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Departments of Surgery and Genetics, Stanford University School of Medicine, Stanford, California 94305
Address all correspondence and requests for reprints to: Zijie Sun, Ph.D., Departments of Surgery and Genetics, R135, Edwards Building, Stanford University School of Medicine, Stanford, California 94305-5328. E-mail: zsun{at}stanford.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In recent years, numerous studies have shown that nuclear hormone receptors mediate specific gene transcription by directly or indirectly interacting with other transcriptional cofactors (4). Several nuclear receptor coactivators possessing intrinsic HAT activities have been identified in recent years, including cAMP response element-binding protein-binding protein (CBP)/P300 (5), the p160/SRC family (6, 7), and pCAF/GCN5 (8, 9). In the presence of ligand, these coactivators bind to the nuclear receptors and acetylate histone in chromatin to facilitate nuclear receptor-mediated, ligand-dependent transcription. The TR, the RAR, and other nuclear hormone receptors can function as potent transcription repressors in the absence of ligand (4). Recent studies on the mechanisms led to the identification of two related proteins, known as nuclear receptor corepressor (NCoR) and silencing mediator for RAR and TR (SMRT) (10, 11, 12). These two proteins mediated the repressive effect of unliganded nuclear receptors through Sin3A-dependent or -independent mechanisms to recruit HDACs in a multisubunit repressor complex leading to a more compact, transcriptionally repressed chromatin structure. Based on these observations, it has been suggested that recruitment of corepressors to the nuclear receptors may be a conversion point from transcription activation to repression.
AR belongs to the nuclear hormone receptor superfamily and plays a
critical role in promoting normal and tumoral cell growth (13, 14). However, an important feature of the AR and some other
steroid hormone receptors that distinguishes them from TR and RAR is
that they are compartmentalized to the cytoplasm as complexes with
heat-shock proteins (HSPs) before exposure to ligand (15, 16). Upon binding to ligand, AR dissociates from the HSPs and
translocates into the nucleus, where it facilitates androgen-regulated
transcription (17, 18). It has been shown that AR can bind
to p160/SRC, CBP/P300, and other coactivators to increase AR-mediated
transcription (19, 20). However, the molecular mechanism
for converting AR from transcriptionally active into silent states is
unknown. For the ER
, it has been suggested that the ubiquitin
proteasome pathway is involved in down-regulation by protein
degradation (21). However, the mechanism by which the
nucleosome is repacked into an inactive state, and whether HDACs are
involved in transcriptional repression of AR and other steroid hormone
receptors, is still unclear.
5'TG3' interacting factor (TGIF) is a homeodomain transcription repressor that was previously shown to bind to the RXR response element and inhibit RXR-mediated transcription by competitive DNA binding to an overlapping site (22). Recently, it has been demonstrated that TGIF interacts with Smad2, the mediator of TGF-ß signaling, and represses TGF-ß-activated transcription (23). Repression of Smad2-mediated transcription by TGIF is mediated through recruitment of HDACs (23, 24). Identification of a protein-protein interaction between TGIF and HDACs suggested a molecular mechanism by which Smad2 switched transcriptional activation to repression by recruiting TGIF to compete with p300/CBP binding.
In this study, we demonstrated that TGIF selectively represses AR-mediated transcription on the androgen-induced promoters. The repression is mediated through binding of TGIF to the DNA binding domain (DBD) of AR and is trichostatin (TSA), a HDAC inhibitor, sensitive. The results provide the first line of evidence showing that AR-mediated transcription is regulated via the HDAC pathway. We also identified a protein-protein interaction between TGIF and a transcriptional corepressor, Sin3A. Interestingly, the region of TGIF that interacts with Sin3A is also responsible for the binding to HDAC1 (24). Using several biochemical approaches, we further demonstrated that Sin3A directly interacts with TGIF, indicating that it may bridge both TGIF and HDAC1 in a protein complex. This study suggests a novel mechanism for TGIF-mediated transcriptional repression.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Cotransfection of TGIF expression
construct showed a dosage-dependent repression of ligand-dependent AR
activation. In contrast, the positive control, the known AR-coactivator
ARA70 (25), brought about a doubling in DHT-dependent
transcription. Thus, this result suggests that TGIF repressed
transcription from an AR-dependent promoter.
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, TGIF reduced AR-mediated
transcription on an MMTV-luc reporter by approximately 50% at TGIF/AR
plasmid ratios of 1:1. The human prostate-specific antigen
(PSA) gene is an AR-regulated target gene that has
been widely used as a prostate-specific tumor marker (26, 27). To determine whether the repression of AR activity by TGIF
could be reproduced on a natural AR-dependent promoter, transient
transfections were repeated in PC3 cells with a luciferase reporter
gene driven by the 7-kb PSA promoter (28). TGIF plasmid
reduced AR-mediated transcription from the PSA promoter by 70% (Fig. 1B).
We next tested repression of AR-dependent transcription by TGIF in a
physiologically relevant cellular context. Transient transfections were
repeated in LNCaP, a human prostate cancer cell line that
expresses endogenous AR protein. Repression of endogenous AR activity
by TGIF is measured with a cotransfected PSA promoter. As seen in Fig. 1C, endogenous AR induces the PSA promoter approximately 25-fold in the
presence of DHT. Overexpression of TGIF showed a dosage-dependent
repression of AR activity. This result demonstrates that TGIF can
repress endogenous AR protein-mediated transcription from the PSA
promoter.
To ensure that the TGIF-mediated repression did not reflect toxic or other nonspecific effects of the cotransfected plasmids, luciferase expression in all experiments was normalized using ß-galactosidase (ß-gal) production from a cotransfected plasmid. We also examined the intracellular steady-state levels of AR protein in the above transfectants and found them to be similar, indicating that the TGIF-mediated repression was not due to reduced levels of expression of AR (data not shown).
TGIF Selectively Represses AR-Mediated Transcription
TGIF is expressed in most human tissues, and it also
functions to repress transcription mediated by other activators such as
Smad2, a transducer of TGFß signals. To assess whether TGIF represses
other steroid hormone receptor activities, we tested GR and PRß along
with AR, using similar experimental conditions. The MMTV promoter
(MMTVpA3-Luc) containing the steroid hormone responsive elements for
the respective receptors (29, 30) were cotransfected with
AR, PRß, or GR expression vector into CV-1 cells. With the
appropriate ligand, the three receptors showed ligand-dependent
transcription from the MMTVpA3-Luc reporter vector (Fig. 2A). As we observed previously, TGIF
showed a dosage-dependent repression of AR activity. However, TGIF only
slightly repressed GR-mediated transcription when 10 ng of the plasmid
were used, and has no repression of PRß activity (Fig. 2A).
Based on these results, we conclude that TGIF mediated a specific
transcriptional repression on AR under identical experimental
conditions.
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, expression of AR produced about
a 10-fold induction on a luciferase reporter driven by duplicated AREs
in the presence of DHT. Induction was reduced by 70% by cotransfection
of TGIF, which is consistent with the results shown in Fig. 1 for both
the MMTV and PSA promoters. In contrast, there was no repression by
TGIF of ER-, TR-, and VDR-controlled promoters driven by the
corresponding responsive elements (Fig. 2B). Taken together, our
results suggest that TGIF selectively represses AR-mediated
transcription.
Repression of AR Activity by TGIF Is Mediated Through HDAC
Pathways
Although an earlier study showed that TGIF represses RXR-mediated
transcription by competing the RXR binding to its target elements
(22), the recent studies have shown that transcriptional
repression by TGIF is mainly mediated through the HDAC pathway
(23, 24). In this regard, we tested whether repression on
AR is also through the HDAC pathway using a HDAC inhibitor, TSA
(31). In transfected CV1 cells with both AR and TGIF
expression plasmids, TGIF strongly repressed AR-mediated transcription
(Fig. 3A). However, when the transfected
cells were treated with TSA at 5 or 10 nM, the repression
was partially or fully reversed, and high luciferase activity was
observed compared with untreated cells (Fig. 3A).
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Bß1 expression plasmid as a control,
which was previously shown to repress transcription mediated by the AR
and other nuclear receptors (32). As shown in Fig. 3B, cotransfection of AR and an equal amount of the TGIF or IBß1
expression construct driven by a cytomegalovirus (CMV) promoter
reduced the ligand-dependent AR transcription. Addition of 10
nM TSA in the transfected cells can reverse TGIF-mediated
repression, but showed no effect on the cells transfected with
pcDNA3-IBß1. These results clearly indicate that TGIF-mediated
repression is TSA sensitive and that the HDAC pathway is involved in
the regulation process.
TGIF Interacts with AR Protein
Our results indicate that repression of AR activity by TGIF is
mediated through the HDAC pathway. One possible mechanism for TGIF
repressing AR on multiple AR-dependent promoters would be by a physical
interaction with the AR. Therefore, a series of glutathione
S-transferase (GST)-AR fusion proteins containing
various functional domains were generated to assess possible
interactions with TGIF (Fig. 4A). Binding
of [35S]methionine-labeled full-length TGIF
protein to GST-AR fusion proteins was analyzed by SDS-PAGE and detected
by autoradiography. As seen in Fig. 4B, a specific retention of TGIF
protein was observed when AR-DBD was present, indicating that TGIF was
binding to a region of the AR in or near the DBD (Fig. 4B). Additional
GST-AR fusion proteins were generated to determine whether the binding
site was in the DBD or in the hinge regions flanking the DBD. The
GST fusion proteins incorporating the amino-terminal
(AR-5'DBD) or carboxyl-terminal hinge regions
(AR-3'DBD) did not bind to TGIF (top panel, Fig. 4C). In contrast, the construct containing precisely the DBD (AR-C'DBD)
showed a strong interaction with TGIF when similar amounts of the GST
fusion proteins were used in the experiments (bottom panel,
Fig. 4C). These results indicate that a physical protein-protein
interaction occurs between AR and TGIF, and that the DBD of AR is
responsible for the binding.
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). These results verify that Sin3A
specifically interacts with TGIF in yeast cells.
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, top and middle panels). Whole-cell lysates were
immunoprecipitated with normal mouse IgG or an anti-Flag monoclonal
antibody. To increase the specificity of protein complexes precipitated
by the Flag antibody, immunoprecipitates were eluted with the Flag
peptide. Eluted protein complexes were then analyzed by Western blot
using a Sin3A N-terminal antibody. As shown in the bottom
panel of Fig. 5C, endogenous Sin3A protein was detected in
anti-Flag immunoprecipitates from cells transfected with the Flag-TGIF
plasmid (lane 4), but not in untransfected cells (lane 2) or when
normal mouse IgG was used (lanes 1 and 3). These results demonstrate
that interaction between the full-length Sin3A and TGIF proteins occurs
in vivo.
To precisely map the interaction region of TGIF protein with Sin3A, GST
pull-down experiments were carried out with a series of GST-TGIF fusion
proteins (Fig. 5D). [35S]Methionine-labeled
full-length Sin3A protein bound to GST-TGIF fusion proteins was
analyzed by SDS-PAGE and detected by autoradiography. As shown in Fig. 5D, a strong retention of Sin3A protein was specifically observed for
the samples with GST-TGIF protein containing amino acids 108–192.
These results are consistent with those of the yeast two-hybrid and
coimmunoprecipitation experiments and suggest that the region between
108 and 192 amino acid residues within TGIF is required for the
interaction with Sin3A.
TGIF Directly Interacts with Sin3A
Previous studies showed that Sin3A functions as a corepressor
through recruitment of HDAC factors to transcriptional complexes
(10, 11, 12). An interaction between TGIF and HDAC1 was
observed previously in coimmunprecipitation experiments using the
overexpressed proteins (24). In this study, we have
identified a protein-protein interaction between Sin3A and TGIF.
Importantly, the interacting region of TGIF for both Sin3A and HDAC1 is
mapped to amino acids 108–192 (24). Therefore, it is
possible that the TGIF-HDAC1 interaction identified previously may be
mediated through Sin3A protein. To test this model, we first examined
the interaction between TGIF and HDAC1 or Sin3A using in
vitro protein pull-down experiments. The truncated GST-TGIF
proteins between amino acids 108 and 192 and the control constructs
were incubated with in vitro-translated HDAC1 or Sin3A
proteins. As observed previously, the GST-TGIF protein containing amino
acids 108–192 showed a strong retention of in
vitro-translated Sin3A protein (right panel, Fig. 6A). In contrast, in the identical
experiment setting, no binding was detected between the GST-TGIF
proteins and HDAC1 (left panel, Fig. 6A). The results from
these experiments suggest that TGIF directly binds Sin3A but not
HDAC1.
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). Blots
were renatured for 48 h and probed with the
32P-labeled TGIF protein fragment (amino acids
108–192). As shown in Fig. 6B, a specific Sin3A band was labeled with
the TGIF probe but not HDAC1. These data confirmed the above-described
GST pull-down results and indicate that Sin3A protein directly
interacts with TGIF.
To further test our hypothesis that Sin3A bridges TGIF and HDAC1
proteins in the same protein complex, we cotransfected both Flag-tagged
HDAC1 and T7-tagged TGIF expression constructs into CV-1 cells. CV-1
cells were chosen because they contain detectable endogenous levels of
Sin3A protein. A Flag-tag antibody was used for coimmunoprecipitations,
and then the coimmunprecipitates with both normal IgG and Flag antibody
were analyzed by Western blots with Flag, T7, and Sin3A antibodies. As
shown in Fig. 6C, both Sin3A and TGIF proteins were detected in the
complex with HDAC1. These results provide an additional line of
evidence showing that Sin3A mediates the interaction between HDAC1 and
TGIF proteins in the cells.
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DISCUSSION |
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and its coactivator is down-regulated through the
ubiquitin proteasome pathway (21). In addition, a recent
report has demonstrated that upon binding an antagonist, tamoxifen,
ER recruits both NCoR and SMRT to form a transcriptional repressor
complex (37). Although both reports have shed light on the
negative regulation of ER-mediated transcription, it is unclear whether
AR and other steroid hormone receptors are also regulated in the same
manner. Moreover, there is still a gap in our knowledge regarding how
an open, active nucleosome with an active transcriptional complex can
turn into a compact, inactive form on AR- and other steroid hormone
receptor-regulated promoters. In this report, we have identified the
transcriptional repressor, TGIF, which functions as a bona
fide repressor to specifically repress AR-mediated transcription.
The repression by TGIF is mediated through a protein complex containing
TGIF, Sin3A, and HDAC1 proteins, which provides evidence for the first
time that the HDAC pathway is involved in AR-mediated
transcription.
TGIF is a homeobox protein that contains multiple transcriptional
repression domains (24). Although it was shown earlier
that TGIF binds to RXR response element and interferes with RXR DNA
binding, recent evidence has suggested that TGIF repression is mediated
through the HDAC pathway (23). In our transient
transfection assays, we found that TGIF substantially represses
AR-mediated transcription on several androgen-induced promoters. A
protein-protein interaction between the DBD of AR and TGIF was
demonstrated. The repression by TGIF was further determined to be TSA
sensitive. In the process of probing the mechanism of TGIF-mediated
repression, using several in vivo and in vitro
approaches, we determined that TGIF physically interacts with a
corepressor, Sin3A. Previous studies have shown that Sin3A is a
component of transcription repression complexes and functions as a
mediator between sequence-specific DNA binding repressors and HDAC
proteins. Our results suggest a potential role for Sin3A in the
TGIF-mediated repression.
Using a series of deletion mutants of both TGIF and Sin3A, we mapped
the interaction regions of the two proteins. The interaction region of
Sin3A with TGIF was mapped to the N-terminal region (Fig. 5A), which
contains the paired amphipathic -helix 1, 2, and 3 domains.
This region was also previously shown to interact with the
transcriptional repressors MAD and NCoR/SMRT (38, 39, 40),
suggesting that a common mechanism may apply to TGIF and these
transcriptional repressors. The binding region of TGIF with Sin3A was
mapped to the middle portion of TGIF (amino acids 108–192), which is
the exact same region that interacts with HDAC1 (24).
Interestingly, in our GST pull-down experiments, we only detected the
interaction between TGIF and Sin3A, but not HDAC1, under identical
experimental conditions. One possibility is that the interaction
between TGIF and HDAC1 is not direct and is possibly mediated through
Sin3A. Using far Western blot, we confirmed that a direct interaction
between TGIF and Sin3A occurs. Taken together, these results suggest
that Sin3A is necessary for TGIF to recruit HDAC1 for the repression of
AR-mediated transcription.
More than seven HDAC proteins have been identified. Based on their sequences, they can be divided into two classes: class I includes HDAC1–3, whereas class II includes HDAC4–6 (36, 41, 42). Several reports have shown that Sin3A appears to play a pivotal role in targeting the class I HDACs to transcriptional repressor complexes (10, 36). However, HDAC4, HDAC5, and an as yet unnamed new member of the class II HDACs are capable of interacting directly with the corepressors NCoR and SMRT to target various transcriptional complexes in a Sin3A-independent manner (43). Our finding that Sin3A allows HDAC1 to complex with TGIF is consistent with the previous observation and suggests that TGIF may function on AR in a Sin3A-dependent manner.
TGIF has been reported previously to be a corepressor of Smad2 protein.
In this report, we show that TGIF interacts with the AR and represses
AR-mediated transcription. However, we found that TGIF probably does
not act broadly as a nuclear hormone corepressor, because it repressed
AR-mediated transcription but not the activities of other nuclear
hormone receptors, including PRß, ER, TR, and VDR. In the context
of the MMTV promoter, we showed that TGIF slightly inhibits GR
activity. Because the DBD of AR, which is composed of two zinc fingers,
was mapped as the interacting region with TGIF, it is likely that the
selective repression by TGIF on AR and GR may be due to the close
sequence similarities in the DBD regions of these proteins.
In this report, we demonstrated that TGIF interacts with the DBD of AR, facilitating binding of Sin3A and HDAC1 to form a repression complex resulting in deacetylation of the core histones. Although it is unclear whether other cellular factors are involved in this process, it is likely that TGIF can trigger this regulation. Repression of AR-mediated transcription by TGIF suggests a potentially significant mechanism to control androgen-induced cell growth, which may play a critical role in the growth and development of normal prostate tissue, and in the development and progression of prostate cancer.
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MATERIALS AND METHODS |
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Transient transfections for CV-1 were carried out by using a LipofectAMINE transfection kit (Life Technologies, Inc.) as described previously (47). About 200 ng of total plasmid DNA per well was used in the transfection. The total amount of DNA per dish was kept constant by adding pBluescript plasmid (Stratagene, La Jolla, CA). Approximately 16 h after transfection, the cells were washed and fed medium containing 5% charcoal-stripped (steroid hormone-free) FCS (HyClone Laboratories, Inc.) in the presence or absence of steroid hormones. LNCaP and PC3 cells were transfected with the LipofectAMINE 2000 reagent. Because we have observed that simian virus 40 (SV40) promoter is transcriptionally inactive in these prostate cancer cell lines, the expression vectors driven by a CMV promoter were used in the experiments. Luciferase activity was measured in relative light units as previously described (48, 49). The relative light units from individual transfections were normalized by measurement of ß-gal activity expressed from a cotransfected plasmid in the same samples. Individual transfection experiments were done in triplicate and the results are reported as mean luciferase/ß-gal (±SD) from representative experiments.
Plasmid Construction
The human TGIF cDNA was a kind gift of Dr. Roger Clerc
(Roche, Basel, Switzerland). GST-TGIF constructs
containing different portions of TGIF and the full-length TGIF
expression vector were generated by PCR with specific primers and
subcloned into the pGEX (Amersham Pharmacia Biotech,
Piscataway, NJ) and pcDNA3-Flag vectors, respectively. For yeast
two-hybrid screening, N-terminal (amino acids 1–700) and C-terminal
segments (amino acids 700–1,219) of human Sin3A were generated by PCR
and fused to the GAL4 DBD in the pGBT9 vector (CLONTECH Laboratories, Inc., Palo Alto, CA). Human HDAC1 expression
vector (pBJ5.1-HDAC1) with a carboxyl-terminal Flag epitope tag was
kindly provided by Dr. Edward Seto (University of South Florida, Tampa,
FL).
The AR expression vector, pSV-hAR, was provided by Dr. Albert Brinkmann
(Erasmus University, Rotterdam, The Netherlands). pMMTV-luc was
provided by Dr. Richard Pestell (Albert Einstein College of Medicine,
New York, NY). The pPSA-luc reporter plasmid was obtained from Dr.
Belldegrun (UCLA, Los Angeles, CA) (28).
pSV-ß-gal, an SV40 driven ß-gal reporter plasmid (Promega Corp., Madison, WI) was used in CV-1 cells as an internal
control. The pSG5-ARA70 plasmid, containing the full-length ARA70 cDNA,
and the reporter plasmid pARE-luc were the kind gifts of Dr. Chawnshang
Chang (University of Rochester, Rochester, NY)
(25). pCMV-VDR, pSV-hGR, and pVDRE-luc were provided by
Dr. David Feldman (Stanford University, Stanford, CA). A human ER
expression construct (pcDNA3-ER) and a luciferase reporter plasmid with
three estrogen responsive elements were kindly provided by Dr. Myles
Brown (Dana-Farber Cancer Institute, Boston, MA). A human TRß
expression vector and a luciferase reporter controlled by two
testosterone responsive elements were kindly provided by Dr. Anthony
Hollenberg (Beth Israel Deaconess Medical Center, Boston, MA). The
human PRß expression vector, pSV-hPRß, was a gift of Dr. Nancy L.
Weigel (Baylor College of Medicine, Houston, TX). The human IBß1
expression vector, pcDNA3-IBß1, was kindly provided by Dr. David
Moore (Baylor College of Medicine, Houston, TX).
Yeast Two-Hybrid Screen
The interaction between Sin3A and TGIF was examined in
a yeast two-hybrid assay. pGBT9-Sin3A-N (amino acids 1–700),
pGBT9-Sin3A-C (amino acids 700-1219), and a myeloid cDNA library
(34) were used in the experiments with a modified yeast
strain, PJ69-4A (50). The experimental conditions for
yeast two-hybrid screenings were previously described
(49). Transformants were selected on Sabouraud dextrose
medium lacking adenine, leucine, and tryptophan. Specific interactions
were measured by the production of adenine and ß-gal. A liquid
ß-gal assay was used to quantify the interaction.
GST Pull-Down Assay
Expression and purification of GST fusion proteins were
performed as described previously (51). The full-length
human TGIF and Sin3A proteins were translated and labeled in
vitro using the TNT-coupled reticulocyte
lysate system (Promega Corp.). Equal amounts of GST-AR or
-TGIF fusion proteins coupled to glutathione Sepharose beads were
incubated with radiolabeled TGIF or Sin3A proteins at 4 C for 2 h.
The buffer used for protein binding is a modified NETN buffer (0.2%
Nonidet P-40 (NP-40), 1 mM EDTA, 20
mM Tris-Cl (pH 8.0), 100 mM
NaCl, 5% glycerol, 4 mM
MgCl2, 0.5 mM
dithiothreitol (DTT), 1 mM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin, and 5 µg/ml aprotinin). Beads
were carefully washed four times in NETN buffer and then analyzed by
SDS-PAGE followed by autoradiography.
Immunoprecipitation and Western Blotting
The human Sin3A expression vector, pIRES1HisSin3A
(33), alone or with a Flag-tagged pcDNA3-TGIF expression
plasmid, were transfected into CV-1 cells. After incubation for 24
h, cells were harvested into a buffer containing 20 mM
HEPES (pH 8.0), 0.5% NP-40, 100 mM NaCl, 1 mM
EDTA, 5 mM MgCl2, 50 mM
NaF, 0.3 mM sodium vanadate, 1 mM DTT, 1
mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and
5% glycerol). Whole-cell lysates were incubated with mouse normal IgG
or Flag monoclonal antibody (Sigma, St Louis, MO) at 4 C
for 2 h. Pre-equilibrated protein A-Sepharose beads were then
added and, after 1 h of incubation, collected by centrifugation
and gently washed three times with the same buffer as described above.
Specific protein complexes were eluted with 100 ng/ml of Flag peptide
in a buffer containing 10 mM HEPES (pH 8.0), 100
mM NaCl, 1 mM EDTA, and 0.1% NP-40. The eluted
samples were boiled in SDS sample buffer and resolved on a 10%
SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane
and probed with a 1:500 dilution of a polyclonal antibody against the N
terminus of mSin3A (Santa Cruz Biotechnology, Santa Cruz,
CA; catalog no. sc-994). Proteins were detected using an ECL kit
(Amersham Pharmacia Biotech, Arlington Heights,
IL).
Far Western Blot Analyses
The overexpressed Sin3A and Flag-tagged HDAC1 proteins were
precipitated with specific antibodies. The precipitated Sin3A and
HDAC1-F were then resolved on an 8% SDS-PAGE and transferred onto
nitrocellulose membranes. One set of membranes was analyzed by Western
blotting with Sin3A or Flag antibodies. The other was first denatured
in 6 M guanidine hydrochloride buffer [50 mM
HEPES (pH7.5), 50 mM NaCl, 0.1 mM EDTA, 10
mM MgCl2, 1 mM DTT, 0.1
mM ZnSO4, and 10% glycerol). The
membrane was renatured by removing the guanidine hydrochloride
gradually (from 6 M to 0.5 mM) in changing the
buffer every 2–3 h and was then blocked in renaturation buffer with
5% fat-free milk overnight. The blocked membrane was then incubated
with 
-P32-labeled TGIF (amino acids 108–192) for
10–14 h in the renaturation buffer with 0.25% fat-free milk. The
membrane was then washed in the same buffer three times at room
temperature. Each wash was for 30 min. The membrane was then air-dried
and exposed to x-ray film using intensifying screens.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ARE, Androgen responsive element; CBP, cAMP response element-binding protein-binding protein; CMV, cytomegalovirus; CREB, cAMP response element-binding protein; DBD, DNA binding domain; DTT, dithiothreitol; ß-gal, ß-galactosidase; GST, glutathione-S-transferase; HAT, histone acetyltransferase; HDAC, histone deacetylase; HSP, heat-shock protein; MMTV, mouse mammary tumor virus; NCoR, nuclear receptor corepressor; NP-40, Nonidet P-40; PSA, prostate-specific antigen; SMRT, silencing mediator for RAR and TR; SRC, steroid receptor coactivator; SV40, simian virus 40; TGIF, 5'TG3' interacting factor; TSA, trichostatin.
Received for publication May 14, 2001. Accepted for publication August 3, 2001.
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J. Shen and C. A. Walsh Targeted Disruption of Tgif, the Mouse Ortholog of a Human Holoprosencephaly Gene, Does Not Result in Holoprosencephaly in Mice Mol. Cell. Biol., May 1, 2005; 25(9): 3639 - 3647. [Abstract] [Full Text] [PDF]
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Y. Liu, B. O. Kim, C. Kao, C. Jung, J. T. Dalton, and J. J. He Tip110, the Human Immunodeficiency Virus Type 1 (HIV-1) Tat-interacting Protein of 110 kDa as a Negative Regulator of Androgen Receptor (AR) Transcriptional Activation J. Biol. Chem., May 21, 2004; 279(21): 21766 - 21773. [Abstract] [Full Text] [PDF]
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C. A. Hyman, L. Bartholin, S. J. Newfeld, and D. Wotton Drosophila TGIF Proteins Are Transcriptional Activators Mol. Cell. Biol., December 15, 2003; 23(24): 9262 - 9274. [Abstract] [Full Text] [PDF]
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 W.A. Schulz, M. Burchardt, and M.V. Cronauer Molecular biology of prostate cancer Mol. Hum. Reprod., August 1, 2003; 9(8): 437 - 448. [Abstract] [Full Text] [PDF]
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 Z. Wang and R. S. Mann Requirement for two nearly identical TGIF-related homeobox genes in Drosophila spermatogenesis Development, July 1, 2003; 130(13): 2853 - 2865. [Abstract] [Full Text] [PDF]
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H.-Y. Kang, K.-E. Huang, S. Y. Chang, W.-L. Ma, W.-J. Lin, and C. Chang Differential Modulation of Androgen Receptor-mediated Transactivation by Smad3 and Tumor Suppressor Smad4 J. Biol. Chem., November 8, 2002; 277(46): 43749 - 43756. [Abstract] [Full Text] [PDF]
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