell
cycle specific manner (43, 42, 47). CK2 phosphorylation of RNA pol III is required
for transcription, with inhibition of CK2 in S phase extracts inhibiting transcription,
whereas CK2 phosphorylation of Bdp1 during mitosis represses transcription (43,
42). CK2 was also found to phosphorylate Brf1 in proliferating cells and thereby
promote the interaction between TFIIIB and TFIIIC (47). The mitogen-activated
protein (MAP) kinase ERK also phosphorylates Brf1; however it does so
independently of the cell cycle (25). ERK phosphorylation of Brf1 increases the
association of TFIIIB with TFIIIC and with RNA pol III as well (47, 25). Blocking
ERK activity in vivo only partially reduces Brf1 phosphorylation and therefore it is
likely that Brf1 phosphorylation by both CK2 and ERK is required for maximal
RNA pol III transcription (47, 25).
10
Ras
The Ras proteins function to convey mitogen induced activation of growth factor
receptor protein tyrosine kinases from the membrane to the nucleus via a cascade of
signal transduction pathways involving serine/threonine kinases that include the
MAP kinases (48). Ras is known to directly interact with 3 effector protein families
— Raf, RalGDS and PI3K — to regulate signal transduction (48). Activated Ras
increases RNA pol III transcription by increasing TBP protein levels through
stimulation of the TBP promoter via Raf and RalGDS signaling (101, 46). The MAP
kinase kinase MEK is required for this induction of TBP promoter activity (46).
PTEN
PTEN is a lipid phosphatase that antagonizes the PI-3 kinase signal
transduction pathway
The tumor suppressor PTEN (phosphatase and tensin homolog deleted on
chromosome 10) is a lipid phosphatase that dephosphorylates the 3-position of the
inositol ring of phosphatidylinositol (3,4,5) phosphate [PI(3,4,5)P
3
]. This
phosphatase activity directly antagonizes the phosphatidylinositol-3 kinase (PI3K)
signal transduction pathway and is required for PTENs function as a tumor
suppressor (71, 60, 117). The PI3K signal transduction pathway, which is stimulated
in response to growth factors, regulates cell growth, proliferation, and survival (7,
11
99). PI3K phosphorylates the membrane lipid phosphatidylinositol (4,5) phosphate
[PI(4,5)P
2
], with this phosphorylation leading to activation of the serine/threonine
kinase Akt which in turn leads to activation of the mTOR kinase (35). Due to its
regulation of protein synthesis, mTOR is a key regulator of cell growth and
proliferation (35).
PTEN, which was first cloned as a tumor suppressor for gliomas, is mutated in ~
50% of glioblastomas, endometrial and prostate carcinomas, and melanomas (88,
59). It is also mutated in leukemias and lymphomas, and breast carcinomas (2). The
genomic locus of PTEN — chromosome 10q23 — undergoes loss of heterozygosity
in many human tumors (59).
PTEN, which is 403 amino acids in length, has an N-terminal phosphatase domain
and a C-terminal C2 domain. (The C2 domain of proteins mediates their binding to
membranes.) The phosphatase domain contains the signature motif — HCXXGXXR
— found in the active sites of protein tyrosine phosphatases and dual-specificity
serine/tyrosine phosphatases, and PTEN was found to dephosphorylate serine,
tyrosine, and threonine residues in vitro (72). However, PTEN has little sequence
homology to protein phosphatases outside of its phosphatase domain, and the
biological consequences of its putative protein phosphatase activity have yet to be
elucidated (29).
12
PTEN is referred to as an interfacial enzyme because it acts at the interface between
the membrane and the aqueous phase of the cytosol and it is thought to have distinct
membrane binding and substrate binding steps (29, 66). [PTEN is a “hopping
enzyme” in that it is readily released from the membrane (66)]. An important means
of regulating PTENs activity is by affecting its association with the plasma
membrane (18). The C-terminal C2 domain of PTEN and its phosphatase domain,
which associate across an extensive interface that is adjacent to the phosphatase
active site, contribute to membrane binding (18, 51). The inner layer of the plasma
membrane contains an ~ 100 to 1000 fold excess of PI(4,5)P
2
over PI(3,4,5)P
3
.
PTEN contains an N-terminal PI(4,5)P
2
binding site, and the binding of PTEN to
PI(4,5)P
2
is thought to productively orient PTENs active site to its substrate PIP
3
(66). Another group found that PI(4,5)P
2
increased the hydrolysis of PI(3,4,5)P
3
by
PTEN in the absence of membrane structures and proposed a positive feedback loop
in which PI(4,5)P
2
activates PTEN through an allosteric conformational change (6).
The phosphatase active site pocket of PTEN is larger than that of protein tyrosine
phosphatases and dual-specificity phosphatases, and the residues responsible for this
increase in size are conserved in PTEN homologs across species (51). The active
site pocket, which has the signature motif — HCKAGKGR (residues 123-130) — at
the bottom, is both wider and deeper than that of protein phosphatases and is
consistent with the larger size of PI(3,4,5)P
3
. The mutant PTENG129E, which is
13
defective for lipid phosphatase activity but still catalyzes tyrosine dephosphorylation,
illustrates the importance of the larger pocket for lipid phosphatase activity. The
glycine to glutamate mutation decreases the size of the active site pocket (51). The 2
positively charged lysine residues, K125 and K128, which are not found in protein
phosphatases, interact electrostatically with the negatively charged phosphates
groups of PI(3,4,5)P
3
and productively orient the D-3 phosphate in the active site
(66)
.
Regulation of PTEN activity
CK2 phosphorylation of serine 380, threonine 382, and threonine 383 in PTENs C-
terminal 50 amino acid “tail” inhibits PTENs phosphatase activity (93, 98). The last
3 amino acids of PTEN (401-403) constitute a PDZ binding domain that can interact
with PDZ domain-containing proteins that serve as scaffolds to assemble membrane-
localized multiprotein complexes (23). Phosphorylation of the PTEN tail results in a
“closed” more stable conformation in which the PDZ binding domain is masked
(97). Phosphorylation of PTENs tail also interferes with membrane targeting by
interfering with electrostatic interactions between PTEN and the membrane (18). A
negative feedback loop has been proposed in which D3-phosphorylated inositol
lipids promote serine 380 phosphorylation of PTEN thereby inhibiting its lipid
phosphatase activity (2).
14
Nuclear localization of PTEN
Although originally thought to be a strictly cytoplasmic protein due to its antagonism
of the PI3K signal transduction pathway, PTEN is now known to be present in the
nucleus. Components of the PI3K signal transduction pathway, PI3K, PDK1,
activated Akt, and the phosphatidylinositol phosphate PI(4,5)P
2
and PI(3,4,5)P
3
substrates of PI3K and PTEN respectively, have been determined to be in the
nucleus as well (55). It is interesting to note that the loss of nuclear PTEN and an
increase in nuclear activated Akt correlates with increased tumorigenicity (53). A
nuclear function of PTEN that is not dependent on its lipid phosphatase activity is
that of maintaining chromosomal integrity (86). The mechanism of PTEN transport
into the nucleus is currently under study. PTEN has been found to associate with the
major vault protein and vault particles may transport PTEN into the nucleus (15),
and PTEN has also been found to enter the nucleus by passive diffusion (55). PTEN
nuclear import is also mediated by ubiquitination (95).
PTEN and the cell cycle
PTEN has been shown to arrest the cell cycle in the G1 phase. In a study performed
using PTEN expressing MCF-7 cells engineered to overexpress PTEN in the absence
of tetracycline, a reduction in the cell number of PTEN overexpressing cells relative
to non-PTEN overexpressing cells was first seen 48 hours following PTEN induction
with cell cycle arrest in G1 first seen 36 hours following PTEN induction (105). G1
15
arrest is seen in PTEN null 786-O renal carcinoma cells 40 hours following transient
transfection with a vector encoding PTEN (79). An increase in G1 arrested cells is
seen in PTEN null U87 cells 48 hours following infection with a retroviral vector
expressing PTEN (52). PTEN null endometrial cancer cells show G1 arrest 48 hours
following infection with an adenoviral vector expressing PTEN and a concomitant
greater than 100 fold increase in p27 expression (63).
PTEN mediates cell cycle arrest via its antagonism of the PI3K pathway (90, 52, 79,
105). Lipid phosphatase defective PTEN does not cause cell cycle arrest and
constitutively active Akt overcomes G1 arrest (31, 52, 79, 105). PTEN increases the
protein stability of the cyclin dependent kinase (CDK) inhibitor p27 which is
required for PTEN-mediated cell cycle arrest (31, 52, 61). The protein stability of
p27 is regulated by an ubiquitin E3 ligase complex. Inhibition of the PI3K pathway
independently of PTEN and expression of PTEN in PTEN null cells increases p27
protein levels by decreasing p27 ubiquitination (52, 61). PTEN expression greatly
reduces the mRNA and protein levels of a subunit (SKP2) of the E3 ligase complex
and thereby reduces p27 ubiquitin-mediated protein degradation (61). Inhibition of
the PI3K pathway leads to the activation of p27 gene transcription by the Forkhead
transcription factors which are phosphorylated and inactivated by Akt (68, 73).
PTEN also affects the cell cycle by decreasing both the protein level and nuclear
localization of cyclin D1 (74, 77). Inhibition of the PI3K signaling pathway by
16
PTEN mediates this effect as phosphatase defective PTEN does not affect cyclin D1
levels (77). Phosphorylation of cyclin D1 on threonine 286 by glycogen synthase
kinase-3 (GSK-3), which is phosphorylated and inactivated by Akt, leads to the
export of cyclin D1 from the nucleus to the cytoplasm and subsequent proteasomal
degradation (20). Reduction of cyclin D1 leads to a reduction in phosphorylation of
Rb on cyclin D/CDK4 specific sites (77). PTEN also decreases Rb phosphorylation
by increasing the recruitment of p27 into cyclin E/CDK2 complexes (12). It is
interesting to note that PTEN does not cause cell cycle arrest in Rb deficient cells
(74).
The tumor suppressors p53 and PTEN enhance each others activity
Antagonism of the PI3K signal transduction pathway by PTEN increases the
transcriptional activity and protein level of p53. Phosphorylated Akt phosphorylates
Mdm2 on serines 166 and 186 which leads to the nuclear translocation of Mdm2 and
subsequent binding to p300 (65, 122). This binding provides a platform for the
assembly of an Mdm2-p300-p53 complex and leads to the ubiquitination and
degradation of p53 (122). Inhibition of Akt phosphorylation by PTEN is a
phosphatase-dependent mechanism of regulating p53 function. It has also been
determined that ectopic expression of phosphatase-dead PTEN induces a significant
increase in p53 protein levels, and that PTEN can stabilize p53 in the absence of
Mdm2 (28). These findings suggest that PTEN can regulate p53 activity in a
17
phosphatase- and Mdm2-independent manner. In support of this, PTEN was found
to associate with p53 and regulate its transcriptional activity by modulating its DNA
binding (28).
A p53 binding site has been identified within the PTEN promoter at -1157 to -1190.
This site is required for p53 activation of the PTEN gene, and p53 induction leads to
an increase in PTEN mRNA and protein (87). Basal levels of PTEN transcription
are regulated by elements outside of the p53 binding element (87). Thus a positive
feedback loop is established in which p53 induction enhances PTEN expression
which then serves to protect p53 from degradation (94).
18
Chapter 1: p53 represses RNA polymerase III transcription by
targeting TBP
p53 selectively represses transcription from a modified tRNA
Arg
gene
p53 has been shown to be a general repressor of RNA pol III transcription (5). To
determine the mechanism by which p53 mediates repression of RNA pol III
transcription we obtained p53 null H1299 cells that are engineered to express p53
from a stably integrated gene in the absence of tetracycline (11). [p53 expression is
approximately 6 times greater in the absence of tetracycline than in the presence of
tetracycline (Fig. 3B).] These cells, which are derived from a human lung
carcinoma, were transiently transfected with the pArg-maxi plasmid that encodes a
modified tRNA
Arg
gene containing a 12 base pair (bp) insert between the A and B
boxes that allows for differentiation from endogenous tRNAs (21). The transfected
cells were grown in the absence and presence of tetracycline and total RNA was
isolated 48 hours following transfection. Ribonuclease protection assays (RPAs)
were performed to quantify the amount of pArg-maxi transcribed in the absence and
presence of p53 induction. An autoradiograph resulting from an RPA is shown in
figure 1A. The arrow denotes the 83 nucleotide fragment of the probe that is
protected from RNase degradation, and the bracket denotes endogenous tRNAs that
are cleaved by RNase. H1299 cells were also transiently transfected
with a plasmid containing a large portion of the RNA pol II transcribed TBP
promoter preceding a luciferase gene (p4500/+66TBP-luc) (27). p53 selectively
19
represses RNA pol III transcription by nearly 70% whereas RNA pol II transcription
of the TBP promoter is not affected (Fig. 1B).
Fig. 1. p53 selectively represses transcription from a modified tRNA
Arg
gene. (A) Induction of p53
expression decreases transcription of a tRNA
Arg
gene. H1299 cells were transiently transfected with 2 μg of
pArg-maxi (a tRNA
Arg
gene reporter) in the absence or presence of p53 induction (presence or absence of
tetracycline). Forty eight hours following transfection, total RNA was isolated and ribonuclease protection
assays (RPAs) were performed (as described in ‘Materials and Methods’) to determine the amount of pArg-maxi
transcribed. A resultant autoradiograph is shown. The arrow designates the 83 nucleotide transcript transcribed
from pArg-maxi, and the bracket denotes the half-size tRNA molecules generated by the cleavage of endogenous
tRNAs that hybridized with the probe. (B) p53 selectively represses RNA polymerase III transcription. H1299
cells were transiently transfected with 2 μg of pArg-maxi in the absence or presence of p53 induction, total RNA
was isolated and RPAs were performed. H1299 cells were also transiently transfected with 5 μg of
p4500/+66TBP-luc (a luciferase reporter containing the TBP promoter) in the absence or presence of p53
induction, total protein was isolated and luciferase assays were performed (as described in ‘Materials and
Methods’) to determine TBP promoter activity. The results of at least 3 independent determinations were
quantified and the mean ± standard deviation (SD) was graphed.
p53
– +
0
0.5
1.0
1.5
– p53
+
p53
pArg–maxi p–4500/+66 hTBP tRNA gene or TBP promoter activity
Fold change
A
B
20
Overexpression of TBP alleviates p53-mediated repression of RNA polymerase
III transcription
We next sought to determine which of the RNA pol III transcription factors are
targeted by p53. The TFIIIB transcription factor complex, which is comprised of
TBP, Brf1, and Bdp1, has been shown to be targeted for regulation by p53 (5). We
determined whether the TBP and Brf1 components of TFIIIB are targets of p53.
RPAs were performed using RNA isolated from H1299 cells transiently
cotransfected with pArg-maxi and a plasmid encoding TBP [pLTR-E2TBP, (45)]
and grown in the absence and presence of tetracycline. Overexpression of TBP
alleviates p53-mediated repression of RNA pol III transcription (Fig. 2A). TBP is
limiting for RNA pol III transcription in the presence, but not the absence of,
induction of p53 expression. Western blot analysis demonstrates equal expression of
ectopic TBP in the absence and presence of p53 expression (Fig. 2A). To determine
whether the rescue of RNA pol III transcription by TBP was a direct effect of TBP
overexpression or an indirect effect of TBP regulation of RNA pol II transcription, a
plasmid encoding a mutant TBP (TBPE284R) [pLTR-E2TBP-E284R, (45)] that is
selectively defective for RNA pol II transcription was used. This mutant was able to
alleviate p53-mediated repression of RNA pol III transcription (Fig. 2A) suggesting
that TBP is a direct target of p53. These results are in agreement with studies
showing direct interaction between p53 and TBP (85, 56, 8, 39, 96, 24).
21
In contrast to TBP, Brf1 does not alleviate p53-mediated repression of RNA pol III
transcription. Transient transfection of increasing amounts of a Brf1 expression
plasmid [p2HABrf1 (91)] does not increase transcription of pArg-maxi (Fig. 2B).
Western blot analysis demonstrates ectopic expression of Brf1 (Fig. 2B). These
results suggest that p53 directly targets TBP, but not Brf1, to mediate repression of
RNA pol III transcription.
22
Fig. 2. Overexpression of TBP alleviates p53-mediated repression of RNA polymerase III transcription.
(A) TBP overexpression increases tRNA gene transcription in cells induced to express p53 but not in non-
induced cells. H1299 cells were transiently cotransfected with 2 μg of pArg-maxi and either 0.2 to 1 μg of the
TBP expression construct pLTR-E2TBP or 1 μg of the RNA pol II defective mutant TBP expression construct
pLTR-E2TBP-E284R in the absence or presence of p53 induction. Following transfection, total RNA was
isolated and RPAs were performed. The percent change in tRNA
Arg
transcription was calculated relative to the
level of tRNA
Arg
transcription in the absence of p53 induction which was set to 1. The results of at least 3
independent determinations were quantified and the mean ± SD was graphed. For the immunoblot analysis,
H1299 cells were transiently transfected with 1 μg of pLTR-E2TBP or the corresponding empty vector in the
absence and presence of p53 induction. Following transfection, total protein was isolated and immunoblot
analysis was performed as described in ‘Materials and Methods’ using an antibody directed against TBP. The
ectopically expressed HA-TBP and the endogenous TBP are indicated. (B) Brf1 overexpression does not
alleviate p53-mediated repression of tRNA gene transcription. H1299 cells were transiently cotransfected with 2
μg of pArg-maxi and 0.1, 0.3, or 1 μg of the Brf1 expression construct containing a double HA tag (p2HABrf1)
in the absence or presence of p53 induction. Following transfection, total RNA was isolated and RPAs were
performed. The results of at least 3 independent determinations were quantified and the mean ± SD was graphed.
For the immunoblot analysis, H1299 cells were transiently transfected with 1 μg of p2HABrf1 or the
corresponding empty vector in the absence or presence of p53 induction. Following transfection, total protein
was isolated and immunoblot analysis was performed using an antibody directed against Brf1. The ectopically
expressed HA-Brf1 and the endogenous Brf1 are indicated.
TBP ––
tRNA gene activity
Fold change
0
0.5
1.0
1.5
+ +
TBP-E284R ––
– p53
+
p53
Brf1 – –
HA–Brf1
HA–Brf1
Brf1
+ –
tRNA gene activity
Fold change
0
0.5
1.0
1.5
– p53
+
p53
+
+
+
+
–
–
–
–
p53
HA-TBP
HA-TBP
TBP
A
B
23
p53 does not disrupt TBP/Brf1 binding
TBP and Brf1 associate directly, with the TFIIB-related N-terminus (aa 1-300) of
Brf1 weakly associating with TBP while the C-terminus (aa 281-675) interacts
strongly with TBP (103). Using coimmunoprecipitation assays, we determined
whether p53 disrupts the association between TBP and Brf1. p53 expression was
induced in H1299 cells for 0, 2, or 6 days and protein lysates were prepared.
Antibodies against TBP and Brf1 were used to immunoprecipitate these proteins
from total protein lysates. Western blot analysis was then performed using the same
antibodies. p53 does not disrupt the association between TBP and Brf1 (Fig 3A).
There is no difference in the amount of Brf1 coimmunoprecipitated with TBP in the
absence and presence of p53 induction (Fig. 3A). Conversely, no difference is seen
in the amount of TBP coimmunoprecipitated with Brf1 (Fig. 3A). Although p53
does not disrupt the TBP/Brf1 complex, it associates with the complex. Thus, p53
coimmunoprecipitates with both TBP and Brf1 (Fig. 3B). Direct immunoblot
analysis (Fig. 3B) shows p53 expression to be approximately 6 times greater in the
absence of tetracycline as compared to the presence of tetracycline. These results
suggest that although p53 associates with the TBP/Brf1 complex it does not disrupt
the interaction between these two proteins.
We have determined that p53 selectively represses RNA pol III transcription by
directly targeting TBP. Overexpression of TBP alleviates p53-mediated repression
24
of RNA pol III transcription with TBP being limiting for RNA pol III transcription in
the presence but not the absence of p53. Brf1 overexpression does not alleviate
repression suggesting that p53 specifically targets TBP. Although p53 associates
with the TBP/Brf1 complex it does not decrease the association between them.
Fig. 3. p53 does not disrupt TBP/Brf1 binding. (A) TBP and Brf1 co-immunoprecipitate both in the absence
and presence of p53 induction. Protein lysates were prepared from H1299 cells that were induced to express p53
for 0, 2, or 6 days. Co-immunoprecipitation assays were then performed as described in ‘Materials and Methods’
using 400 μg of protein and antibodies directed against TBP and Brf1. Mock controls designate assays in which
no antibodies were used. Immunoblot analysis was performed using antibodies directed against TBP and Brf1.
Input designates protein lysates (40 μg) that were directly subjected to immunoblot analysis. (B) p53 associates
with TBP-Brf1 complexes. Protein lysates were prepared from non-induced H1299 cells and cells induced to
express p53. Co-immunoprecipitation assays were performed using 50 μg of protein and antibodies directed TBP
and Brf1. Immunoblot analysis was performed using antibodies directed against p53. Input designates protein
lysates (10 μg) that were directly subjected to immunoblot analysis. The p53 band was determined by using
recombinant p53 as a standard. CRM designates bands that represent non-specific cross-reacting material that is
immunoprecipitated with IgG.
A
B
IP
TBP
IP
Brf1
CRM
++ +
25
Chapter 2: PTEN represses RNA pol III transcription via it lipid
phosphatase activity
PTEN represses RNA polymerase III transcription via its lipid phosphatase
activity and independently of p53
Previous work performed in our lab determined that the tumor suppressor p53
represses RNA pol III transcription by directly targeting TBP and limiting promoter
occupancy of the TFIIIB transcription factor complex (16). As PTEN is a tumor
suppressor that directly antagonizes the PI3K signal transduction pathway, which is
stimulated in response to growth factors, we determined whether PTEN could also
repress RNA pol III transcription. We first determined whether overexpression of
PTEN in cells lacking endogenous PTEN protein could repress RNA pol III
transcription. As p53 and PTEN enhance each others function (28, 87) we used both
cells expressing endogenous p53 and cells lacking endogenous p53 to determine
whether PTEN could repress RNA pol III transcription in the absence of p53. The
glioblastoma-derived cells lines A172 (p53 /) and U87 (p53 +/+) were transiently
transfected with an RNA pol III reporter plasmid (pArg-maxi) encoding a modified
tRNA
Arg
gene containing a 12 bp insert between the A and B boxes that allows for
distinction from endogenous tRNAs (21). Expression plasmids for PTEN or a
mutant PTEN G129E (98) that lacks lipid phosphatase activity were transiently co-
transfected with pArg-maxi. To quantify the amount of pArg-maxi transcribed in the
absence of PTEN and in the presence of PTEN or lipid phosphatase defective PTEN,
ribonuclease protection assays (RPAs) were performed using total RNA isolated
26
from the cells 24 hours following transfection. As shown in figure 4A, expression of
PTEN but not PTEN G129E reduced RNA pol III transcription by more than half in
both the A172 and U87 cell lines. The results of at least 3 RPAs are shown
graphically with a representative autoradiograph. Western blot analysis (performed
by Dr. Cheng Zhang) confirms robust expression from the PTEN and PTEN G129E
expression plasmids (Fig. 4B). The data show that ectopic expression of PTEN in
PTEN null cells represses transcription from pArg-maxi by more than half.
Transcription is equally repressed in the p53 null A172 cells as compared to the p53
expressing U87 cells suggesting that repression of RNA pol III transcription by
PTEN does not require p53. In contrast however, the lipid phosphatase activity of
PTEN is required for repression of pArg-maxi transcription. The PTEN mutant
G129E, which lacks lipid phosphatase activity due to the diminished size of the
active site pocket (51), is unable to repress RNA pol III transcription.
27
Fig. 4. PTEN represses RNA polymerase III transcription via its lipid phosphatase activity and
independently of p53. (A) Ectopic PTEN expression in PTEN null cell lines decreases RNA pol III
transcription. Glioblastoma A172 (p53 /) and U87 (p53 +/+) cell lines were transiently cotransfected with 2
μg of pArg-maxi (a tRNA
Arg
gene reporter) and with 4 μg of expression vector for PTEN or PTEN G129E, a
lipid phosphatase defective mutant. Twenty four hours following transfection, total RNA was isolated and
ribonuclease protection assays (RPAs) were performed (as described in Materials and Methods) to determine the
amount of pArg-maxi transcribed. The resultant autoradiographs were scanned and quantified by densitometry.
The results of at least 3 independent determinations were quantified and the mean ± standard deviation (SD) was
graphed. A representative autoradiograph is shown above each graph. (B) Expression from PTEN vectors.
Immunoblot analysis shows expression from transiently transfected PTEN and PTENG129E expression vectors
in U87 cells. Cells were transfected as in (A), total protein was isolated, and 100 μg of protein was subjected to
SDS-PAGE. Immunoblot analysis was performed using an antibody that recognizes both PTEN and PTEN
G129E. Further immunoblot analysis was performed on the same membrane using an antibody against actin.
U87
PTEN
PTEN G129E
PTEN
Control
actin
p53 –/– p53 +/+
A172 U87
tRNA gene activity
Fold change
PTEN
G129E
0
0.5
1.0
1.5
tRNA gene activity
Fold change
PTEN
0
0.5
1.0
1.5
Control PTEN
PTEN
G129E Control
A
B
28
PTEN selectively represses endogenous RNA polymerase III transcription
Further confirming the requirement of the lipid phosphatase activity of PTEN for the
repression of RNA pol III transcription is work performed using U87 cells
engineered to express PTEN or phosphatase defective PTEN C124S in the presence
of doxycycline (77). Western blot analysis (performed by Dr. Cheng Zhang)
demonstrates the complete absence of PTEN expression in the absence of
doxycycline, an increase in PTEN expression following 6 hours of doxycycline
induction, and robust PTEN expression following 24 hours of doxycycline induction
(Fig. 5A.) Consistent with the results shown in figure 4A, RPA analysis
demonstrates that repression of RNA pol III transcription is seen with the induction
of PTEN but not phosphatase defective PTEN C124S (Fig. 5B). Paralleling the
induction of PTEN expression demonstrated by immunoblot analysis, a reduction in
RNA pol III transcription is seen following 6 hours of PTEN induction, with a
greater decrease seen following 24 hours of PTEN induction (Fig. 5B).
The results discussed so far have determined the level of transcription from a
transiently transfected RNA pol III reporter plasmid in the absence and presence of
wild type or mutant PTEN expression. We next determined the effect of PTEN on
the transcription of the endogenous RNA pol III genes, 7SL, tRNA
Tyr
, and tRNA
Leu
.
The 7SL gene, which contains both intragenic and extragenic promoter elements,
does not fall into the 3 main categories of RNA pol III promoters (115). The 7SL
29
gene product is a small untranslated RNA that is a component of the signal
recognition particle that targets both membrane and secretory proteins to the
endoplasmic reticulum (49). RT-PCR analysis was performed using total RNA
isolated from non-induced and 24 hour doxycycline induced U87 cells, and primers
directed against the reverse-transcribed 7SL gene product and precursor tRNA
Tyr
and
tRNA
Leu
. Intron-specific primers for tRNA precursors were used because these
transcripts are processed rapidly and therefore provide an assay of ongoing
transcriptional activity (116). Induction of PTEN expression reduced endogenous
7SL, precursor tRNA
Tyr
, and precursor tRNA
Leu
transcription by more than half (Fig.
5C). No reduction is seen in the transcription of the RNA pol II transcribed GAPDH
gene (Fig. 5C). These results suggest that PTEN selectively represses endogenous
RNA pol III transcription from a variety of RNA pol III promoters.
30
Fig. 5. PTEN selectively represses endogenous RNA polymerase III transcription. (A) Doxycycline induces
PTEN expression. PTEN null U87 cells, engineered to express PTEN or the phosphatase defective mutant PTEN
C124S in the presence of doxycycline, were treated with doxycycline for 0, 6, or 24 hours. Total protein was
isolated, 100 μg of protein was subjected to SDS-PAGE, and immunoblot analysis was performed using an
antibody that recognizes both PTEN and PTEN C124S. Further immunoblot analysis was performed on the same
membrane using an antibody against actin. (B) Induction of PTEN, but not phosphatase defective PTEN C124S,
decreases RNA pol III transcription. U87 cells were transiently transfected with 2 μg of pArg-maxi and treated
with doxycycline for 0, 6, or 24 hours, total RNA was isolated, and RPAs were performed. The results of at least
3 independent determinations were quantified by densitometry and the mean ± SD was graphed. A representative
autoradiograph is shown above each graph. (C) PTEN selectively represses endogenous RNA pol III
transcription. U87 cells were treated with doxycycline for 0 or 24 hours and total RNA was isolated. RT-PCR
analysis was performed (as described in Materials and Methods) to quantify transcription from endogenous RNA
pol III genes. cDNA was synthesized using random hexamers and 3 μg of total RNA. Primers specific for the
genes indicated were used for PCR analysis and the products of the reactions were subjected to agarose gel
electrophoresis. The results of at least 3 independent determinations were quantified by densitometry and
graphed ± SD.
A
C
GAPDH or RNA pol III gene activity
Fold change
GAPDH 7SL RNA
0
0.5
1.0
1.5
tRNA
Tyr
pre- tRNA
Leu
pre-
Doxycycline
Control
PTEN
actin
Doxycycline (hr)
66 00 24 24
B
PTEN
PTEN
tRNA gene activity
Fold change
PTEN C124S
PTEN C124S
tRNA gene activity
Fold change
Doxycycline (hr)
6 0 24
Doxycycline (hr)
6 0 24
0
0.5
1.0
1.5
0
0.5
1.0
1.5
31
Decreasing PTEN expression selectively enhances RNA polymerase III
transcription
We have demonstrated that expression of PTEN in PTEN null glioblastoma cells
reduces both endogenous RNA pol III transcription and transcription from the
transiently transfected RNA pol III reporter plasmid pArg-maxi by more than half.
We next determined whether reduction of PTEN expression in cell lines that contain
endogenous PTEN could enhance RNA pol III transcription. The glioblastoma-
derived cell line LN18 and the breast epithelial-derived cell line MCF-7 express wild
type endogenous PTEN. These cells were transiently cotransfected with pArg-maxi
and either an siRNA directed against PTEN or a control siRNA. Total protein or
RNA was isolated 24 hours following transfection. Western blot analysis illustrates
the decrease in PTEN protein seen using the PTEN siRNA (Fig. 6A). The
accompanying actin immunoblot demonstrates equal protein loading.
RPA analysis reveals a greater than 2.5 fold increase in transcription from the
transiently transfected pArg-maxi reporter plasmid when PTEN expression is
reduced using an siRNA directed against PTEN (Fig. 6B). This increase in RNA pol
III transcription is seen in both the LN18 and MCF-7 cell lines. RT-PCR analysis
performed using reverse-transcribed total RNA isolated 24 hours following
cotransfection of pArg-maxi and PTEN or control siRNA, indicates that transcription
of the endogenous precursor tRNA
Tyr
gene is increased more than 2 fold when PTEN
32
expression is reduced (Fig. 6C). The concomitant increase in transcription seen with
a reduction in PTEN expression is specific for RNA pol III transcription as no
change is seen in the transcription of the RNA pol II transcribed -actin gene. These
results demonstrate that reducing PTEN expression via transient transfection of an
siRNA increases both endogenous RNA pol III transcription and transcription from a
transiently transfected RNA pol III reporter plasmid.
We have determined that PTEN expression in PTEN null cells represses transcription
from a tRNA
Arg
gene reporter plasmid. This repression is independent of p53 and
requires PTENs lipid phosphatase activity. Conversely, reduction of PTEN
expression in PTEN expressing cells increases transcription. PTEN was also found
to selectively repress transcription from a variety of endogenous RNA pol III genes.
33
Fig. 6. Decreasing PTEN expression selectively enhances RNA polymerase III transcription. (A) An siRNA
directed against PTEN decreases PTEN expression. PTEN expressing LN18 and MCF-7 cell lines were
transiently transfected with an siRNA (final concentration 100 nM) directed against PTEN or a control siRNA as
described in ‘Materials and Methods’. Twenty four hours following transfection, total protein was isolated, 100
μg of protein was subjected to SDS-PAGE, and immunoblot analysis was performed using antibodies against
PTEN and actin. (B) Decreasing PTEN expression enhances RNA polymerase III transcription. PTEN
expressing LN18 and MCF-7 cell lines were transiently cotransfected with 2 μg of pArg-maxi and an siRNA
directed against PTEN or a control siRNA. Twenty four hours following transfection, total RNA was isolated
and RPAs were performed. The results of at least 3 independent determinations were quantified by densitometry
and graphed ± SD. A representative autoradiograph is shown above each graph. (C) Decreasing PTEN
expression selectively enhances endogenous RNA polymerase III transcription. LN18 cells were transiently
transfected with an siRNA directed against PTEN or a control siRNA and total RNA was isolated 24 hours
following transfection. cDNA was synthesized using random hexamers and 3 μg of total RNA, and PCR analysis
was performed using primers against the genes indicated. The products of the reactions were subjected to
agarose gel electrophoresis. The results of at least 3 independent determinations were quantified by densitometry
and graphed ± SD.
A
LN18 MCF-7
siRNA
PTEN
siRNA
control
PTEN PTEN
siRNA
PTEN
siRNA
control
Cell line:
actin actin
B
tRNA gene activity
Fold change
siRNA
PTEN
siRNA
control
siRNA
PTEN
siRNA
control
tRNA gene activity
Fold change
siRNA
PTEN
siRNA
control
siRNA
PTEN
siRNA
control
0
1
2
3
0
1
2
3
C
0
1
2
3
control siRNA
PTEN siRNA
-actin
or tRNA gene activity -actin
tRNA
Tyr
pre-
Fold change
34
Chapter 3: The PI-3 kinase signal transduction pathway regulates
RNA polymerase III transcription
Inhibition of the PI-3 kinase signal transduction pathway decreases RNA
polymerase III transcription
The lipid phosphatase activity of PTEN, which is required for its function as a tumor
suppressor, directly antagonizes the phosphatidylinositol-3 kinase (PI3K) signal
transduction pathway by dephosphorylating the membrane lipid that is
phosphorylated by PI3K (71). To determine whether PTEN represses RNA pol III
transcription via its antagonism of the PI3K signal transduction pathway, we
inhibited activation of the pathway by targeting a number of pathway components.
Glioblastoma-derived LN18 cells transiently transfected with pArg-maxi were either
treated with the PI3K inhibitor wortmannin at 0.5 μM for 6 hours, treated with the
mTOR inhibitor rapamycin at 100 nM for 3 hours, or transiently cotransfected with
expression plasmids encoding dominant negative mutants of the pathway
components PI3K or Akt (kindly provided by David Ann, City of Hope, Duarte,
California). Total RNA was isolated 24 hours following transfection, and RPA
analysis was performed to quantify transcription from pArg-maxi. The data shown
in figure 7A demonstrate a marked decrease in RNA pol III transcription with PI3K
pathway inhibition. The decrease in transcription is specific for RNA pol III as
luciferase assays (performed by Dr. Cheng Zhang) demonstrate that transcription
from the RNA pol II transcribed TBP promoter is not affected by inhibition of the
PI3K pathway (Fig. 7A).
35
Activation of the PI-3 kinase signal transduction pathway increases RNA
polymerase III transcription
Primary hepatocytes, freshly plated from the livers of 6 to 12 week old male
Sprague-Dawley rats, were used to further determine whether PI3K signaling
regulates RNA pol III transcription. Primary hepatocytes are more physiologically
relevant than cell lines as they are nontransformed and do not have the genomic
alterations found in most cell lines derived from tumors. Primary hepatocytes do not
replicate when cultured in standard medium and therefore any alterations seen in
RNA pol III transcription would be due solely to manipulation of Ras activated
signal transduction pathways. The hepatocytes were transiently cotransfected with
pArg-maxi and an expression plasmid for either the constitutively active Ras mutant
RasV12 or the Ras effector mutant RasV12C40 that specifically activates PI3K (108,
80). The cells were also treated with wortmannin at 0.5 μM for 6 hours. RPA
analysis was performed using total RNA isolated 24 hours following transfection. In
keeping with the quiescent nature of primary cells, the PI3K inhibitor wortmannin
had little to no inhibitory effect on RNA pol III transcription as compared to non-
treated cells (Fig. 7B). In contrast, activation of the PI3K signal transduction
pathway via constitutively activated RasV12 increases RNA pol III transcription
over 3 fold. Treatment of RasV12 transfected cells with wortmannin reduces RNA
pol III transcription by more than 50% indicating that stimulation of the PI3K
pathway is a primary mechanism by which RasV12 is activating RNA pol III
36
transcription. Further confirmation of this is seen with the activation of RNA pol III
transcription by the Ras effector mutant RasV12C40 that specifically activates PI3K
(Fig. 7B).
We have determined that the PI3K signal transduction pathway regulates RNA pol
III transcription, with inhibition of the pathway decreasing transcription and
activation of the pathway increasing transcription. These findings suggest that one
mechanism by which PTEN represses RNA pol III transcription is through its
antagonism of the PI3K signal transduction pathway.
37
Fig. 7. The PI-3 kinase signal transduction pathway regulates RNA polymerase III transcription. (A)
Inhibiting the PI3K pathway decreases RNA pol III transcription. LN18 cells were transiently transfected with 2
μg of pArg-maxi and either treated with wortmannin, at a final concentration of 0.5 μM, for 6 hours,
cotransfected with 2 μg of an expression vector for a dominant negative mutant of PI3K or with 2 μg of an
expression vector for a dominant negative mutant of Akt, or treated with rapamycin, at a final concentration of
100 nM, for 3 hours. Total RNA was isolated 24 h following transfection and immediately following inhibitor
treatment, and RPAs were performed. The results of at least 3 independent determinations were quantified by
densitometry and graphed ± SD. A representative autoradiograph is shown above the graph. LN18 cells were
also transiently transfected with 5 μg of a vector containing the TBP promoter preceding a luciferase gene
(p4500/+66hTBP-luc) and treated as described above. Twenty four hours following transfection, total protein
was isolated and luciferase assays were performed as described in ‘Materials and Methods’. The results of at
least 3 independent determinations were graphed ± SD. (B) Activating the PI3K pathway increases RNA pol III
transcription. Primary hepatocytes were transiently transfected (as described in ‘Materials and Methods’) with 4
μg of pArg-maxi, treated with wortmannin at a final concentration of 0.5 μM for 6 hours, and/or cotransfected
with 2 μg of either an expression vector for the constitutively active mutants RasV12 or RasV12C40. Twenty
four hours following transfection, total RNA was isolated and RPAs were performed. The results of at least 3
independent determinations were quantified by densitometry and graphed ± SD.
TBP promoter activity
Fold change
0
0.5
1.0
1.5
Control
PI3K-DN
Akt-DN
Rapamycin
Control
PI3K-DN
Akt-DN
Rapamycin
tRNA gene activity
Fold change
0
0.5
1.0
1.5
Primary Hepatocytes
LN18
0
1
tRNA gene activity
Fold change
Control
RasV12
RasV12
+
RasV12C40
4
2
3
A
B
Wortmannin
Wortmannin
Wortmannin
Wortmannin
38
Chapter 4: PTEN-mediated repression of RNA polymerase III
transcription in LN18 and U87 cells is not due to cell cycle affects
Reduction of PTEN levels in LN18 cells does not induce cell cycle changes
RNA pol III transcription is at a minimum in early G1 phase of the cell cycle (110).
PTEN has been found to mediate cell cycle arrest in G1 by increasing the protein
stability of the CDK inhibitor p27 and, conversely, decreasing the protein level and
nuclear localization of cyclin D1 (31, 52, 61, 74); therefore, we determined whether
the changes seen in RNA pol III transcription with manipulation of PTEN levels
were simply due to changes in the cell cycle. LN18 cells, which contain endogenous
wild type PTEN, were transiently transfected with an siRNA directed against PTEN
or a control siRNA. Twenty four hours following transfection, total protein was
isolated and Western blot analysis was performed. As shown in figure 8A,
transfection of an siRNA directed against PTEN decreases PTEN protein levels but
does not affect the protein levels of the cell cycle regulatory proteins cyclin D1 and
p27.
Fluorescence activated cell sorting (FACS) analysis (Fig. 8B), performed with LN18
cells fixed with ethanol 24 hours following transient transfection of a PTEN siRNA,
demonstrates no change in cell cycle profile between non-transfected cells or cells
transfected with either a control siRNA or an siRNA directed against PTEN. These
results suggest that under our experimental conditions PTEN is not affecting cell
39
cycle changes and thus any PTEN mediated changes in RNA pol III transcription
seen are independent of PTEN-mediated affects on the cell cycle.
Fig. 8. Reduction of PTEN levels in LN18 cells does not induce cell cycle changes. (A) Reduction of PTEN
levels does not affect the protein levels of cyclin D1 or p27. LN18 cells were transiently transfected with an
siRNA directed against PTEN or a control siRNA. Twenty four hours following transfection, total protein was
isolated, 100 μg of protein was subjected to SDS-PAGE, and immunoblot analysis was performed using
antibodies against cyclin D1, p27, and PTEN. (B) Reduction of PTEN levels does not alter the cell cycle profile.
LN18 cells were transfected with an siRNA directed against PTEN, a control siRNA, or left untransfected.
Twenty four hours following transfection, the cells were prepared for FACS analysis as described in ‘Materials
and Methods’, and FACS analysis was performed.
A
G1/G0: 52.2%
S: 39.5%
G2/M: 8.3%
non-transfected
G1/G0: 52.1%
S: 37.3%
G2/M: 10.6%
PTEN siRNA
G1/G0: 50.9%
S: 39.5%
G2/M: 9.6%
control siRNA
B
cyclin D1
siRNA
PTEN
siRNA
control
PTEN
p27
40
Cyclin D1 and nucleus persistent cyclin D1 T286A do not alleviate PTEN-
mediated repression of RNA polymerase III transcription in U87 cells
Cell cycle analysis of LN18 cells determined that there were no PTEN induced
changes in the cell cycle profile of cells that had been transfected with an siRNA
directed against PTEN (Fig. 8B). As PTEN has been shown to decrease both cyclin
D1 protein levels and its nuclear localization (77), we next determined whether
expression of cyclin D1 or a nucleus persistent cyclin D1 mutant T286A could
alleviate PTEN-mediated repression of RNA pol III transcription in U87 cells.
Twenty four hours following transfection, total RNA was isolated from cells
transiently cotransfected with pArg-maxi and/or expression vectors for PTEN, cyclin
D1 or cyclin D1 T286A. RPA analysis was then performed. Reiterating results seen
in figure 4A, expression of PTEN in PTEN null U87 cells decreases RNA pol III
transcription by more than 50% (Fig. 9A). Expression of cyclin D1 or cyclin D1
T286A does not alleviate this repression indicating that PTENs affect on RNA pol III
transcription is independent of its affect on cyclin D1. Cyclin D1 and cyclin D1
T286A also do not affect RNA pol III transcription in the absence of PTEN
expression (Fig. 9A).
Western blot analysis indicates that under our experimental conditions PTEN does
not affect cyclin D1 expression. Figure 9B shows that PTEN expression in PTEN
null U87 cells does not decrease endogenous cyclin D1 protein levels, and indicates
41
equal expression of FLAG-tagged cyclin D1 in the absence and presence of ectopic
PTEN expression and the equal expression of FLAG-tagged cyclin D1 T286A in the
absence and presence of ectopic PTEN expression.
Fig. 9. Cyclin D1 & nucleus-persistent cyclin D1 T286A do not alleviate PTEN-mediated repression of
RNA polymerase III transcription in U87 cells. (A) Expression of cyclin D1 or cyclin D1 T286A does not
alleviate PTEN-mediated repression of RNA pol III transcription. U87 cells were transiently cotransfected with 2
μg of pArg-maxi and 4 μg of an expression vector for PTEN, or 2 μg of an expression vector for FLAG epitope
tagged cyclin D1 or FLAG epitope tagged cyclin D1 T286A in combination or alone as indicated. Total RNA
was isolated 24 h following transfection and RPAs were performed. The results of at least 3 independent
determinations were quantified by densitometry and graphed ± SD. (B) PTEN expression does not alter cyclin
D1 expression. U87 cells were transfected as in (A) and total protein was isolated 24 h following transfection.
Immunoblot analysis was performed using antibodies against PTEN, cyclin D1, the FLAG epitope, and actin.
A
0
0.5
1.0
1.5
tRNA gene activity
Fold change
PTEN + cyc D1
Control
PTEN
cyc D1
cyc D1 T286A
PTEN + cyc D1 T286A
B
PTEN
PTEN
Endogenous cyclin D1
+
_
+ +
+
+ +
+
FLAG
PTEN
FLAG-cyclin D1
FLAG-cyclin D1 T286A
_ _
_ _
_ _
actin
42
Chapter 5: PTEN mediates repression of RNA polymerase III
transcription by decreasing the promoter occupancy of TFIIIB
We have determined that PTEN mediates repression of transcription from both
transiently expressed and endogenous RNA pol III genes. The lipid phosphatase
activity of PTEN, which directly antagonizes the PI3K signal transduction pathway,
is required for this repression. We have also determined that the PI3K pathway
regulates RNA pol III transcription, which suggests that one mechanism by which
PTEN regulates RNA pol III transcription is through its antagonism of the PI3K
pathway. To further examine the mechanism by which PTEN mediates repression of
RNA pol III transcription, we sought to determine the affect of PTEN on TFIIIB
which has been shown to be targeted by the tumor suppressors Rb and p53 (84, 5,
16).
PTEN does not alter the protein levels of the TFIIIB components
Western blot analysis indicates that PTEN does not alter the protein levels of the
TFIIIB components (Fig. 10A). LN18 cells were transfected with an siRNA directed
against PTEN or a control siRNA, and U87 cells were induced to express PTEN via
doxycycline treatment. Total protein was isolated 24 hours following transfection or
after 24 hours of doxycycline treatment. Although PTEN levels are decreased in the
LN18 cells and increased in the U87 cells, no change is seen in TBP, Brf1, or Bdp1
protein levels. No change is seen in TFIIIC
102
protein levels in the U87 cells as well
(Fig. 10A).
43
PTEN disrupts TBP/Brf1 binding
As PTEN does not decrease the protein levels of the TFIIIB components, we next
determined whether PTEN decreases the association between TBP and Brf1. LN18
cells were transiently transfected with either an siRNA directed against PTEN or a
control siRNA and total protein was isolated 24 hours following transfection.
Coimmunoprecipitation analysis was then performed using antibodies against TBP
and Brf1. Protein lysates were incubated with an antibody against TBP and
immunoblot analysis was performed using an antibody against Brf1, and conversely,
protein lysates were incubated with a Brf1 antibody and a TBP antibody was used
for immunoblot analysis. As shown in figure 10B, decreasing PTEN expression
increases the association between TBP and Brf1 and thus it can be concluded that
PTEN mediates the disruption of the association between TBP and Brf1. This
disruption is not caused by a PTEN-mediated change in the protein levels of TBP
and Brf1 (Fig. 10A). Experiments performed using the same antibody for both the
immunoprecipitation and immunoblot analysis show that equal amounts of TBP and
Brf1 were immunoprecipitated from both the control siRNA and PTEN siRNA
protein lysates (Fig. 10B).
PTEN decreases Brf1 serine phosphorylation
Having determined that PTEN disrupts the TBP/Brf1 complex, we determined
whether PTEN alters Brf1 serine phosphorylation. Phosphorylation of serine residues
44
is a means of regulating protein binding and function, with phosphorylation of Brf1
by CK2 and ERK having been shown to activate RNA pol III transcription (47, 25).
Twenty four hours following transfection, protein lysates were prepared from LN18
cells transfected with either a control siRNA or an siRNA directed against PTEN.
Brf1 was immunoprecipitated from the lysates and immunoblot analysis was
performed using an antibody against phosphorylated serine. As shown in figure
10C, decreasing PTEN expression increases Brf1 serine phosphorylation.
We next determined the effect of PTEN expression in PTEN null cells on Brf1 serine
phosphorylation. U87 cells were treated with doxycycline for 0, 6, or 24 hours to
induce PTEN expression and protein lysates were prepared. Brf1 was
immunoprecipitated from the lysates and immunoblot analysis was performed using
an antibody against phosphorylated serine. As shown in figure 10C, increasing
PTEN expression decreases Brf1 serine phosphorylation. Serine phosphorylation is
reduced to almost half following 6 hours of PTEN induction, with a further reduction
seen following 24 hours of induction. Experiments performed using the Brf1
antibody for both the immunoprecipitation and immunoblot analysis show that equal
amounts of Brf1 were immunoprecipitated from the 0, 6, and 24 hour protein lysates
(Fig. 10C). These results suggest that the reduction of Brf1 serine phosphorylation
by PTEN is a dynamic process in which a substantial decrease in phosphorylation is
45
seen following a few hours of PTEN induction, with a smaller decrease seen with
prolonged PTEN induction.
PTEN increases Bdp1 serine phosphorylation
CK2 phosphorylation of Bdp1 (which contains a number of putative CK2 serine
phosphorylation sites) during mitosis has been shown to repress RNA pol III
transcription by decreasing the association of Bdp1 with RNA pol III promoters (42).
Therefore we determined whether PTEN alters Bdp1 serine phosphorylation.
Twenty four hours following transfection, protein lysates were prepared from LN18
cells transfected with either a control siRNA or an siRNA directed against PTEN.
Protein lysates were also prepared from U87 cells that had been treated with
doxycycline to induce PTEN expression for 0, 6, or 24 hours. Bdp1 was
immunoprecipitated from the lysates and immunoblot analysis was performed using
an antibody against phosphorylated serine. Reducing PTEN expression in LN18
cells decreases Bdp1 serine phosphorylation by 50% (Fig. 10D). U87 cells induced
to express PTEN for 6 hours show no change in Bdp1 serine phosphorylation,
however induction of PTEN expression for 24 hours leads to a 4-fold increase in
Bdp1 serine phosphorylation (Fig. 10D). For both the LN18 and U87 cells, analysis
performed using a Bdp1 antibody for the immunoprecipitation as well as the
immunoblot shows that equal amounts of Bdp1 were immunoprecipitated from the
various lysates (Fig. 10D). These results indicate that PTEN increases Bdp1 serine
46
phosphorylation with a prolonged period of PTEN induction required for this
change.
47
Fig. 10. The affect of PTEN on TFIIIB. (A) PTEN does not alter the protein levels of the TFIIIB components.
LN18 cells were transfected with a PTEN or control siRNA, and PTEN-inducible U87 cells were treated with
doxycycline for 0 or 24 h, and total protein was isolated. Immunoblot analysis was performed using antibodies
against the proteins indicated. (B) PTEN disrupts TBP/Brf1 binding. LN18 cells were transfected with a PTEN
or control siRNA, and total protein was isolated. Co-immunoprecipitation assays were performed as described in
‘Materials and Methods’. Antibodies against TBP and Brf1 were used for both the immunoprecipitation and the
immunoblot. (IP: immunoprecipitation, IB: immunoblot) The results of at least 3 independent determinations
were quantified by densitometry and graphed ± SD. A representative film is shown above each graph. (C)
PTEN decreases Brf1 serine phosphorylation. LN18 cells were transfected with a PTEN or control siRNA, and
PTEN-inducible U87 cells were treated with doxycycline for 0, 6, or 24 h and total protein was isolated. An
antibody against Brf1 was used for the immunoprecipitation and antibodies against Brf1 and phosphoserine for
the immunoblot. (D) PTEN increases Bdp1 serine phosphorylation. LN18 cells were transfected with a PTEN or
control siRNA, and PTEN-inducible U87 cells were treated with doxycycline for 0, 6, or 24 h, and total protein
was isolated. An antibody against Bdp1 was used for the immunoprecipitation and antibodies against Bdp1 and
phosphoserine for the immunoblot.
D
0
0.5
1.0
1.5
Bdp1 Fold change
Bdp1
Doxycycline (hr) 6 0
Bdp1 serine phosphorylation
Fold change
p-Ser
IP: Bdp1
IB: p-Ser
IP:
IB:
Bdp1
Bdp1
U87
5
4
3
2
1
0
24 Doxycycline (hr) 6 0 24
Bdp1 serine phosphorylation
Fold change
siRNA
PTEN
siRNA
control
0
0.5
1.0
1.5
IP: Bdp1
IB: p-Ser
p-Ser
siRNA
control
siRNA
PTEN
Bdp1
0.5
1.0
1.5
0
Bdp1 Fold change
IP:
IB:
Bdp1
Bdp1
LN18
C
LN18
IP: Brf1
IB: p-Ser
Brf1 serine phosphorylation
Fold change
siRNA
PTEN
siRNA
control
0
1
2
3
U87
p-Ser p-Ser
B
LN18
0
1
2
3
Brf1 Fold change
siRNA
control
siRNA
PTEN
Brf1
IP: TBP
IB: Brf1
A
LN18 U87
siRNA
control
siRNA
PTEN
TBP
Brf1
Bdp1
PTEN
actin
TFIIIC 102
Doxycycline (hr) 0 24
0
1
2
3
TBP Fold change
siRNA
control
siRNA
PTEN
TBP
IP: Brf1
IB: TBP
TBP Fold change
0
0.5
1.0
1.5
TBP
siRNA
control
siRNA
PTEN
IP:
IB:
TBP
TBP
Brf1 Fold change
Brf1
IP: Brf1
IB: Brf1
0
0.5
1.0
1.5
siRNA
control
siRNA
PTEN
IP: Brf1
IB: p-Ser
Brf1 serine phosphorylation
Fold change
0
0.5
1.0
1.5
Doxycycline (hr)
Brf1
IP: Brf1
IB: Brf1
0.5
1.0
1.5
0
Brf1 Fold change
6 0 24
Doxycycline (hr) 6 0 24
48
PTEN decreases the promoter occupancy of TFIIIB but not TFIIIC
102
Having found that PTEN disrupts the association between TBP and Brf1, and that
PTEN decreases Brf1 serine phosphorylation while increasing Bdp1 serine
phosphorylation, we performed chromatin immunoprecipitation (ChIP) analysis to
determine whether PTEN affects the promoter occupancy of the TFIIIB components.
Chromatin was isolated from U87 cells that had been treated with doxycycline for 0,
6, or 24 hours to induce PTEN expression. Antibodies against TBP, Brf1, Bdp1, or
TFIIIC
102
were used to immunoprecipitate chromatin with which these proteins were
associated and primers specific for the tRNA
Leu
promoter were used in qPCR to
quantify the amount of these proteins present in the absence and presence of PTEN
expression. Induction of PTEN expression for 6 hours reduces the tRNA
Leu
occupancy of TBP, Brf1, and Bdp1 by approximately 50% (Fig. 11). A further
reduction in promoter occupancy is seen when PTEN expression is induced for 24
hours. The 3 components of TFIIIB show similar reduction in occupancy upon
induction of PTEN expression. The reduction in promoter occupancy is specific for
TFIIIB as the occupancy of TFIIIC
102
is unchanged upon induction of PTEN
expression (Fig. 11).
We examined the effect of PTEN on the serine phosphorylation of Brf1 and Bdp1,
and on the promoter occupancy of TFIIIB kinetically. PTEN-mediated change in
serine phosphorylation and TFIIIB promoter occupancy is a dynamic process. A
49
large reduction in Brf1 serine phosphorylation is seen following 6 hours of PTEN
induction, with phosphorylation continuing to decrease slightly over the next 18
hours (Fig. 10C). Paralleling this decrease in serine phosphorylation is the promoter
occupancy of Brf1 (Fig. 11), suggesting that the PTEN-mediated decrease in serine
phosphorylation may reduce Brf1 promoter occupancy. The decrease in Brf1 serine
phosphorylation may also cause the PTEN-mediated disruption of the
TBP/Brf1complex which in turn could account for the decrease seen in TBP
promoter occupancy. No change in Bdp1 serine phosphorylation is seen following 6
hours of PTEN induction while a substantial decrease in promoter occupancy is seen
(Fig. 10D; Fig. 11). The promoter occupancy of Bdp1 decreases slightly over the
next 18 hours while a substantial increase in phosphorylation is seen (Fig. 11; Fig.
10D). These results suggest that the PTEN-mediated increase in Bdp1 serine
phosphorylation may not affect Bdp1 promoter occupancy. The decrease in TFIIIB
promoter occupancy is not due to a decrease in the protein levels of the components
as we have determined that PTEN does not alter the protein levels of the TFIIIB
components.
50
Fig. 11. PTEN expression selectively decreases the promoter occupancy of TFIIIB. U87 cells were treated
with doxycycline for 0, 6, or 24 h to induce PTEN expression, chromatin was isolated, and ChIP analysis was
performed as described in ‘Materials and Methods’. Antibodies against TBP, Brf1, Bdp1, and TFIIIC
102
were
used to immunoprecipitate the cross-linked chromatin. QPCR analysis was then performed as described in
‘Materials and Methods’ to quantify promoter occupancy. The results of at least 3 independent determinations
were quantified and graphed ± SD.
Control
Doxycycline 6 hr
Doxycycline 24 hr
TBP Brf1
Bdp1
0
0.5
1.0
1.5
tRNA
Leu
promoter occupancy
Fold change
TFIIIC
102
51
Chapter 6: Discussion
p53 represses RNA polymerase III transcription by targeting TBP
It has been demonstrated that p53 binds to TBP and represses RNA pol II
transcription of genes that lack p53 response elements by interfering with initiation
complex formation (56, 58, 96, 85, 10, 78, 39, 24). p53 has been shown to be a
general repressor of RNA pol III transcription by targeting the TFIIIB complex (5).
p53 has also been shown to repress the U6 snRNA gene that utilizes an alternate
form of TFIIIB (13, 32). We have determined a mechanism by which p53 mediates
repression of RNA pol III transcription. Overexpression of TBP alleviates p53-
mediated repression of a tRNA gene. Importantly, TBP is limiting for RNA pol III
transcription in the presence, but not in the absence, of induction of p53 expression.
These data indicate that TBP is not limiting for tRNA gene transcription in non-
induced cells, but that TBP becomes limiting in response to induction of p53
expression. Overexpression of Brf1 does not alleviate repression, further indicating
that p53 specifically targets TBP. The alleviation of p53-mediated repression by
TBP overexpression is not due to the ability of TBP to support RNA pol II
transcription, as overexpression of a mutant that is selectively defective for RNA pol
II transcription (45) alleviates p53-mediated repression. These results indicate that it
is not the effect of p53 on RNA pol II transcription that is leading to repression of
RNA pol III transcription, rather p53 is targeting TBP directly to repress RNA pol III
transcription.
52
TBP and Brf1 associate to form a stable complex, and therefore we sought to
determine whether the binding of p53 to TBP disrupts this complex. A precedent for
this disruption is seen with the transcriptional repressor Dr1 which binds to TBP and
disrupts the TBP/Brf1 interaction (112). In contrast to this, however,
coimmunoprecipitation analysis determined that although p53 associates with the
TBP/Brf1 complex, p53 does not disrupt this complex. The TFIIIB component Bdp1
interacts weakly with the TBP/Brf1 complex (103), and therefore we were not able
to determine through coimmunoprecipitation analysis whether p53 disrupts this weak
interaction.
Our work demonstrated that p53 disrupts the binding of Brf1 to TFIIIC and to RNA
pol III and thereby inhibits formation of a transcription initiation complex (16).
Expanding upon these findings, p53 was also found to selectively decrease the
promoter occupancy of TBP, Brf1, Bdp1, and RNA pol III on tRNA genes. The
promoter occupancy of the TFIIIC subunits C63, C102, and C110 on tRNA genes is
not decreased by p53, and p53 itself is not present at the tRNA promoters (16). This
mechanism is in contrast to a study that determined that p53 represses transcription
of the RNA pol III transcribed U6 snRNA gene by binding to its gene external
promoter (32). p53 was also found to target TBP, as well as SNAP
C
, to mediate this
repression (32). Our study has shown a functional consequence of the p53/TBP
interaction — that of p53 targeting TBP to repress RNA pol III transcription.
53
PTEN represses RNA polymerase III transcription by mediating a decrease in
TFIIIB promoter occupancy
We sought to determine whether PTEN mediated repression of RNA pol III
transcription because, like p53, PTEN is a tumor suppressor that is lost or mutated in
~ 50% of human cancers. In contrast to p53, which is a transcription factor, PTEN is
a lipid phosphatase that directly antagonizes the PI3K signal transduction pathway.
Our studies have elucidated a new function for PTEN — that of mediating repression
of RNA pol III transcription.
Changes in PTEN levels in a number of human cell lines affected the transcription of
an ectopically expressed tRNA gene as well as a variety of endogenous RNA pol III
genes. The repression of RNA pol III transcription by PTEN is not dependent on
p53, but is dependent on the lipid phosphatase activity of PTEN. Previous studies
have shown that activated Ras and activated Raf-1, a downstream Ras effector,
stimulate RNA pol III transcription (101). Our studies have determined that another
downstream Ras effector, PI3K, regulates RNA pol III transcription. Activation of
the PI3K signal transduction pathway via a constitutively activated Ras mutant that
specifically activates PI3K increases RNA pol III transcription, while inhibition of
the pathway components PI3K, Akt and mTOR represses RNA pol III transcription.
These findings suggest that inhibition of the PI3K pathway by PTEN is a mechanism
by which PTEN represses RNA pol III transcription. Through its antagonism of the
54
PI3K pathway PTEN affects the activity of the pathway component mTOR which
plays a critical role in coordinating nutrient availability with ribosome biogenesis
and, thereby, protein biosynthetic capacity (64). This is the first demonstration of
regulation of mammalian RNA pol III transcription by mTOR.
RNA pol III transcription varies according to the cell cycle, being lowest in early
G1phase (110). Ectopic overexpression of PTEN has been shown to cause cell cycle
arrest in the G1 phase with PTEN increasing the protein stability of the CDK p27
while decreasing both the protein level and nuclear localization of cyclin D1 (77, 31,
52, 61). FACS analysis showed no change in cell cycle profile with reduction in
PTEN expression, and the protein levels of p27 and cyclin D1 also remain
unchanged. These findings suggest that, under our experimental conditions, the
increase in RNA pol III transcription seen upon reduction of PTEN expression is not
due to cell cycle changes. Expression of cyclin D1 and the nucleus persistent mutant
cyclin D1 T286A has been shown to reverse PTEN-mediated cell cycle arrest (77).
We found that ectopic expression of cyclin D1 and cyclin D1 T286A did not reverse
PTEN-mediated repression of RNA pol III transcription suggesting that this
repression is not due to the reduction of both cyclin D1 protein levels and nuclear
localization by PTEN.
55
We determined that PTEN disrupts the interaction between TBP and Brf1, and thus
does not allow for initiation complex formation. This disruption is also seen with the
tumor suppressor Dr1 which binds to TBP (112). These findings are in contrast to
those for p53 which does not disrupt the TBP/Brf1 complex although it associates
with it. To determine how PTEN facilitates the disruption of the TBP/Brf1 complex,
we considered that changes in the phosphorylation state of Brf1 might cause
dissociation of the complex. A previous study determined that PTEN does not
change the serine phosphorylation state of TBP (120). This same study was unable
to detect any tyrosine phosphorylation of TBP (120). Our study determined that
PTEN decreases the serine phosphorylation of Brf1, but found no change in the
tyrosine phosphorylation of Brf1. It is likely that the PTEN-mediated decrease in
Brf1 serine phosphorylation contributes to disruption of the TBP/Brf1 complex.
This is the first demonstration that Brf1 serine phosphorylation affects the
association of the TBP/Brf1 complex. It is known that phosphorylation of Brf1 by
CK2 and the MAP kinase ERK leads to an increase in RNA pol III transcription (47,
25), and although it has not been determined which Brf1 serine residues are affected
by PTEN, it is possible that PTEN mediates dephosphorylation of all or some of the
serine residues phosphorylated by CK2 and/or ERK. Due to the weak interaction of
Bdp1 with the TBP/Brf1 complex, we were not able to determine whether PTEN
affects the interaction between Bdp1 and this complex.
56
We examined PTEN-mediated repression of RNA pol III transcription and the effect
of PTEN on tRNA
Leu
promoter occupancy of TFIIIB and TFIIIC following a time
course of PTEN induction. A decrease in RNA pol III transcription is seen 6 hours
following PTEN induction, while a more considerable decrease is seen 24 hours
following induction. A greater than 50% decrease in transcription is seen overall at
24 hours. A decrease in the promoter occupancy of TBP, Brf1, and Bdp1 is seen
following PTEN induction, with the greatest change in occupancy occurring 6 hours
following PTEN induction, and a lesser change in occupancy occurring 24 hours
following PTEN induction. Overall the decrease in occupancy is ~ 50% at 24 hours.
The decrease in occupancy is strikingly similar for all 3 components of TFIIIB. A
simultaneous decrease in occupancy of all 3 components of TFIIIB is seen with
induction of p53 expression as well (16). A decrease in protein levels cannot
account for this decrease in occupancy as no change in the protein levels of the
TFIIIB components is seen with induction of PTEN expression.
We also examined changes in Brf1 and Bdp1 serine phosphorylation following a
time course of PTEN induction. Paralleling the decrease seen in Brf1 promoter
occupancy, a substantial decrease in Brf1 serine phosphorylation is seen 6 hours
following induction of PTEN expression, with a smaller decrease in phosphorylation
seen following 24 hours of induction. As with the decrease in promoter occupancy
of Brf1, the overall decrease in Brf1 serine phosphorylation is ~50% at 24 hours. As
57
the levels of Brf1 serine phosphorylation parallel the promoter occupancy of Brf1, it
is likely that the PTEN-mediated decrease in Brf1 serine phosphorylation leads to the
reduction in Brf1 promoter occupancy seen upon induction of PTEN expression. A
concomitant decrease in TBP promoter occupancy is seen with the decrease in Brf1
occupancy, and it is likely that PTEN-mediated disruption of the TBP/Brf1 complex
contributes to this decrease in TBP occupancy. It has been shown that
phosphorylation of Brf1 by CK2 and ERK promotes the interaction of TFIIIB with
TFIIIC (47, 25); therefore, the PTEN-mediated decrease in Brf1 serine
phosphorylation may further reduce the promoter occupancy of Brf1 by decreasing
its association with TFIIIC. A substantial decrease in Bdp1 promoter occupancy is
seen 6 hours following PTEN induction while no change in Bdp1 serine
phosphorylation is seen. A large increase in Bdp1 serine phosphorylation is seen 24
hours following PTEN induction while only a small further decrease in occupancy is
seen. Thus, Bdp1 serine phosphorylation does not correlate with promoter
occupancy. We also examined the effect of PTEN on Bdp1 tyrosine phosphorylation
but were unable to detect any phosphorylated tyrosine residues.
While the promoter occupancy of the TFIIIB components decreases in response to
PTEN induction, the occupancy of TFIIIC
102
remains unchanged. These findings are
consistent with earlier determinations that TFIIIC stably associates with the internal
58
promoter of tRNA genes in the absence of any other transcription factors (19). Thus,
PTEN does not appear to affect the function of TFIIIC or its promoter occupancy.
As there is a clear correlation between the PTEN-mediated decrease in Brf1 serine
phosphorylation, disruption of the TBP/Brf1 complex, reduction in TFIIIB promoter
occupancy, and decrease in RNA pol III transcription, we propose a model in which
PTEN-mediated repression of RNA pol III transcription is driven by the reduction in
Brf1 serine phosphorylation and subsequent disruption of the TBP/Brf1 complex.
We suggest that the association between TBP and Brf1 is required for their
recruitment to the promoter and that they come to the promoter as a complex. Thus,
due to the disruption of the TBP/Brf1 complex, the recruitment of Brf1 and TBP to
the tRNA promoter is simultaneously reduced. The promoter occupancy of Bdp1 is
dependent on the promoter occupancy of the TBP/Brf1complex, and while the serine
phosphorylation state of Bdp1 does not contribute to the initial decrease in Bdp1
promoter occupancy, it may contribute to the later reduction in Bdp1 promoter
occupancy. The possibly reduced association between Brf1 and TFIIIC, may
contribute to a further reduction in Brf1 occupancy on the promoter. This model is
illustrated graphically in Figure 12.
59
Fig. 12. Model of PTEN-mediated repression of RNA polymerase III transcription. In the absence of PTEN
expression, Brf1 is phosphorylated on serine residues, the TBP/Brf1 complex is intact, the TFIIIB complex is
present on the tRNA promoter, and transcription is at a maximum. Six hours following induction of PTEN
expression, Brf1 serine phosphorylation is decreasing, the TBP/Brf1 complex is beginning to dissociate, the
TFIIIB complex is beginning to dissociate from the promoter, and transcription is reduced. Twenty four hours
following induction of PTEN expression, Brf1 serine phosphorylation is greatly reduced, the TBP/Brf1 complex
is dissociated, Bdp1 serine phosphorylation is greatly increased, the occupancy of the TFIIIB complex on the
promoter is substantially reduced, and transcription is at a minimum.
Bdp1
TFIIIC
TBP
Brf1
P
Bdp1
TFIIIC
TBP
Brf1
P
TFIIIC
TBP
P
Brf1
Bdp1
P
PTEN
6 h
PTEN
24 h
60
RNA polymerase III transcription and biosynthetic capacity
The growth rate of cells is directly proportional to the accumulation of protein (1);
therefore, the protein biosynthetic capacity of cells is an important determinant of
growth rate. As the products of RNA pol III transcription are essential for protein
synthesis, RNA pol III transcriptional activity is tightly linked to the rate of cell
growth (4). In the absence of cell growth, cell cycle progression and proliferation
cannot occur (109). Relatively high levels of RNA pol III transcription products are
seen in transformed and tumor cells (4). The importance of regulating RNA pol III
transcription is evidenced by the fact that the 2 cardinal tumor suppressors, Rb and
p53, repress RNA pol III transcription (5, 16, 114). We have shown that the tumor
suppressor PTEN represses RNA pol III transcription as well. The function of PTEN
as a lipid phosphatase that inhibits the PI3K signal transduction pathway
distinguishes it from p53 and Rb which function as transcription factors.
The transcription products of RNA pol I, the large rRNAs, are also essential for
protein synthesis, and many of the proteins that regulate RNA pol III transcription
regulate RNA pol I transcription as well. c-Myc, Ras, ERK, and CK2 have all been
shown to activate both RNA pol I and pol III transcription (102, 89, 121, 33, 100, 62,
54), while Rb, p53 and PTEN have been shown to repress both RNA pol I and pol III
transcription (119, 5, 16, 120 ). Paralleling the disruption of the TBP/Brf1 complex
by PTEN, the SL1 complex was also found to be disrupted by PTEN (120).
61
Coordinate regulation of RNA pol I and pol III transcription ensures efficient
production of components necessary for protein synthesis.
Future directions
As the PTEN-mediated decrease in Brf1 serine phosphorylation contributes to
disruption of the TBP/Brf1 complex, it would be of interest to determine which Brf1
serine residues are affected by PTEN and whether these same residues are
phosphorylated by CK2 and/or ERK. As ERK has been shown to phosphorylate a
Brf1 threonine residue (25), the affect of PTEN on Brf1 threonine phosphorylation
could also be determined. Brf1 mutants that prevent and mimic phosphorylation at
this site are available. Studies have determined that PTEN is present in the nucleus
(15, 53, 55), thus, PTEN may directly affect the promoter occupancy of TFIIIB. To
determine whether this is so, ChIP analysis could be performed to determine whether
PTEN is present at the tRNA
Leu
promoter.
62
Chapter 7: Materials & Methods
Cell lines & culture
The H1299 cell line, which is derived from a human lung carcinoma, contains a
stably integrated p53 gene under the control of tetracycline (11). p53 is expressed in
the absence of tetracycline. The cells were grown in RPMI 1640 medium
(Mediatech) supplemented with 10% fetal bovine serum (FBS), G418 at 300 μg/ml,
puromycin at 2 μg/ml, and tetracycline at 4.5 μg/ml. H1299 cells were engineered to
stably express the Arg-maxi gene by co-transfecting pArg-maxi and pCEP4, which
encodes a hygromycin B resistance gene. The cells were selected with hygromycin
B at a concentration of 250 μg/ml.
The glioblastoma derived A172, U87, and LN18 cell lines, were obtained from the
American Type Culture Collection (ATCC) and grown in high glucose Dulbecco’s
Modification of Eagle’s Medium (DMEM) (Mediatech) supplemented with 10%
FBS. The breast epithelial derived MCF-7 cell line was obtained from the ATCC
and grown in high glucose DMEM supplemented with 10% FBS.
The PTEN-inducible and PTEN C124S-inducible U87 cell lines were obtained from
Maria-Magdalena Georgescu (MD Anderson Cancer Center, Houston, Texas) and
grown in DMEM supplemented with 10% FBS, G418 at 1mg/ml, and blasticidine at
63
10 μg/ml. The cells were engineered to express PTEN or PTEN C124S in the
presence of doxycycline at 1 μg/ml (77).
Primary hepatocyte cultures from male Sprague-Dawley rats (225 to 400 g; 6 to 12
weeks old) were obtained from the University of Southern California Liver Tissue
Culture Core Facility. Cells were isolated and cultured in William’s E Medium
supplemented with 5% FBS, 2 mM L-glutamine, 0.1× ITS-X (insulin at 1 μg/ml,
sodium transferrin at 0.55 μg/ml, 3.9 μM sodium selenite, ethanolamine at 0.2
μg/ml), penicillin at 200 U/ml, streptomycin at 200 μg/ml, amphotericin B
(Fungizone) at 0.25 μg/ml, and gentamicin at 50 μg/ml. Cells were plated on
Primaria plates (60 by 15 mm, Becton Dickinson) at 10
6
per plate (46).

Plasmids
The pArg-maxi construct, which is a tRNA
Arg
gene reporter that contains an
additional 12 base pairs inserted between the internal A and B blocks, was
constructed as previously described (21). The 12 additional base pairs allow for
differentiation from endogenous tRNAs. The hTBP promoter luciferase construct
p4500/+66hTBP-luc was kindly provided by Diane Hawley (University of Oregon,
Eugene) and is previously described (27). The TBP expression constructs pLTR-
E2TBP and pLTR-E2TBP-E284R were constructed in our laboratory as previously
described (45). TBP E284R is a mutant of TBP that is specifically defective for
64
RNA pol II transcription. The Brf1 expression construct p2HABrf1 is previously
described (91). The expression constructs for PTEN (pCMV-PTEN) and the lipid
phosphatase defective mutant PTEN G129E (pCMV-PTEN G129E) are previously
described (98). The expression constructs for FLAG epitope tagged cyclin D1 and
the nucleus persistent mutant cyclin D1 T286A (also FLAG epitope tagged) are
previously described (20). Dominant negative PI3K (PI3K-DN) and dominant
negative Akt (Akt-DN) expression constructs were kindly provided by David Ann
(City of Hope, Duarte, California). The expression constructs for the constitutively
active Ras mutants pDCR-RasV12 and pDCR-RasV12C40 were kindly provided by
Michael White (University of Texas, Southwestern Medical Center, Dallas) and are
previously described (108). For all control transfections, the appropriate amount of
empty vector corresponding to the respective expression construct was added.
Transient transfection
H1299 cells were transiently transfected using a lipid-based reagent obtained from
the USC/Norris Cancer Center Reagent Core facility. Approximately 8 X 10
5
cells
were seeded on 100 mm dishes in 10 ml of RPMI 1640 medium containing 10%
FBS, G418, puromycin and tetracycline as described above. The cells were
transfected ~ 18 h later with 2 μg of pArg-maxi and other plasmids, as indicated in
the figure legends, with pSK for a total of 10 μg of DNA. For luciferase assays, the
cells were transfected with 0.5 μg of p4500/+66hTBP-luc. For each plate, 10 μl of
65
the lipid reagent was incubated with 600 μl of OPTI-MEM 1 (Invitrogen) for 45 min
at 25°C. The plasmids were incubated with 600 μl of OPTI-MEM 1 for 10 min at
25°C, added to the lipid reagent/OPTI-MEM 1 mix, incubated at 25°C for 15 min,
and added to a dish containing 4 ml of RPMI 1640 medium with tetracycline at 4.5
μg/ml. The H1299 cells were incubated with the transfection mix for 4 h at 37°C.
The transfection mix was then replaced with 10 ml of RPMI 1640 containing 10%
FBS, G418, puromycin, and, for the cells not expressing p53, tetracycline. Forty
eight hours following transfection, total RNA or protein was harvested and
ribonuclease protection assays (RPAs), luciferase assays, or immunoblot analyses
were performed.
The A172, U87, U87-inducible, and LN18 cells were transiently transfected using
lipofectin (Invitrogen). Approximately 8 X 10
5
cells were seeded on 100 mm dishes
in 10 ml of 10% FBS/DMEM. The cells were transfected ~ 18 h later with 2 μg of
pArg-maxi and other plasmids, as indicated in the figure legends, with pSK for a
total of 10 μg of DNA. For each plate, 10 μl of lipofectin was incubated with 500 μl
of OPTI-MEM 1 (Invitrogen) for 45 min at 25°C. The plasmids were incubated with
500 μl of OPTI-MEM 1 for 10 min at 25°C, added to the lipofectin/OPTI-MEM 1
mix, incubated at 25°C for 20 min, and added to a dish containing 9 ml of DMEM.
The cells were incubated with the transfection mix for 4 h at 37°C. The transfection
mix was then replaced with 10 ml of 10% FBS/DMEM. Twenty four hours
66
following transfection, total RNA or protein was harvested and RPAs, luciferase
assays, or immunoblot analyses were performed.
Transient transfection of siRNA
Lipofectamine 2000 was used for transient transfection of a PTEN or control siRNA
into LN18 and MCF-7 cells. The siRNAs were obtained from the USC/Norris
Cancer Center Microchemical Core facility. The sequence of the siRNA directed
against PTEN is as follows: sense strand — CAA AUC CAG AGG CUA GCA GTT;
antisense strand — CUG CUA GCC UCU GGA UUU GTT. The sequence of the
control siRNA is as follows: sense strand — CAA AUC CGG ACG CUA GCA
GTT; antisense strand — CUG CUA GCG UCC GGA UUU GTT. The separate
siRNA strands were reconstituted to a concentration of 50 μM and annealed using
annealing buffer (100 mM KOAc, 30 mM HEPES KOH, 2 mM MgOAc, pH 7.4,
Xeragon) in a ratio of 1:1:0.5 for a final annealed siRNA concentration of 20 μM.
Approximately 8 X 10
5
cells were seeded on 100 mm dishes in 10 ml of 10%
FBS/DMEM. The cells were transfected ~ 18 h later with 2 μg of pArg-maxi, pSK,
and 25 μl of 20 μM siRNA for a total of 10 μg of DNA/siRNA and a final siRNA
concentration of 100 nM. For each plate 20 μl of Lipofectamine 2000 was incubated
with 500 μl of OPTI-MEM 1 for 5 min at 25°C. The 10 μg of DNA/siRNA was
added to 500 μl of OPTI-MEM 1, mixed with the Lipofectamine 2000/ OPTI-MEM
67
1, incubated for 20 min at 25°C and then added to a 100 mm dish containing 4 ml of
10% FBS/DMEM. The cells were incubated with the transfection mix for 4 h at
37°C. The transfection mix was then replaced with 10 ml of 10% FBS/DMEM.
Twenty four hours following transfection, total RNA or protein was harvested and
RPAs, immunoblot analyses, co-immunoprecipitation analyses, RT-PCR analyses,
and fluorescence activated cell sorting (FACS) analyses were performed.
Transient transfection of primary rat hepatocytes
Primary rat hepatocytes were transfected 4 hours after isolation. Hepatocytes were
rinsed with phosphate buffered saline (PBS) and 4 ml of transfection medium
(William’s E Medium supplemented with 2 mM L-glutamine, and 0.1× ITS-X) was
put on the plates. Using Targefect F1 (Targeting Systems; 1 μg/μg DNA) according
to the manufacturer’s specifications, cells were transfected with a total of 10 μg of
DNA including 4 μg of pArg-maxi and, where indicated, 2 μg of pDCR-RasV12 or
pDCR-RasV12C40. pSK was added to maintain a total of 10 μg. Cells were
transfected overnight (~ 18 h) after which the medium was changed to isolation
medium which is described above. Where indicated, wortmannin was added to the
medium at a final concentration of 0.5 μM 6 h before harvesting. Total RNA was
isolated from the cells 65 h following transfection and RPAs were performed.
68
Ribonuclease protection assay
The MAXIscript T7 kit (Ambion) was used, according to the manufacturer’s
protocol, to synthesize a radioactive probe using
32
P-CTP (MP Biomedicals) and
pArg-maxi that had been digested/linearized with Xba1 (New England Biolabs).
Total RNA was isolated from transfected cells using TRIzol (Invitrogen) or RNA
STAT-60 (Tel-Test, Inc., Friendswood, Texas) according to the manufacturers’
protocol, and the RNA concentration was determined using a spectrophotometer. To
determine the amount of pArg-maxi transcribed under the various experimental
conditions, ribonuclease protection assays (RPAs) were performed using the RPA III
kit (Ambion) according to the manufacturer’s protocol. RNA (2 μg) was hybridized
with an excess of
32
P-labeled probe and incubated overnight at 42°C. Unhybridized
RNA was digested using a RNase A/T1 mix at a 1:100 dilution, and total RNA was
precipitated. (Due to the small size of the fragment of interest (83 nucleotides) 150
μl of ethanol was added to the precipitation mix.) 8 μl of gel loading buffer II was
added to the pellets which were run on an 8% polyacrylamide/ 8M urea denaturing
gel which was then exposed to Kodak X-OMAT AR film. The resultant
autoradiographs were scanned and quantified using UN-Scan-It software (Silk
Scientific) and graphs were prepared.
69
Immunoblot analysis
H1299 cells were grown in the absence or presence of tetracycline for 2 or 6 days
and were also transfected with pLTR-E2TBP or p2HABrf1. A172 and U87 cells
were transfected with either 2 μg of empty vector, pCMV-PTEN, or pCMV-PTEN
G129E. The U87 cells were also transfected with expression constructs for cyclin
D1 or the nucleus persistent mutant cyclin D1 T286A. The PTEN-inducible and
PTEN C124S-inducible U87 cell lines were treated with doxycycline for 0, 6, or 24
hours. The LN18 and MCF-7 cells were transfected with an siRNA directed against
PTEN. Following transfection, total protein was isolated from the cells using 400 to
600 μl of immunoprecipitation buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1mM
EDTA, 1mM EGTA, 0.1% Triton X-100, 2.5 mM sodium pyrophosphate, 1mM -
glycerol phosphate, 1 mM Na
3
VO
4
, 1 mM PMSF, and Protease Inhibitor Cocktail
Set III (Calbiochem)] per 100 mm plate. The cells were scraped off the plates,
incubated for 15 min on ice, sonicated for 15 sec, and centrifuged for 20 min at
10,000 g at 4°C. The supernatant was collected and the protein concentration of the
lysates was determined by the Bradford method using the Bio-Rad Protein Assay.
100 to 150 μg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). Hybond ECL nitrocellulose membrane (GE
Healthcare, Amersham) was used for protein transfer. Bound primary antibody was
visualized using biotinylated IgG secondary antibody and the standard Vectastain
ABC kit (Vector Laboratories) or horseradish peroxidase-conjugated secondary
70
antibody (Pierce), and enhanced chemiluminescence (ECL) (GE Healthcare,
Amersham) or SuperSignal West Pico Chemiluminescent Substrate (Pierce). The
primary antibodies (all of which are anti-human) were used as indicated and are as
follows: mouse biotinylated p53 (Oncogene Research Products), mouse polyclonal
PTEN (Axel Schönthal, University of Southern California), rabbit polyclonal TBP
(Upstate), rabbit polyclonal Brf1 (Robert White, University of Glasgow), goat
polyclonal Brf1 (Santa Cruz Biotechnology), rabbit polyclonal Bdp1 (Robert White,
University of Glasgow), TFIIIC
102
, (Martin Teichmann, Children’s Hospital, Boston,
Massachusetts ), goat polyclonal p27 (Santa Cruz Biotechnology), rabbit polyclonal
cyclin D1 (Santa Cruz Biotechnology), rabbit polyclonal phosphoserine (Chemicon),
mouse monoclonal FLAG (Sigma-Aldrich), and mouse monoclonal actin
(Chemicon). The membranes were exposed to Kodak X-OMAT AR film.
Co-immunoprecipitation assay
Protein was isolated as described under ‘Immunoblot analysis’. 400 μg of protein
was incubated with 4 μl of anti-TBP, Brf1, or Bdp1 antibodies overnight at 4°C. 25
μl of Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) were then
added and incubation was continued for 3 h. The beads were washed 3 times with
cold PBS, 40 μl of Sample Buffer, Laemmli 2x Concentrate (Sigma) was added, the
beads were boiled for 10 min and subjected to SDS-PAGE. Immunoblot analysis
was then performed using the antibodies indicated. The resultant autoradiographs
71
were scanned and quantified using UN-Scan-It software (Silk Scientific) and graphs
were prepared.
Luciferase assay
H1299 cells were transfected with 5 μg of p4500/+66hTBP-luc, and LN18 cells
were transfected with 5 μg of p4500/+66hTBP-luc and 2 μg of an expression
construct for either PI3K-DN or Akt-DN; or treated with wortmannin (final
concentration 0.5 μM, Calbiochem) for 6 h, or rapamycin (100 nM final
concentration, Sigma-Aldrich) for 3 h. The luciferase assays were performed as
previously described (46). For luciferase activity measurements, protein was
isolated 24 h following transfection using 30 μl of Promega reporter lysis buffer per
60 mm plate. The cells were scraped off the plates, incubated for 10 min on ice,
frozen and thawed twice, and centrifuged for 20 min at 10,000 g at 4°C. The
supernatant was collected and the protein concentration of the lysates was
determined by the Bradford method using the Bio-Rad Protein Assay. The lysates
were analyzed for luciferase activity using a luminometer and the Promega
Luciferase Assay System according to the manufacturer’s protocol. Resultant
luciferase activities were normalized to the amount of protein in each lysates. For
the H1299 cells, the fold change in promoter activity was calculated by determining
the level of luciferase activity in the absence of p53 expression and setting this value
at 1 for each independent experiment. For the LN18 cells, the fold change in
72
promoter activity was calculated by determining the level of luciferase activity in the
presence of the empty vector plasmid and in the absence of inhibitor treatment, and
setting this value at 1 for each independent experiment.
Fluorescence activated cell sorting analysis
LN18 cells were transfected with an siRNA against PTEN, a control siRNA, or left
untransfected. Twenty four hours following transfection, the cells were trypsinized
and then fixed by resuspension in 500 μl of PBS followed by 5 ml of 100% ethanol.
For cell cycle analysis, the cells were washed with PBS, resuspended in 1 ml of PBS
containing DNase free RNase at 40 μg/ml and propidium iodide (Sigma-Aldrich) at
20 μg/ml, and incubated at room temperature for up to 3 h. The analysis was
performed by the USC/Norris Cancer Center Core Facility.
RT-PCR analysis
Total RNA was isolated from cells as described under ‘Ribonuclease protection
assay’. cDNA was synthesized using the SuperScript First-Strand Synthesis System
for RT-PCR (Invitrogen). 3 μg of RNA was mixed with 100 ng of random hexamers
in a total volume of 24 μl, and incubated at 80°C for 10 min. A master mix was then
added to the RNA/random hexamers reaction for a total reaction volume of 40 μl
containing 1
st
Strand buffer at 1X, 10 mM DTT, 0.5 mM dNTPs, and 100 U of
73
SuperScript reverse transcriptase. The reaction mix was incubated at 42°C for 1 h
and then at 70°C for 15 min.
PCR was performed with Invitrogen reagents using serial dilutions of cDNA in a
total reaction volume of 20 μl with PCR buffer at 1X, forward and reverse primers at
20 picomoles, dNTPs at 0.2 mM, and 0.5 U of Platinum Taq DNA polymerase
(Invitrogen). The primers were synthesized by the USC Norris Cancer Center
Microchemical Core. The primer sequences and the PCR conditions are as follows:
All PCR reactions started with 1 cycle of 3 min at 95°C and ended with 1 cycle of 5
min at 72°C.
pre-tRNA
Tyr
forward 5-CCTTCGATAGCTCAGCTGGTAGAGCGGAGG-3
reverse 5-CGGAATTGAACCAGCGACCTAAGGATGTCC-3
84 bp product
30 cycles of: 95°C for 1 min, 65°C for 30 sec, 72°C for 15 sec
pre-tRNA
Leu
forward 5-GTCAGGATGGCCGAGTGGTCTAAG-3
reverse 5-CCACGCCTCCATACGGAGAACCAGAAGACCC-3
88 bp product
32 cycles of: 95°C for 1 min, 65°C for 30 sec, 72°C for 15 sec
74
7SL forward 5-GTGTCCGCACTAAGTTCGGCATCAATATGG-3
reverse 5-TATTCACAGGCGCGATCCCACTACTGATC-3
150 bp product
25 cycles of: 94°C for 1 min, 70 °C for 30 sec, 72°C for 30 sec
-actin forward 5-CGACAACGGCTCCGGCATG-3
reverse 5-CTGGGGTGTTGAAGGTCTCAAACATG-3
317 bp product
33 cycles of: 94°C for 45 sec, 55°C for 30 sec, 72°C for 20 sec
GAPDH forward 5-TCCACCACCCTGTTGCTGTA-3
reverse 5-ACCACAGTCCATGCCATCAC-3
452 bp product
28 cycles of: 95°C for 30 sec, 66°C for 40 sec, 72°C for 1 min
The reaction products were resolved on an agarose gel containing ethidium bromide,
visualized using an AlphaImager 2000 (Alpha Innotech), quantified using Un-Scan-
It software (Silk Scientific), and graphed.
Chromatin immunoprecipitation (ChIP) assay
U87 PTEN-inducible cells were treated with doxycycline for 0, 6, or 24 hours.
Chromatin was isolated from 150 mm plates at ~ 45% confluency. Cells were fixed
75
with 1% formaldehyde at 25°C for 10 min, the reaction was stopped with 0.125M
glycine at 25°C for 5 min. The cells were scraped and washed with cold PBS
containing PMSF, resuspended in 1 ml of hypotonic lysis buffer (10 mM Hepes-
KOH pH 7.8, 10 mM KCl, 1.5 mM MgCl
2
) containing fresh PMSF and Protease
Inhibitor Cocktail Set III (Calbiochem), drawn up into and expelled from a syringe
with a 23 G needle 5 times, centrifuged for 5 min at 10,000 g at 4°C, resuspended in
500 μl to 1000 μl (depending on pellet size) of nucleus lysis buffer (50 mM Tris-Cl
pH 8.1, 10 mM EDTA, and 1% SDS) containing fresh PMSF and Protease Inhibitor
Cocktail Set III (Calbiochem), sonicated 8 times, and centrifuged for 10 min at
10,000 g at 4°C. The supernatant is the chromatin.
For the antibody pull-down of cross-linked chromatin, 900 μl of dilution buffer
(0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl pH 8.1, and 167
mM NaCl), 10 μl of Protease Inhibitor Cocktail Set III (Calbiochem), and 30 μl of
Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) were added to 100 μl
of chromatin, incubated on a rotating platform at 4°C for 30 min, and centrifuged for
5 min at 10,000 g at 4°C. 5 μl of antibodies against TBP, Brf1, Bdp1, or TFIIIC
102
were added to the supernatant and incubated on a rotating platform at 4°C overnight.
50 μl of Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) were then
added and incubated on a rotating platform at 4°C for 2 h. The beads were
centrifuged for 5 min at 10,000 g at 4°C, washed twice with wash buffer 1 (0.1%
76
SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl pH 8, and 150 mM NaCl),
washed once with wash buffer 2 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20
mM Tris-Cl pH 8, and 150 mM NaCl), washed once with wash buffer 3 (0.25 M
LiCl, 1% NP-40, 1% deoxycholic acid, 1 mM EDTA, and 10 mM Tris-Cl pH 8), and
washed 3 times with TE (10 mM Tris-Cl pH 8 and 1mM EDTA). 150 μl of elution
buffer was added to the beads which were then shaken on a vortex for 15 min and
centrifuged for 5 min at 10,000 g at 4°C. The supernatant was transferred to a new
tube and the step was repeated. To reverse the protein/DNA cross-linking, 19 μl of 5
M NaCl was added to the 300 μl of supernatant for a final concentration of 0.3 M
NaCl. For the input, 10 μl of chromatin was added to 290 μl of dilution buffer and
19 μl of 5 M NaCl. The samples were then incubated in a 65°C water bath overnight.
To digest the proteins, 80 μl of 5X proteinase K buffer (50 mM Tris-Cl pH 7.5, 25
mM EDTA, and 1.25% SDS) and 20 μg of proteinase K were added to the samples
which were then incubated in a 45°C water bath for 2 h.
To prepare the DNA for use in PCR, the ‘Desalting and Concentrating DNA from
Solutions’ protocol from the QIAEX II gel extraction kit (QIAGEN) was used.
Three volumes (1200 μl) of Buffer QX1 and 40 μl of QIAEX II beads were added to
the samples and incubated at 25°C for 10 min. The beads were washed twice with
Buffer PE and air dried. The following step was performed twice, 30 to 35 μl of 10
77
mM Tris-Cl pH 8.5 was added and incubated at 25°C for 5 min, the beads were spun
down and the supernatant collected. The DNA was used in real time PCR analysis.
Real time PCR (QPCR) analysis
QPCR analysis was performed using iQ SYBR Green Supermix (Bio-Rad) and the
Stratagene Mx3000P QPCR System according to the manufacturers’ instructions.
The pre-tRNA
Leu
primers were used at a final reaction concentration of 0.15 μM with
2 μl of DNA. Data analysis was performed by normalizing each time point (0, 6, or
24 h of doxycycline induction) to its respective input and then setting the value for
the control time point (0 h) to 1. This was done for each antibody separately. The
results were then graphed.
78
References
1. Baxter, G. C., and C. P. Stanners. 1978. The effect of protein degradation
on cellular growth characteristics. J Cell Physiol 96:139-45.
2. Birle, D., N. Bottini, S. Williams, H. Huynh, I. deBelle, E. Adamson, and
T. Mustelin. 2002. Negative feedback regulation of the tumor suppressor PTEN by
phosphoinositide-induced serine phosphorylation. J Immunol 169:286-91.
3. Boulanger, P. A., S. K. Yoshinaga, and A. J. Berk. 1987. DNA-binding
properties and characterization of human transcription factor TFIIIC2. J Biol Chem
262:15098-105.
4. Brown, T. R., P. H. Scott, T. Stein, A. G. Winter, and R. J. White. 2000.
RNA polymerase III transcription: its control by tumor suppressors and its
deregulation by transforming agents. Gene Expr 9:15-28.
5. Cairns, C. A., and R. J. White. 1998. p53 is a general repressor of RNA
polymerase III transcription. EMBO J 17:3112-23.
6. Campbell, R. B., F. Liu, and A. H. Ross. 2003. Allosteric activation of
PTEN phosphatase by phosphatidylinositol 4,5-bisphosphate. J Biol Chem
278:33617-20.
7. Cantley, L. C. 2002. The phosphoinositide 3-kinase pathway. Sci 296:1655-
7.
8. Chang, J., D. H. Kim, S. W. Lee, K. Y. Choi, and Y. C. Sung. 1995.
Transactivation ability of p53 transcriptional activation domain is directly related to
the binding affinity to TATA-binding protein. J Biol Chem 270:25014-9.
9. Chen, W., W. Bocker, J. Brosius, and H. Tiedge. 1997. Expression of
neural BC200 RNA in human tumours. J Pathol 183:345-51.
10. Chen, X., G. Farmer, H. Zhu, R. Prywes, and C. Prives. 1993.
Cooperative DNA binding of p53 with TFIID (TBP): a possible mechanism for
transcriptional activation. Genes Dev 7:1837-49.
11. Chen, X., L. J. Ko, L. Jayaraman, and C. Prives. 1996. p53 levels,
functional domains, and DNA damage determine the extent of the apoptotic response
of tumor cells. Genes Dev 10:2438-51.
79
12. Cheney, I. W., S. T. Neuteboom, M. T. Vaillancourt, M. Ramachandra,
and R. Bookstein. 1999. Adenovirus-mediated gene transfer of MMAC1/PTEN to
glioblastoma cells inhibits S phase entry by the recruitment of p27Kip1 into cyclin
E/CDK2 complexes. Cancer Res 59:2318-23.
13. Chesnokov, I., W. M. Chu, M. R. Botchan, and C. W. Schmid. 1996. p53
inhibits RNA polymerase III-directed transcription in a promoter-dependent manner.
Mol Cell Biol 16:7084-8.
14. Chu, W. M., Z. Wang, R. G. Roeder, and C. W. Schmid. 1997. RNA
polymerase III transcription repressed by Rb through its interactions with TFIIIB and
TFIIIC2. J Biol Chem 272:14755-61.
15. Chung, J. H., and C. Eng. 2005. Nuclear-cytoplasmic partitioning of
phosphatase and tensin homologue deleted on chromosome 10 (PTEN) differentially
regulates the cell cycle and apoptosis. Cancer Res 65:8096-100.
16. Crighton, D., A. Woiwode, C. Zhang, N. Mandavia, J. P. Morton, L. J.
Warnock, J. Milner, R. J. White, and D. L. Johnson. 2003. p53 represses RNA
polymerase III transcription by targeting TBP and inhibiting promoter occupancy by
TFIIIB. EMBO J 22:2810-20.
17. Dang, C. V. 1999. c-Myc target genes involved in cell growth, apoptosis, and
metabolism. Mol Cell Biol 19:1-11.
18. Das, S., J. E. Dixon, and W. Cho. 2003. Membrane-binding and activation
mechanism of PTEN. Proc Natl Acad Sci U S A 100:7491-6.
19. Dean, N., and A. J. Berk. 1988. Ordering promoter binding of class III
transcription factors TFIIIC1 and TFIIIC2. Mol Cell Biol 8:3017-25.
20. Diehl, J. A., F. Zindy, and C. J. Sherr. 1997. Inhibition of cyclin D1
phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-
proteasome pathway. Genes Dev 11:957-72.
21. Dingermann, T., S. Sharp, B. Appel, D. DeFranco, S. Mount, R.
Heiermann, O. Pongs, and D. Soll. 1981. Transcription of cloned tRNA and 5S
RNA genes in a Drosophila cell free extract. Nucleic Acids Res 9:3907-18.
80
22. Fairley, J. A., P. H. Scott, and R. J. White. 2003. TFIIIB is phosphorylated,
disrupted and selectively released from tRNA promoters during mitosis in vivo.
EMBO J 22:5841-50.
23. Fanning, A. S., and J. M. Anderson. 1996. Protein-protein interactions:
PDZ domain networks. Curr Biol 6:1385-8.
24. Farmer, G., J. Colgan, Y. Nakatani, J. L. Manley, and C. Prives. 1996.
Functional interaction between p53, the TATA-binding protein (TBP), andTBP-
associated factors in vivo. Mol Cell Biol 16:4295-304.
25. Felton-Edkins, Z. A., J. A. Fairley, E. L. Graham, I. M. Johnston, R. J.
White, and P. H. Scott. 2003. The mitogen-activated protein (MAP) kinase ERK
induces tRNA synthesis by phosphorylating TFIIIB. EMBO J 22:2422-32.
26. Felton-Edkins, Z. A., N. S. Kenneth, T. R. Brown, N. L. Daly, N. Gomez-
Roman, C. Grandori, R. N. Eisenman, and R. J. White. 2003. Direct regulation of
RNA polymerase III transcription by RB, p53 and c-Myc. Cell Cycle 2:181-4.
27. Foulds, C. E., and D. K. Hawley. 1997. Analysis of the human TATA
binding protein promoter and identification of an ets site critical for activity. Nucleic
Acids Research 25:2485-94.
28. Freeman, D. J., A. G. Li, G. Wei, H. H. Li, N. Kertesz, R. Lesche, A. D.
Whale, H. Martinez-Diaz, N. Rozengurt, R. D. Cardiff, X. Liu, and H. Wu.
2003. PTEN tumor suppressor regulates p53 protein levels and activity through
phosphatase-dependent and -independent mechanisms. Cancer Cell 3:117-30.
29. Gericke, A., M. Munson, and A. H. Ross. 2006. Regulation of the PTEN
phosphatase. Gene 374:1-9.
30. Gomez-Roman, N., C. Grandori, R. N. Eisenman, and R. J. White. 2003.
Direct activation of RNA polymerase III transcription by c-Myc. Nat 421:290-4.
31. Gottschalk, A. R., D. Basila, M. Wong, N. M. Dean, C. H. Brandts, D.
Stokoe, and D. A. Haas-Kogan. 2001. p27Kip1 is required for PTEN-induced G1
growth arrest. Cancer Res 61:2105-11.
32. Gridasova, A. A., and R. W. Henry. 2005. The p53 tumor suppressor
protein represses human snRNA gene transcription by RNA polymerases II and III
independently of sequence-specific DNA binding. Mol Cell Biol 25:3247-60.
81
33. Hannan, R. D., W. M. Hempel, A. Cavanaugh, T. Arino, S. I. Dimitrov,
T. Moss, and L. Rothblum. 1998. Affinity purification of mammalian RNA
polymerase I. Identification of an associated kinase. J Biol Chem 273:1257-67.
34. Hartl, P., J. Gottesfeld, and D. J. Forbes. 1993. Mitotic repression of
transcription in vitro. J Cell Biol 120:613-24.
35. Hay, N., and N. Sonenberg. 2004. Upstream and downstream of mTOR.
Genes Dev 18:1926-45.
36. Hernandez, N. 2001. Small nuclear RNA genes: a model system to study
fundamental mechanisms of transcription. J Biol Chem 276:26733-6.
37. Hirsch, H. A., L. Gu, and R. W. Henry. 2000. The retinoblastoma tumor
suppressor protein targets distinct general transcription factors to regulate RNA
polymerase III gene expression. Mol Cell Biol 20:9182-91.
38. Hollstein, M., D. Sidransky, B. Vogelstein, and C. C. Harris. 1991. p53
mutations in human cancers. Sci 253:49-53.
39. Horikoshi, N., A. Usheva, J. Chen, A. J. Levine, R. Weinmann, and T.
Shenk. 1995. Two domains of p53 interact with the TATA-binding protein, and the
adenovirus 13S E1A protein disrupts the association, relieving p53-mediated
transcriptional repression. Mol Cell Biol 15:227-34.
40. Hsieh, Y. J., T. K. Kundu, Z. Wang, R. Kovelman, and R. G. Roeder.
1999. The TFIIIC90 subunit of TFIIIC interacts with multiple components of the
RNA polymerase III machinery and contains a histone-specific acetyltransferase
activity. Mol Cell Biol 19:7697-704.
41. Hsieh, Y. J., Z. Wang, R. Kovelman, and R. G. Roeder. 1999. Cloning and
characterization of two evolutionarily conserved subunits (TFIIIC102 and TFIIIC63)
of human TFIIIC and their involvement in functional interactions with TFIIIB and
RNA polymerase III. Mol Cell Biol 19:4944-52.
42. Hu, P., K. Samudre, S. Wu, Y. Sun, and N. Hernandez. 2004. CK2
phosphorylation of Bdp1 executes cell cycle-specific RNA polymerase III
transcription repression. Mol Cell 16:81-92.
43. Hu, P., S. Wu, and N. Hernandez. 2003. A minimal RNA polymerase III
transcription system from human cells reveals positive and negative regulatory roles
for CK2. Mol Cell 12:699-709.
82
44. Huang, Y., and R. J. Maraia. 2001. Comparison of the RNA polymerase III
transcription machinery in Schizosaccharomyces pombe, Saccharomyces cerevisiae
and human. Nucleic Acids Res 29:2675-90.
45. Johnson, S. A., L. Dubeau, M. Kawalek, A. Dervan, A. H. Schonthal, C.
V. Dang, and D. L. Johnson. 2003. Increased expression of TATA-binding protein,
the central transcription factor, can contribute to oncogenesis. Mol Cell Biol
23:3043-51.
46. Johnson, S. A., N. Mandavia, H. D. Wang, and D. L. Johnson. 2000.
Transcriptional regulation of the TATA-binding protein by Ras cellular signaling.
Mol Cell Biol 20:5000-9.
47. Johnston, I. M., S. J. Allison, J. P. Morton, L. Schramm, P. H. Scott, and
R. J. White. 2002. CK2 forms a stable complex with TFIIIB and activates RNA
polymerase III transcription in human cells. Mol Cell Biol 22:3757-68.
48. Khosravi-Far, R., S. Campbell, K. L. Rossman, and C. J. Der. 1998.
Increasing complexity of Ras signal transduction: involvement of Rho family
proteins. Adv Cancer Res 72:57-107.
49. Kuglstatter, A., C. Oubridge, and K. Nagai. 2002. Induced structural
changes of 7SL RNA during the assembly of human signal recognition particle. Nat
Struct Biol 9:740-4.
50. Kundu, T. K., Z. Wang, and R. G. Roeder. 1999. Human TFIIIC relieves
chromatin-mediated repression of RNA polymerase III transcription and contains an
intrinsic histone acetyltransferase activity. Mol Cell Biol 19:1605-15.
51. Lee, J. O., H. Yang, M. M. Georgescu, A. Di Cristofano, T. Maehama, Y.
Shi, J. E. Dixon, P. Pandolfi, and N. P. Pavletich. 1999. Crystal structure of the
PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity
and membrane association. Cell 99:323-34.
52. Li, D. M., and H. Sun. 1998. PTEN/MMAC1/TEP1 suppresses the
tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells. Proc
Natl Acad Sci U S A 95:15406-11.
53. Lian, Z., and A. Di Cristofano. 2005. Class reunion: PTEN joins the nuclear
crew. Oncogene 24:7394-400.
83
54. Lin, C. Y., S. Navarro, S. Reddy, and L. Comai. 2006. CK2-mediated
stimulation of Pol I transcription by stabilization of UBF-SL1 interaction. Nucleic
Acids Res 34:4752-66.
55. Liu, F., S. Wagner, R. B. Campbell, J. A. Nickerson, C. A. Schiffer, and
A. H. Ross. 2005. PTEN enters the nucleus by diffusion. J Cell Biochem 96:221-34.
56. Liu, X., C. W. Miller, P. H. Koeffler, and A. J. Berk. 1993. The p53
activation domain binds the TATA box-binding polypeptide in Holo-TFIID, and a
neighboring p53 domain inhibits transcription. Mol Cell Biol 13:3291-300.
57. Lundberg, A. S., and R. A. Weinberg. 1998. Functional inactivation of the
retinoblastoma protein requires sequential modification by at least two distinct
cyclin-cdk complexes. Mol Cell Biol 18:753-61.
58. Mack, D. H., J. Vartikar, J. M. Pipas, and L. A. Laimins. 1993. Specific
repression of TATA-mediated but not initiator-mediated transcription by wild-type
p53. Nat 363:281-3.
59. Maehama, T., and J. E. Dixon. 1999. PTEN: a tumour suppressor that
functions as a phospholipid phosphatase. Trends Cell Biol 9:125-8.
60. Maehama, T., and J. E. Dixon. 1998. The tumor suppressor,
PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol
3,4,5-trisphosphate. J Biol Chem 273:13375-8.
61. Mamillapalli, R., N. Gavrilova, V. T. Mihaylova, L. M. Tsvetkov, H. Wu,
H. Zhang, and H. Sun. 2001. PTEN regulates the ubiquitin-dependent degradation
of the CDK inhibitor p27(KIP1) through the ubiquitin E3 ligase SCF(SKP2). Curr
Biol 11:263-7.
62. Mateyak, M. K., A. J. Obaya, S. Adachi, and J. M. Sedivy. 1997.
Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous
recombination. Cell Growth Differ 8:1039-48.
63. Matsushima-Nishiu, M., M. Unoki, K. Ono, T. Tsunoda, T. Minaguchi,
H. Kuramoto, M. Nishida, T. Satoh, T. Tanaka, and Y. Nakamura. 2001.
Growth and gene expression profile analyses of endometrial cancer cells expressing
exogenous PTEN. Cancer Res 61:3741-9.
84
64. Mayer, C., and I. Grummt. 2006. Ribosome biogenesis and cell growth:
mTOR coordinates transcription by all three classes of nuclear RNA polymerases.
Oncogene 25:6384-91.
65. Mayo, L. D., and D. B. Donner. 2001. A phosphatidylinositol 3-kinase/Akt
pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc
Natl Acad Sci U S A 98:11598-603.
66. McConnachie, G., I. Pass, S. M. Walker, and C. P. Downes. 2003.
Interfacial kinetic analysis of the tumour suppressor phosphatase, PTEN: evidence
for activation by anionic phospholipids. Biochem J 371:947-55.
67. McCulloch, V., P. Hardin, W. Peng, J. M. Ruppert, and S. M. Lobo-
Ruppert. 2000. Alternatively spliced hBRF variants function at different RNA
polymerase III promoters. EMBO J 19:4134-43.
68. Medema, R. H., G. J. Kops, J. L. Bos, and B. M. Burgering. 2000. AFX-
like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB
through p27kip1. Nat 404:782-7.
69. Miller, J., A. D. McLachlan, and A. Klug. 1985. Repetitive zinc-binding
domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J
4:1609-14.
70. Mittnacht, S. 1998. Control of pRB phosphorylation. Curr Opin Genet Dev
8:21-7.
71. Myers, M. P., I. Pass, I. H. Batty, J. Van der Kaay, J. P. Stolarov, B. A.
Hemmings, M. H. Wigler, C. P. Downes, and N. K. Tonks. 1998. The lipid
phosphatase activity of PTEN is critical for its tumor supressor function. Proc Natl
Acad Sci U S A 95:13513-8.
72. Myers, M. P., J. P. Stolarov, C. Eng, J. Li, S. I. Wang, M. H. Wigler, R.
Parsons, and N. K. Tonks. 1997. P-TEN, the tumor suppressor from human
chromosome 10q23, is a dual-specificity phosphatase. Proc Natl Acad Sci U S A
94:9052-7.
73. Nakamura, N., S. Ramaswamy, F. Vazquez, S. Signoretti, M. Loda, and
W. R. Sellers. 2000. Forkhead transcription factors are critical effectors of cell death
and cell cycle arrest downstream of PTEN. Mol Cell Biol 20:8969-82.
85
74. Paramio, J. M., M. Navarro, C. Segrelles, E. Gomez-Casero, and J. L.
Jorcano. 1999. PTEN tumour suppressor is linked to the cell cycle control through
the retinoblastoma protein. Oncogene 18:7462-8.
75. Paule, M. R., and R. J. White. 2000. Survey and summary: transcription by
RNA polymerases I and III. Nucleic Acids Res 28:1283-98.
76. Prescott, D. M., and M. A. Bender. 1962. Synthesis of RNA and protein
during mitosis in mammalian tissue culture cells. Exp Cell Res 26:260-8.
77. Radu, A., V. Neubauer, T. Akagi, H. Hanafusa, and M. M. Georgescu.
2003. PTEN induces cell cycle arrest by decreasing the level and nuclear localization
of cyclin D1. Mol Cell Biol 23:6139-49.
78. Ragimov, N., A. Krauskopf, N. Navot, V. Rotter, M. Oren, and Y. Aloni.
1993. Wild-type but not mutant p53 can repress transcription initiation in vitro by
interfering with the binding of basal transcription factors to the TATA motif.
Oncogene 8:1183-93.
79. Ramaswamy, S., N. Nakamura, F. Vazquez, D. B. Batt, S. Perera, T. M.
Roberts, and W. R. Sellers. 1999. Regulation of G1 progression by the PTEN
tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-
kinase/Akt pathway. Proc Natl Acad Sci U S A 96:2110-5.
80. Rodriguez-Viciana, P., P. H. Warne, A. Khwaja, B. M. Marte, D. Pappin,
P. Das, M. D. Waterfield, A. Ridley, and J. Downward. 1997. Role of
phosphoinositide 3-OH kinase in cell transformation and control of the actin
cytoskeleton by Ras. Cell 89:457-67.
81. Schmidt, E. V. 1999. The role of c-myc in cellular growth control. Oncogene
18:2988-96.
82. Schramm, L., P. S. Pendergrast, Y. Sun, and N. Hernandez. 2000.
Different human TFIIIB activities direct RNA polymerase III transcription from
TATA-containing and TATA-less promoters. Genes Dev 14:2650-63.
83. Scott, M. R., K. H. Westphal, and P. W. Rigby. 1983. Activation of mouse
genes in transformed cells. Cell 34:557-67.
84. Scott, P. H., C. A. Cairns, J. E. Sutcliffe, H. M. Alzuherri, A. McLees, A.
G. Winter, and R. J. White. 2001. Regulation of RNA polymerase III transcription
during cell cycle entry. J Biol Chem 276:1005-14.
86
85. Seto, E., A. Usheva, G. P. Zambetti, J. Momand, N. Horikoshi, R.
Weinmann, A. J. Levine, and T. Shenk. 1992. Wild-type p53 binds to the TATA-
binding protein and represses transcription. Proc Natl Acad Sci U S A 89:12028-32.
86. Shen, W. H., A. S. Balajee, J. Wang, H. Wu, C. Eng, P. P. Pandolfi, and
Y. Yin. 2007. Essential role for nuclear PTEN in maintaining chromosomal integrity.
Cell 128:157-70.
87. Stambolic, V., D. MacPherson, D. Sas, Y. Lin, B. Snow, Y. Jang, S.
Benchimol, and T. W. Mak. 2001. Regulation of PTEN transcription by p53. Mol
Cell 8:317-25.
88. Steck, P. A., M. A. Pershouse, S. A. Jasser, W. K. Yung, H. Lin, A. H.
Ligon, L. A. Langford, M. L. Baumgard, T. Hattier, T. Davis, C. Frye, R. Hu, B.
Swedlund, D. H. Teng, and S. V. Tavtigian. 1997. Identification of a candidate
tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in
multiple advanced cancers. Nat Genet 15:356-62.
89. Stefanovsky, V. Y., G. Pelletier, R. Hannan, T. Gagnon-Kugler, L. I.
Rothblum, and T. Moss. 2001. An immediate response of ribosomal transcription to
growth factor stimulation in mammals is mediated by ERK phosphorylation of UBF.
Mol Cell 8:1063-73.
90. Sun, H., R. Lesche, D. M. Li, J. Liliental, H. Zhang, J. Gao, N. Gavrilova,
B. Mueller, X. Liu, and H. Wu. 1999. PTEN modulates cell cycle progression and
cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein
kinase B signaling pathway. Proc Natl Acad Sci U S A 96:6199-204.
91. Sutcliffe, J. E., T. R. Brown, S. J. Allison, P. H. Scott, and R. J. White.
2000. Retinoblastoma protein disrupts interactions required for RNA polymerase III
transcription. Mol Cell Biol 20:9192-202.
92. Sutcliffe, J. E., C. A. Cairns, A. McLees, S. J. Allison, K. Tosh, and R. J.
White. 1999. RNA polymerase III transcription factor IIIB is a target for repression
by pocket proteins p107 and p130. Mol Cell Biol 19:4255-61.
93. Torres, J., and R. Pulido. 2001. The tumor suppressor PTEN is
phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN
stability to proteasome-mediated degradation. J Biol Chem 276:993-8.
94. Trotman, L. C., and P. P. Pandolfi. 2003. PTEN and p53: who will get the
upper hand? Cancer Cell 3:97-9.
87
95. Trotman, L. C., X. Wang, A. Alimonti, Z. Chen, J. Teruya-Feldstein, H.
Yang, N. P. Pavletich, B. S. Carver, C. Cordon-Cardo, H. Erdjument-Bromage,
P. Tempst, S. G. Chi, H. J. Kim, T. Misteli, X. Jiang, and P. P. Pandolfi. 2007.
Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128:141-
56.
96. Truant, R., H. Xiao, C. J. Ingles, and J. Greenblatt. 1993. Direct
interaction between the transcriptional activation domain of human p53 and the
TATA box-binding protein. J Biol Chem 268:2284-7.
97. Vazquez, F., S. R. Grossman, Y. Takahashi, M. V. Rokas, N. Nakamura,
and W. R. Sellers. 2001. Phosphorylation of the PTEN tail acts as an inhibitory
switch by preventing its recruitment into a protein complex. J Biol Chem 276:48627-
30.
98. Vazquez, F., S. Ramaswamy, N. Nakamura, and W. R. Sellers. 2000.
Phosphorylation of the PTEN tail regulates protein stability and function. Mol Cell
Biol 20:5010-8.
99. Vivanco, I., and C. L. Sawyers. 2002. The phosphatidylinositol 3-Kinase
AKT pathway in human cancer. Nat Rev Cancer 2:489-501.
100. Voit, R., and I. Grummt. 2001. Phosphorylation of UBF at serine 388 is
required for interaction with RNA polymerase I and activation of rDNA
transcription. Proc Natl Acad Sci U S A 98:13631-6.
101. Wang, H. D., A. Trivedi, and D. L. Johnson. 1997. Hepatitis B virus X
protein induces RNA polymerase III-dependent gene transcription and increases
cellular TATA-binding protein by activating the Ras signaling pathway. Mol Cell
Biol 17:6838-46.
102. Wang, H. D., A. Trivedi, and D. L. Johnson. 1998. Regulation of RNA
polymerase I-dependent promoters by the hepatitis B virus X protein via activated
Ras and TATA-binding protein. Mol Cell Biol 18:7086-94.
103. Wang, Z., and R. G. Roeder. 1995. Structure and function of a human
transcription factor TFIIIB subunit that is evolutionarily conserved and contains both
TFIIB- and high-mobility-group protein 2-related domains. Proc Natl Acad Sci U S
A 92:7026-30.
88
104. Wang, Z., and R. G. Roeder. 1997. Three human RNA polymerase III-
specific subunits form a subcomplex with a selective function in specific
transcription initiation. Genes Dev 11:1315-26.
105. Weng, L. P., W. M. Smith, P. L. Dahia, U. Ziebold, E. Gil, J. A. Lees, and
C. Eng. 1999. PTEN suppresses breast cancer cell growth by phosphatase activity-
dependent G1 arrest followed by cell death. Cancer Res 59:5808-14.
106. Weser, S., C. Gruber, H. M. Hafner, M. Teichmann, R. G. Roeder, K. H.
Seifart, and W. Meissner. 2004. Transcription factor (TF)-like nuclear regulator,
the 250-kDa form of Homo sapiens TFIIIB", is an essential component of human
TFIIIC1 activity. J Biol Chem 279:27022-9.
107. Weser, S., J. Riemann, K. H. Seifart, and W. Meissner. 2003. Assembly
and isolation of intermediate steps of transcription complexes formed on the human
5S rRNA gene. Nucleic Acids Res 31:2408-16.
108. White, M. A., C. Nicolette, A. Minden, A. Polverino, L. Van Aelst, M.
Karin, and M. H. Wigler. 1995. Multiple Ras functions can contribute to
mammalian cell transformation. Cell 80:533-41.
109. White, R. J. 2005. RNA polymerases I and III, growth control and cancer.
Nat Rev Mol Cell Biol 6:69-78.
110. White, R. J., T. M. Gottlieb, C. S. Downes, and S. P. Jackson. 1995. Cell
cycle regulation of RNA polymerase III transcription. Mol Cell Biol 15:6653-62.
111. White, R. J., T. M. Gottlieb, C. S. Downes, and S. P. Jackson. 1995.
Mitotic regulation of a TATA-binding-protein-containing complex. Mol Cell Biol
15:1983-92.
112. White, R. J., B. C. Khoo, J. A. Inostroza, D. Reinberg, and S. P. Jackson.
1994. Differential regulation of RNA polymerases I, II, and III by the TBP-binding
repressor Dr1. Sci 266:448-50.
113. White, R. J., D. Stott, and P. W. Rigby. 1990. Regulation of RNA
polymerase III transcription in response to Simian virus 40 transformation. EMBO J
9:3713-21.
114. White, R. J., D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides.
1996. Repression of RNA polymerase III transcription by the retinoblastoma protein.
Nat 382:88-90.
89
115. Willis, I. M. 1993. RNA polymerase III. Genes, factors and transcriptional
specificity. Eur J Biochem 212:1-11.
116. Winter, A. G., G. Sourvinos, S. J. Allison, K. Tosh, P. H. Scott, D. A.
Spandidos, and R. J. White. 2000. RNA polymerase III transcription factor
TFIIIC2 is overexpressed in ovarian tumors. Proc Natl Acad Sci U S A 97:12619-24.
117. Wu, X., K. Senechal, M. S. Neshat, Y. E. Whang, and C. L. Sawyers.
1998. The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative
regulator of the phosphoinositide 3-kinase/Akt pathway. Proc Natl Acad Sci U S A
95:15587-91.
118. Zarkowska, T., and S. Mittnacht. 1997. Differential phosphorylation of the
retinoblastoma protein by G1/S cyclin-dependent kinases. J Biol Chem 272:12738-
46.
119. Zhai, W., and L. Comai. 2000. Repression of RNA polymerase I
transcription by the tumor suppressor p53. Mol Cell Biol 20:5930-8.
120. Zhang, C., L. Comai, and D. L. Johnson. 2005. PTEN represses RNA
Polymerase I transcription by disrupting the SL1 complex. Mol Cell Biol 25:6899-
911.
121. Zhao, J., X. Yuan, M. Frodin, and I. Grummt. 2003. ERK-dependent
phosphorylation of the transcription initiation factor TIF-IA is required for RNA
polymerase I transcription and cell growth. Mol Cell 11:405-13.
122. Zhou, B. P., Y. Liao, W. Xia, Y. Zou, B. Spohn, and M. C. Hung. 2001.
HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation.
Nat Cell Biol 3:973-82.

Abstract (if available)

Abstract For cells to grow and proliferate they must be capable of protein synthesis. As RNA polymerase (pol) III transcription products are required for protein synthesis, RNA pol III transcriptional activity is tightly linked to the rate of cell growth. High levels of RNA pol III transcription products are seen in transformed and tumor cells, suggesting that aberrant regulation of RNA pol III transcription contributes to transformation and tumorigenesis. The tumor suppressor p53 represses RNA pol III transcription by directly targeting the RNA pol III transcription factor complex TFIIIB which consists of TBP, Brf1, and Bdp1. TBP and Brf1 associate to form a stable complex while Bdp1 associates reversibly with the complex. Our studies determined that p53 mediates repression of RNA pol III transcription by directly targeting TBP which is limiting for RNA pol III transcription in the presence but not the absence of p53 expression. In contrast, Brf1 is not limiting for RNA pol III transcription. Although p53 binds to the TBP/Brf1 complex, it does not disrupt the complex. As p53 mediates repression of RNA pol III transcription, we determined whether another tumor suppressor, PTEN, represses RNA pol III transcription. Whereas p53 is a transcription factor that functions in the nucleus, PTEN is a lipid phosphatase that functions largely in the cytoplasm. The lipid phosphatase activity of PTEN directly antagonizes the PI-3 kinase (PI3K) signal transduction pathway. We determined that PTEN mediates repression of RNA pol III transcription via its lipid phosphatase activity.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

RNA polymerase III-dependent transcription is repressed under prolonged hypoxic conditions

PDF

Mechanism of repression of RNA polymerase III-dependent transcription under hypoxia

PDF

SUMOylation regulates RNA polymerase III -- dependent transcripton via MAF1

PDF

Positive regulation of RNA polymerase III-mediated transcription of tRNA genes by the Mediator kinase submodule

PDF

Novel roles for Maf1 in embryonic stem cell differentiation and adipogenesis

PDF

Maf1 is a novel target of the tumor suppressor PTEN and a negative regulator of lipid metabolism

PDF

Vpr-binding protein negatively regulates p53 by site-specific phosphorylation through intrinsic kinase activity

PDF

Steatosis induces Wnt/β-catenin pathway to stimulate proliferation of hepatic tumor initiating cells and promote liver cancer development

Asset Metadata

Creator Woiwode, Annette (author)

Core Title Repression of RNA polymerase III-dependent transcription by the tumor suppressors p53 and PTEN

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology

Publication Date 04/22/2007

Defense Date 03/19/2007

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

Tag gene transcription,OAI-PMH Harvest,p53,PTEN,RNA polymerase III,tumor suppressors

Language English

Advisor Johnson, Deborah L. (committee chair), Ann, David K. (committee member), Comai, Lucio (committee member)

Creator Email woiwode@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m437

Unique identifier UC1470084

Identifier etd-Woiwode-20070422 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-493020 (legacy record id),usctheses-m437 (legacy record id)

Legacy Identifier etd-Woiwode-20070422.pdf

Dmrecord 493020

Document Type Thesis

Rights Woiwode, Annette

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu