From the George Whipple Laboratory for Cancer Research, Department of Pathology, Urology, Radiation Oncology, and the Cancer Center, University of Rochester Medical Center, Rochester, New York 14642
Received for publication, March 17, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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Androgen receptor (AR) may communicate with the
general transcription machinery on the core promoter to exert its
function as a transcriptional modulator. Our previous report
demonstrated that the AR interacted with transcription factor IIH
(TFIIH) under physiological conditions and that overexpression of
Cdk-activating kinase, the kinase moiety of TFIIH,
enhanced AR-mediated transcription in prostate cancer cells. In an
effort to further dissect the mechanisms implicated in AR
transactivation, we report here that AR interacts with PITALRE, a
kinase subunit of positive elongation factor b (P-TEFb). Cotransfection
of the plasmid encoding the mutant PITALRE (mtPITALRE), defective in
its RNA polymerase II COOH-terminal domain (CTD)-kinase activity,
resulted in preferential inhibition of AR-mediated transactivation.
Indeed, AR transactivation in PC-3 cells was preferentially inhibited
at the low concentration of
5,6-dicloro-1- Molecular studies of eukaryotic transcription suggest that the
process of transcription can be divided into the following steps:
preinitiation complex assembly on the core promoter, initiation, promoter clearance, elongation, and termination (1). To initiate transcription, general transcription factors need to be recruited to
the promoter either in a stepwise fashion or in a form of holoenzyme (1). The promoter clearance is defined as a point when RNA polymerase
II leaves the initiation complex to start elongation of transcripts
(2). Phosphorylation of the COOH-terminal domain (CTD)1 of the largest subunit
of RNA polymerase II is required to establish and maintain the
elongation complex (3, 4).
Activators have been demonstrated to stimulate one or more steps of the
transcription cycle by direct or indirect communication with the
general transcription factors (1, 5). In addition, activators may also
interact with auxiliary factors, called coregulators, to enhance
recruitment of the general transcription machinery on the promoter (6,
7). Direct interactions of activators with coregulators and/or general
transcription factors have been suggested to be mechanisms for
transcriptional activation (5-7). Nuclear run-on transcription and
RNase protection analyses revealed three classes of activation domains
(8). Type 1 activators, such as Sp1 and CTF, stimulate an initiation
stage of transcription. Type 2A activators, such as Tat encoded by
human immunodeficiency virus type 1, stimulate an elongation stage,
thus type 2A activators may prevent abortive elongation by arrest of
RNA polymerase II at poorly defined sites. Type 2B activators, such as
VP16 and p53, stimulate both an initiation and an elongation stage.
The AR is a member of the steroid receptor superfamily that is
composed of a variable amino-terminal domain, a highly conserved DNA-binding domain, and a ligand-binding domain (9).
Ligand-dependent transcriptional activation of steroid
receptors is mediated by the COOH-terminal domain that includes a
ligand-binding domain and activation function-2 (10). Crystallographic
studies show that ligand-bound steroid receptors undergo a
conformational change in the activation function-2 core motif (11, 12).
The ligand-induced conformational change presumably recruits
coregulators as well as the basal transcriptional machinery for the
target gene expression (6, 7). The coregulators of nuclear receptors
(ARA24, ARA54, ARA55, ARA70, ARA160, CBP/p300, p/CIP/ACTR/AIB1, Rb,
RIP140, SRC-1/NCoA-1, TIF-2/GRIP1, and TRAPs/DRIPs) have recently been
cloned and characterized (6, 7, 13-17). It has been proposed that
coregulators function as a bridge between activators and the basal
transcription machinery (5-7). They may potentiate transactivation of
nuclear receptors in transient transfection or in in vitro
transcription assays through the modification of nucleosomal structure
or the efficient recruitment of basal transcription machinery (6, 7). A
growing number of coregulators, such as SRC-1, ACTR, and PCAF, of
steroid receptors have been reported to possess and/or recruit histone acetyltransferase activity to induce modification of nucleosomal structures leading to activation of transcription (6). In contrast to
coactivators, corepressors, such as NCoR-1 and SMRT, bind to the
nuclear receptors in the absence of ligands, recruit histone deacetyltransferase, and lead to condensation of nucleosomal structures for repression of transcription (6, 18).
The amino-terminal domain of steroid receptors contains a
ligand-independent activation function-1, which is under the control of
activation function-2 (10). The amino-terminal domain of steroid
receptors has been reported to interact with general transcription factors, as exemplified by AR interaction with transcription factor IIF
(TFIIF) (19) and transcription factor IIH (TFIIH) (20). Transcription
factor IIB has been reported to interact with thyroid receptor (21),
vitamin D receptor (22), and hepatocyte nuclear factor 4 (23). However,
the molecular mechanism by which activation function-1 synergistically
activates transcription remains unclear.
We reported previously that the AR interacted with TFIIH under
physiological conditions and that overexpression of
Cdk-activating kinase, the kinase moiety of TFIIH,
enhanced AR-mediated transactivation in prostate cancer cells (20). In
an effort to further dissect the mechanisms implicated in AR
transactivation, we found that AR interacts with PITALRE, a kinase
subunit of positive elongation factor b (P-TEFb) (24), and that
cotransfection of the plasmid encoding the mutant PITALRE (mtPITALRE),
which is defective in its CTD kinase activity (25), results in
preferential inhibition of AR-mediated transactivation. AR
transactivation is also preferentially inhibited at the low
concentration of DRB, a CTD kinase inhibitor. In addition, a nuclear
run-on transcription assay of the PSA gene, an androgen-inducible gene,
using LNCaP nuclei showed that the transcription efficiency of the
distal region of the gene was enhanced upon androgen induction. These
results suggest that AR interacts with TFIIH and P-TEFb and enhances
the elongation stage of transcription.
Plasmids--
The complementary DNA fragments for PITALRE and
mtPITALRE were generous gifts (25) and subcloned into the eukaryotic
expression vector pSG5 (Stratagene). The complementary DNA fragment for
negative elongation factor-D (NELF-D) (26) was generated by polymerase chain reaction and subcloned into pSG5.
Cell Culture and Transfection Assay--
DU145 and PC-3 cells
were maintained in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% heat inactivated fetal
bovine serum (FBS) and Dulbecco's modified Eagle's medium/F-12
supplemented with 7% FBS, respectively. Non-prostate cancer H1299 and
HeLa cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% FBS. All media contain 50 units/ml penicillin
and 50 µg/ml streptomycin. Cells were seeded to be a density of
50-60% confluence for transfection. Cells in 35-mm dishes were refed
with fresh medium 2 h before transfection and transfected with 2 µg of DNA according to the "SuperFect transfection" instructions
(Qiagen). After 2-3-h incubation, cells were treated with medium
supplemented with charcoal-dextran-treated FBS containing either
ethanol or ligands. Cells were further incubated at 37 °C for
24 h, washed with PBS, and harvested. Cell lysates were prepared
and used for luciferase assay according to the manufacturer's instructions (Promega). Relative luciferase activities were plotted using the activity of AR in the absence of ligand and coactivator as 1. The results were obtained from at least three sets of transfection and
presented as mean ± S.D.
Coimmunoprecipitation--
LNCaP whole cell extracts were
prepared as described previously (27) and aliquots stored at
Biochemical Binding Assay--
A recombinant protein, 6 histidine-tagged AR amino-terminal and DNA-binding domain (AR-NDBD),
amino acids from 38 to 643, was expressed in Escherichia
coli. Bacterial cells were lysed in 5 ml of binding buffer (20 mM HEPES (pH 7.5), 500 mM NaCl, 20 mM imidazole, 5 mM
Proteins obtained from 50 ml of culture were incubated with 100 µl
bed volume of Ni2+ resins. The resins were incubated with
35S-labeled TNT-expressed PITALRE for 4 h and washed
extensively with 20 mM HEPES (pH 7.5), 0.5 mM
EDTA, 20% glycerol, and 400 mM NaCl. The bound proteins
were eluted by 2× SDS loading buffer (100 mM Tris·HCl
(pH 6.8), 4% SDS, 20% glycerol, and 200 mM
Nuclear Run-on Transcription--
Nuclear run-on transcription
assay was performed as described elsewhere (28). Briefly, LNCaP cells
were maintained in RPMI 1640 supplemented with 10% FBS. Cells at
~60-70% confluence were treated with medium supplemented with
charcoal-dextran-treated FBS containing either ethanol or 1 nM DHT. Cells were further incubated at 37 °C for
16 h, washed with PBS, and harvested. Cells were resuspended in 10 mM Tris·HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40 and incubated on
ice for 10 min. The nuclei pellets were spun down at 500 × g and resuspended in 10 mM Tris·HCl (pH 8.0),
40% glycerol, 5 mM MgCl2, and 0.1 mM EDTA. Nuclei were frozen and stored in liquid nitrogen
in portions of 100 µl corresponding to 2 × 107
nuclei. The nuclei were mixed with 100 µl of 10 mM
Tris·HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl, 0.5 mM each ATP, GTP, UTP, and 100 µCi of [
The DNA fragments containing the PSA exons 1 and 2 were obtained
by polymerase chain reaction of the plasmid containing the genomic PSA.
The DNA fragments were gel-purified and digested with EcoRI.
The distal and proximal PSA DNA fragments (Fig. 6A) were
gel-purified. The plasmid-containing 7SK gene (29) was digested with
PstI. Each DNA fragment was denatured and immobilized onto
the nylon membrane using a slot blot (Schleicher & Schuell) as
described elsewhere (28). The membrane was prehybridized for 2 h
at 60 °C in 6× SSC, 10× Denhardt's reagent, 1% SDS, and 100 µg/ml denatured salmon sperm DNA. Hybridization was carried out at
60 °C for 24 h in 6× SSC, 1.0% SDS, 100 µg/ml denatured salmon sperm DNA, and 1 × 106 cpm of labeled RNA
transcripts. The filters were washed twice in 2× SSC for 30 min at
60 °C and then treated in 2× SSC containing RNase A (5 µg/ml) for
20 min at 30 °C to remove unhybridized regions of RNA. The filters
were washed twice in 2× SSC for 10 min at 37 °C. Signals were
detected using a Molecular Dynamics PhosphorImager.
AR Interaction with TFIIH--
Biochemical studies of
protein-protein interactions between AR and the general transcription
factors indicated that AR may interact with TFIIH and TFIIF under
physiological conditions (19, 20). Recruitment of TFIIH completes the
assembly of the preinitiation complex on the promoter and results in
promoter opening and/or the early elongation/promoter clearance steps
(1, 3). The kinase activity in TFIIH has been reported to phosphorylate
the CTD of the largest subunit of RNA polymerase II (3) and to stimulate the elongation stage of transcription by several activators (8). TFIIH-mediated CTD phosphorylation could lead to promoter clearance by dissociation of proteins recruited for the initiation steps of the preinitiation complex assembly, resulting in
establishing an elongation-competent transcription complex (30). The
significance of CTD phosphorylation for the elongation stage of
transcription was demonstrated by Yankulov et al. (31). The
elongation stage of transcription in Xenopus oocytes was
inhibited by microinjection of antibodies against TFIIH subunits, but
not by microinjection of antibodies against TFIIB, a general
transcription factor specific for transcription initiation. In summary,
phosphorylation of the CTD of RNA polymerase II may be associated with
the transition from the initiation to the elongation stage of
transcription (30). Based on intensive molecular and biochemical
studies of transcription mechanisms, interaction of AR with TFIIH
reported in our previous study led us to analyze whether AR enhances
transcription mainly at the elongation stage of transcription.
The Mutant PITALRE (mtPITALRE) Inhibits AR Transactivation in
Prostate Cancer Cells--
Given the fact that general elongation
factors, such as P-TEFb, transcription factor IIS, and Elongins, also
regulate the elongation stage of transcription (4), we analyzed the
effects of positive and negative elongation factors on AR-mediated
transcription. P-TEFb is composed of 124- and 43-kDa polypeptides and a
key regulator controlling RNA polymerase II in the elongation stage of
transcription (24). The small subunit of P-TEFb, PITALRE, possesses
protein kinase activity capable of phosphorylating the CTD of the
largest subunit of RNA polymerase II, which has been known to be a key step required to enter an elongation mode from the preinitiation complex formation on the promoter (4). Recent studies show that
the Tat protein encoded by the human type 1 immunodeficiency virus
(HIV-1) genome, a notable transcriptional modulator, which activates
the elongation stage of transcription, requires P-TEFb kinase activity
for the efficient transactivation both in vivo and in
vitro (25, 32).
Since P-TEFb is an abundant general elongation factor, cotransfection
of the PITALRE wild type expression plasmid showed little, if any,
effect on transcription of the reporter gene (data not shown). Thus, we
used mtPITALRE, defective in its kinase activity (25), to analyze the
effect of P-TEFb on AR-mediated transcription. It is necessary to
compare the inhibitory effect of mtPITALRE on transcription with the
promoter containing AR-responsible elements to the effect on
transcription with the other promoters. We took advantage of a dual
luciferase assay (Promega) using pMMTV-luciferase as a reporter gene
and pRLSV40-luciferase as an internal control. Cells were cotransfected
with pMMTV-luciferase, pRLSV40-luciferase, the AR expression plasmid,
and variable amounts of the mtPITALRE expression plasmid.
Overexpression of mtPITALRE inhibited AR-mediated transcription from
the MMTV-reporter DNA ~10-fold (lane 6 versus lane 9 in Fig. 1) and SV40
enhancer-mediated transcription from the pRLSV40-luciferase less than
3-fold (lane 10 versus lane 13 in Fig.
1) in both prostate cancer PC-3 and DU145 cells. These results indicate
that mtPITALRE preferentially inhibited AR-mediated transcription over
SV40 enhancer-mediated transcription.
However, when we used non-prostate cancer H1299 (Fig.
2) and HeLa cells (data not shown),
cotransfection of the mtPITALRE expression plasmid inhibited
AR-mediated transcription and SV40 enhancer-mediated transcription to
the same degree, resulting in little preferential inhibition of
AR-mediated transcription by mtPITALRE. These results suggest that
there might be another factor(s), which modulates activity of P-TEFb in
prostate cancer PC-3 and DU145 cells and enhances AR-mediated
transcription more efficiently. Since our preliminary data indicate
that AR activates androgen-responsive genes mainly at the elongation
stage of transcription, we propose that the specific factor(s)
modulating activity of PITALRE may play a role in prostate cancer
progression. To analyze whether AR utilizes a specific set of general
elongation factors for regulation of AR transactivation, a
cotransfection assay of the expression plasmid encoding NELF-D, a
subunit of a recently identified negative elongation factor (26), was
performed. Cotransfection of the NELF expression plasmid inhibited both
AR-mediated transcription and SV40 enhancer-mediated transcription to
about the same degree in PC-3 (Fig. 3)
and DU145 (data not shown) cells, resulting in no preferential
inhibition of AR transactivation by NELF. Since cotransfection of the
NELF expression plasmid did not show preferential inhibition of
AR-mediated transcription in prostate cancer cells, we expect that AR
does not utilize NELF to regulate AR transactivation and that the
specific factor(s) modulating activity of PITALRE may not interact with
NELF.
AR Interaction with PITALRE--
Results demonstrating
preferential inhibition of AR-mediated transcription by mtPITALRE led
us to analyze whether PITALRE interacts with AR in prostate cancer
cells. The whole cell extract of AR-positive LNCaP prostate cancer
cells was prepared and used for a coimmunoprecipitation assay with
protein A-Sepharose beads coupled with anti-PITALRE antibody. The
immunoprecipitated samples were analyzed by a Western blot assay using
anti-hAR antibody (NH27). As shown in Fig.
4A, AR was detected in the
immunoprecipitated samples obtained using protein A-Sepharose beads
coupled with anti-PITALRE antibody, but not in the samples obtained
using protein A-Sepharose beads alone, indicating AR interaction with
PITALRE in a ligand-independent manner under physiological conditions. This interaction was also analyzed using AR-negative prostate cancer
PC-3 cells with cotransfection of plasmids encoding AR and PITALRE
(Fig. 4B). AR interaction with P-TEFb in a
ligand-independent manner was further confirmed by a biochemical
binding assay (Fig. 4C). Since glutathione
S-transferase fused with the AR NH2-terminal plus DNA-binding domain (AR-NDBD) gave low purity due to protein degradation, histidine-tagged AR-NDBD was used. Consistent with the
results of coimmunoprecipitation assay, 35S-labeled PITALRE
was retained by the resins containing histidine-tagged AR-NDBD, but not
by the resins containing E. coli proteins prepared by the
same method used for the histidine-tagged AR-NDBD.
Effect of DRB, a CTD Kinase Inhibitor, on AR
Transactivation--
The purine nucleotide analog, DRB, has been known
to preferentially reduce the synthesis of promoter-distal transcripts
and has a minimal effect on the synthesis of promoter-proximal
transcripts both in vitro and in vivo, thus it is
an inhibitor for the elongation stage of transcription by RNA
polymerase II (33, 34). The mechanism of DRB inhibition at the
transcriptional elongation stage was well characterized by the finding
that DRB is an inhibitor for CTD kinases (35). P-TEFb has been reported
to possess a DRB-sensitive CTD kinase activity. A cotransfection assay
was performed in the presence of various concentrations of DRB to analyze the significance of AR modulation during transcriptional elongation. As shown in Fig. 5,
transcription from both the reporter pMMTV-luciferase and the control
pRL-luciferase was slightly enhanced at the concentration of DRB lower
than 10 Nuclear Run-on Transcription Assay of PSA Gene--
Since the
specific sequences of certain genes play a role in pausing or premature
termination of RNA polymerase II (36), a nuclear run-on transcription
assay of PSA gene, an androgen-inducible gene, was performed to exclude
a possibility that preferential inhibition of AR-mediated
transcription, either by mtPITALRE or DRB in a reporter gene assay, was
due to the structure of the reporter gene. Radioactively labeled RNAs
were prepared using LNCaP nuclei and hybridized with the
single-stranded 7SK gene probe as a control, the proximal probe of PSA
gene containing an exon 1 and the distal probe containing an exon 2 (Fig. 6A). Although eukaryotic
RNA polymerase II pausing or arrest signals are poorly characterized,
RNA polymerase II pausing or arrest are frequently caused within a few
hundred nucleotides from the initiation. The exons 1 and 2 of the PSA
gene are separated by 1200 nucleotides, thus the exon 1 and 2 were
chosen as a proximal and distal probe, respectively. As shown in Fig.
6B, the ratio of the signal detected by the distal probe to
that detected by the proximal probe increased ~2-3-fold upon
androgen induction. This result clearly indicates that preferential
inhibition of AR-mediated transcription was due to bona fide
characteristics of AR-mediated transcription.
AR is required to communicate with the general transcription
machinery on the core promoter to exert its function as a
transcriptional modulator. AR interaction with TFIIH and P-TEFb
reported in our previous (20) and current studies indicates that AR may
utilize TFIIH and P-TEFb to regulate the general transcription
machinery on the core promoter. The functional significance of
protein-protein interaction between AR and P-TEFb was analyzed using a
transient transfection assay. Cotransfection of the expression plasmid
encoding mtPITALRE, defective in its kinase activity (25), showed
preferential inhibition of AR-mediated transcription in prostate cancer
PC-3 and DU145 cells, when we compared the effect of mtPITALRE on
AR-mediated transcription with that on SV40 enhancer-mediated
transcription. SV40 enhancer-mediated transcription is a good control
for analyzing the effect of mtPITALRE on AR-mediated transcription,
because SV40 enhancer-mediated transcription is stimulated by several activators, such as AP-2, PU.1, Sp-1, and TEF-1 (37). However, preferential inhibition of AR-mediated transcription by mtPITALRE was
not observed when we used non-prostate cancer cells. These results
suggest that specific factor(s) may interact with P-TEFb in prostate
cancer cells and that cotransfection of the plasmid encoding mtPITALRE
may squelch out the specific factor(s) that may regulate AR-mediated
transcription by modulating the activity of P-TEFb. In addition, this
preferential inhibition of AR-mediated transcription was not observed
with cotransfection of the expression plasmid encoding NELF, a recently
identified negative elongation factor (26). These results indicate that
AR utilizes a specific set of elongation factors for efficient
AR-mediated transcription. The working model for AR-mediated
transcription based on our results is shown in Fig.
7.
-D-ribofuranosylbenzimidazole (DRB),
a CTD kinase inhibitor. These results suggest that CTD phosphorylation
may play an important role in AR-mediated transcription. Furthermore, a
nuclear run-on transcription assay of the prostate-specific antigen gene, an androgen-inducible gene, showed that
transcription efficiency of the distal region of the gene was enhanced
upon androgen induction. Taken together, our reports suggest that AR interacts with TFIIH and P-TEFb and enhances the elongation stage of transcription.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 °C. For immunoprecipitation, protein A-Sepharose
(Amersham Pharmacia Biotech) resins were incubated with bovine
serum albumin (1 mg/ml) overnight, washed with PBS, and coupled to
anti-human PITALRE polyclonal antibody (Santa Cruz) as follows. Swollen
protein A-Sepharose (100 µl) was incubated with 50 µl of
anti-PITALRE antibody (Santa Cruz). The conjugated resins (30 µl bed
volume) were incubated with 1 mg of whole cell extract for 4 h at
4 °C, then extensively washed with 20 mM potassium phosphate (pH 8.0) and 100 mM KCl. The resins were
incubated with 0.2 M glycine HCl (pH 2.5) to elute
proteins. The resins were extensively washed with 20 mM
potassium phosphate (pH 8.0) and were incubated with 0.2 M
ethanolamine (pH 11.5) to elute proteins. The eluted proteins were
combined and analyzed on SDS-polyacrylamide gels. Western blotting was
performed using the ECL system (Amersham Pharmacia Biotech).
-mercaptoethanol, and
10% glycerol). Recombinant proteins were purified using
Ni2+ resin (Novagen) affinity chromatography according to
the manufacturer's instructions. Purity of the proteins was over 90%,
judged by Commassie Blue staining of the gel.
-mercaptoethanol) separated on 12% SDS-polyacrylamide gels, and
analyzed using a Molecular Dynamics PhosphorImager.
-32P]CTP (800 Ci/mmol) and incubated for 30 min at 30 °C. RNase free DNase I was added, and the incubation was
continued for 10 min at 37 °C. Protease K was added to a final
concentration of 300 µg/ml in 0.1% SDS, and the reaction mixture was
incubated 30 min at 37 °C. The labeled RNA transcripts were isolated
by phenol extraction, phenol/chloroform extraction, and ethanol
precipitation. Nuclear transcripts were separated from unincorporated
nucleotides using Sephadex G-50 columns equilibrated with 10 mM Tris·HCl (pH 7.8), 0.5 mM EDTA, and 0.3% SDS.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Preferential inhibition of AR-mediated
transactivation by the mutant PITALRE in prostate cancer PC-3 and DU145
cells. A, AR-negative PC-3 cells were transiently
transfected using SuperFect transfection reagent (Qiagen) with 600 ng
of MMTV-luciferase reporter plasmid, 10 ng of pRLSV40-luciferase as an
internal control, 30 ng of AR expression plasmid, and without or with
increasing amounts of the mutant PITALRE plasmid as indicated. The
total amounts of plasmids were adjusted to 2 µg with vector plasmid
pSG5. The luciferase activities with the MMTV-luciferase reporter
plasmid and pRLSV40-luciferase plasmid are shown as shaded
and hatched bars, respectively. The ratios of luciferase
activity with pMMTV-luciferase over that with pRLSV40-luciferase are
shown as closed bars. The result obtained in the absence of
1 nM DHT is shown as an open bar. Relative
luciferase activities were plotted using the activity without mutant
PITALRE as 1. B, experiments were performed and analyzed as
described above using prostate cancer DU145 cells.
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Fig. 2.
No preferential inhibition of AR-mediated
transactivation by mutant PITALRE in non-prostate cancer cell line
H1299 cells. Experiments were performed and analyzed as described
in the legend to Fig. 1 using non-prostate cancer H1299 cells.
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Fig. 3.
No preferential inhibition of AR-mediated
transactivation by the negative elongation factor NELF in PC-3
cells. PC-3 cells were transiently transfected with 600 ng of
pMMTV-luciferase, 10 ng of pRLSV40-luciferase, 30 ng of AR expression
plasmid, and without or with increasing amounts of NELF expression
plasmids as indicated. Total amounts of plasmids were adjusted to 2 µg using pSG5. Experiments were analyzed as described in the legend
to Fig. 1.
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Fig. 4.
AR interaction with PITALRE.
A, whole cell extracts of LNCaP (1 mg each) were prepared as
described elsewhere (27) and used for coimmunoprecipitation.
Lanes 1 and 4 were obtained with whole cell
extract from ethanol-treated cells. Lanes 2 and 5 were obtained with whole cell extracts from 1 nM
DHT-treated cells. About 0.5% of input was loaded in lanes
4 and 5. The immunoprecipitated samples with
anti-PITALRE antibody-bound protein A-Sepharose were loaded in
lanes 1 and 2. The immunoprecipitated sample with
protein A-Sepharose was loaded in lane 3 as a control.
B, PC-3 cells were transiently transfected with the plasmids
encoding AR and wild type PITALRE as described in the legend to Fig. 1.
Whole cell extracts were prepared as described elsewhere (27) and used
for coimmunoprecipitation. Lanes 1 and 2 were
obtained with whole cell extract from ethanol-treated cells.
Lanes 3 and 4 were obtained with whole cell
extracts from 1 nM DHT-treated cells. About 7.5% of input
was loaded in lanes 1 and 3 as a control.
Immunoprecipitated samples were loaded in lanes 2 and
4. C, bacterial lysate containing
histidine-tagged AR amino-terminal plus DNA-binding domain (AR-NDBD)
was incubated with Ni2+ resins. The resins were incubated
with 35S-labeled TNT-expressed PITALRE and extensively
washed with 20 mM HEPES (pH 7.8), 20% glycerol, 0.5 mM EDTA, and 400 mM NaCl. Proteins were eluted
and analyzed on SDS-polyacrylamide gels, followed by a PhosphorImager.
For the control, bacterial lysate without histidine-tagged AR-NDBD was
used in parallel (lane 2). About 5% of TNT-expressed
samples was loaded on lane 1.
6 M, presumably due to the
inhibition of nonspecific random initiation of RNA polymerase II
resulting in an increase in specific initiation. However, AR-mediated
transcription was markedly inhibited from 4 × 10
6 M DRB, while SV40
enhancer-mediated transcription was not inhibited. These results
indicate that efficient AR-mediated transcription is highly dependent
on the CTD phosphorylation of RNA polymerase II, which is a key step
required to enter the elongation stage (3). Together with
cotransfection results obtained with the mtPITALRE, preferential
inhibition of AR-mediated transcription by DRB indicates that AR may
enhance androgen responsible genes mainly at the elongation stage of
transcription by communicating with P-TEFb and TFIIH.
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Fig. 5.
Preferential inhibition of AR-mediated
transcription by DRB, a CTD kinase inhibitor. DU145 cells were
transiently transfected with pMMTV-luciferase, pRLSV40-luciferase, and
pSG5-AR as described in the legend to Fig. 1. The result obtained in
the absence of DHT is shown as an open bar. Various
concentrations of DRB were treated as shown in the figure. Relative
luciferase activities were plotted using the activity without DRB
treatment as 1.
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Fig. 6.
Nuclear run-on transcription assay of the PSA
gene. The schematic diagram of proximal and distal probes for PSA
RNA transcripts is shown in A. The introns and exons are
shown as lines and open boxes, respectively. The
numbers for the nucleotide positions of the PSA transcript are shown.
The results of the nuclear run-on transcription assay are shown in
B. LNCaP nuclei were prepared from cells incubated in the
presence of 1 nM DHT or EtOH. Radioactively labeled RNA
transcripts were prepared and hybridized with the denatured 7SK,
proximal, and distal probes. The signals were analyzed using a
Molecular Dynamics PhosphorImager.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
A working model for AR-mediated
transcription. Nuclear-specific coregulators and general
coregulators are omitted in the figure to simplify the relative
positions of TFIIH and P-TEFb with AR.
Both TFIIH and P-TEFb possess subunits that can phosphorylate the CTD domain of RNA polymerase II (3, 4). However, TFIIH and P-TEFb function at different stages of transcription. TFIIH is required for promoter clearance, which is defined as a point when RNA polymerase II leaves the initiation complex to start formation of transcripts (30), while P-TEFb is required to prevent arrest of RNA polymerase II within a few hundred nucleotides of the promoter (4). P-TEFb has been reported to be required for the efficient transcription of many, but not all, genes, which explains inhibition of both SV40 enhancer-mediated transcription and AR-mediated transcription. However, AR-mediated transcription appears to suffer more severely from the frequent arrest of RNA polymerase II than SV40 enhancer-mediated transcription. This difference may reflect the possibility that the preinitiation complexes on the pRLSV40 promoter for SV40 enhancer-mediated transcription differ from those on the pMMTV promoter for AR-mediated transcription. Development of a well defined transcription system may be necessary to characterize the mechanisms by which AR enhances transcription.
Since AR interacts with both TFIIH and P-TEFb, it is plausible to
speculate that AR activates transcription mainly at the elongation
stage. Given the fact that PITALRE is a DRB-sensitive CTD kinase (4),
the effect of DRB on AR-mediated transcription was analyzed to
demonstrate that CTD phosphorylation is a rate-limiting step for
efficient AR transactivation. DRB dramatically increases the frequency
of RNA polymerase II arrest within a few hundred nucleotides from the
transcription initiation site by inhibiting phosphorylation of the CTD
(32, 33). SV40 enhancer-mediated transcription was not inhibited in the
presence of 106 M DRB, while
AR-mediated transcription was severely inhibited. Transcriptional
activators, such as AP-2, PU.1, Sp1, and TEF-1, modulate SV40
enhancer-mediated transcription (37), thus phosphorylation of the CTD
by P-TEFb may not be a rate-limiting step for efficient transcription
by these activators. This result is consistent with the observation
that most transcriptional activators enhance the rate of
transcriptional initiation (8). SV40 enhancer-mediated transcription
was inhibited only at the high concentration (5 × 10
5 M) of DRB (data not shown).
This result indicates that AR-mediated transcription requires efficient
CTD phosphorylation. Consistent with the results obtained by a reporter
gene assay using mtPITALRE and DRB, a nuclear run-on transcription
assay of the PSA gene, an androgen-inducible gene, using LNCaP nuclei
indicated that transcription efficiency of the distal region of the PSA
gene was enhanced upon androgen induction (Fig. 6). This result clearly suggests that preferential inhibition of AR-mediated transcription by
mtPITALRE or DRB was not due to the artifact of a reporter gene assay
(e.g. RNA polymerase II pausing or arrest signal in a
reporter gene). The preinitiation transcription complex activated by AR
may require a high level of CTD phosphorylation for efficient transcription. All together, AR may increase the processivity of RNA
polymerase II upon androgen induction. A reporter gene assay with a
reporter gene containing only the multicopy of AR-responsible elements
did not give detectable induction by androgens (data not shown). This
phenomenon may result from the possibility that AR enhances
transcriptional elongation. AR may need other activators to enhance
transcriptional initiation. A recent study of androgen regulation of
the p21 gene indicated that binding sites for AR and Sp1 on the p21
promoter showed synergistic activation (38). Given the fact that Sp1
enhances the rate of transcriptional initiation (8), this study
suggests a cooperation between an activator for elongation and an
activator for initiation for efficient transcription of p21 gene.
Characterization of mechanisms implicated in AR transactivation may
facilitate identification of additional coregulators required for
efficient AR transactivation as well as development of potential therapeutic drugs for effective prevention of prostate cancer.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Grants CA55639 and CA71570.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
Rochester Medical Center, 601 Elmwood Ave., Box 626, Rochester, NY
14642. Tel.: 716-273-4500; Fax: 716-756-4133; E-mail: chang@ urmc.rochester.edu.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M002285200
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ABBREVIATIONS |
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The abbreviations used are:
CTD, COOH-terminal domain of RNA polymerase II largest subunit;
AR, androgen
receptor;
DHT, dihydrotestosterone;
DRB, 5,6-dicloro-1--D-ribofuranosylbenzimidazole;
NELF, negative elongation factor;
PSA, prostate-specific antigen;
P-TEFb, positive-transcription elongation factor b;
TFIIF and TFIIH, transcription factor IIF and IIH, respectively;
FBS, fetal bovine
serum;
MMTV, murine mammary tumor virus;
AR-NDBD, AR amino-terminal and
DNA-binding domain.
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