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Originally published In Press as doi:10.1074/jbc.M111486200 on December 31, 2001

J. Biol. Chem., Vol. 277, Issue 10, 8004-8011, March 8, 2002
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Functional Interaction of STAT3 Transcription Factor with the Coactivator NcoA/SRC1a*

Sandrine Giraud, Frédéric Bienvenu, Sylvie Avril, Hugues Gascan, David M. Heery§, and Olivier CoqueretDagger

From the INSERM EMI-U 9928, 4 rue Larrey, CHU Angers, Angers Cedex 49033, France and the § Department of Biochemistry, University of Leicester, University Rd., Leicester LE1 7RH, United Kingdom

Received for publication, December 3, 2001, and in revised form, December 17, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Signal transducer and activator of transcription 3 (STAT3) transcription factors are cytoplasmic proteins that induce gene activation in response to cytokine receptor stimulation. Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate to the nucleus, and activate specific target genes. This transcriptional activation by STAT3 proteins has been shown to require the recruitment of coactivators such as CREB-binding protein (CBP)/p300. In the present study, we show that steroid receptor coactivator 1, NcoA/SRC1a, originally identified as a nuclear receptor coactivator, also functions as a coactivator of STAT3 proteins. In coimmunoprecipitations, NcoA/SRC1a was found to associate with STAT3 following IL-6 stimulation of HepG2 hepatoma cells. Pull-down experiments indicated that the N-terminal part of NcoA/SRC1a associates with the activation domain of STAT3. Overexpression of NcoA/SRC1a or its SRC1e isoform enhanced transcriptional activation by STAT3 proteins in transient transfection experiments. This ability of NcoA/SRC1a to enhance STAT3 activity is dependent upon the presence of the CBP-interacting domain, activation domain 1. Using chromatin immunoprecipitation assays, we found that STAT3, NcoA/SRC1a, and CBP/p300 are simultaneously recruited to the p21waf1 promoter following interleukin-6 stimulation. Taken together, these data suggest that CBP/p300 and NcoA/SRC1a may function in a common pathway to regulate STAT3 transcriptional activity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STAT31 proteins are cytoplasmic transcription factors that become phosphorylated on a single tyrosine residue (Tyr705) by receptor-associated tyrosine kinases such as JAK kinases (1). Each STAT3 protein contains an Src homology 2 domain close to the C terminus that induces the formation of an active dimer upon Src homology 2 domain-phosphotyrosine interaction. Activated STAT3 transcription factors then translocate into the nucleus to activate target genes. Among these genes, STAT3 proteins can recognize a conserved element in the promoter of p21waf1 and increase the mRNA expression of this cell cycle regulatory gene (2, 3). STAT3 is also required for the regulation of other genes such as c-myc, cyclin D1, Bcl2, Bcl-xL, and beta 2-macroglobulin (4-8). Thus, many STAT3 target genes are key components of the regulation of cell cycle progression from G1 to S phase. Accordingly, STAT3 activation is often associated with cell growth or transformation, and disruption of the stat3 gene causes embryonic lethality around day E7.5 (9), confirming a role for STAT3 in cell survival and proliferation during embryonic development. Recent experiments also indicate that STAT3 transcription factors induce cell transformation and can be considered as oncogenes (5). Tumor-derived cell lines or samples from human cancer frequently contain activated forms of STAT3. Moreover, src-transformed cell lines exhibit activated STAT3, and co-expression of a dominant negative form of STAT3 is sufficient to block cell transformation by src (4, 10-13). Inhibition of STAT3 transcriptional activity up-regulates fas transcription (14), whereas it decreases Bcl-xL expression and induces apoptosis in U266 cells as well as in cultures of primary human myeloma cells (15). Altogether, these results indicate that STAT3 is an important mediator of cell proliferation.

One of the important questions to be resolved is what molecular basis governs gene activation by STAT3 proteins. The molecular basis of gene activation by DNA binding transcription factors involves the recruitment of different coactivator complexes that modify or remodel chromatin at target promoters or recruit the RNA Pol II holoenzyme. Chromatin remodeling machines such as ISWI- or SWI/SNF-containing complexes influence nucleosome positioning. In addition, proteins such as CBP/p300, P/CAF, and TAF250 appear to be required for their ability to acetylate histones and other proteins (16-22). Increased acetylation is often associated with activation of gene transcription and is believed to loosen chromatin structure and facilitate remodeling. Among these histone acetylases, the CBP/p300 protein functions as a coactivator for many different transcription factors. Its HAT activity is important for transcriptional stimulation (23); however, CBP/p300 can also be tightly associated with the RNA polymerase II holoenzyme (24). Thus, histone acetylases allow greater access to DNA but also function as bridging factors to the transcriptional machinery. There are now several families of histone acetylases, related to the GCN5, CBP, TAF250, and NcoA/SRC1a proteins (21, 22), respectively, and, surprisingly, a number of acetylases from different families bind to each other. Initially discovered as a nuclear receptor-binding protein (25), the NcoA/SRC1a coactivator is involved in transcriptional activation by various proteins such as AP-1, SRF, NF-kappa B, and STAT6 (26-30). Although possessing a HAT activity (31), NcoA/SRC1a is thought to contribute to transcriptional activation mainly through the recruitment of CBP/p300 to transcription factors (32-34). This suggests that the assembly of a NcoA/SRC1a-CBP/p300 complex is an important step in the regulation of transcription.

Transcriptional activation by STAT proteins relies on interactions with the coactivator CBP/p300 and requires its HAT activity (23, 35-37). In line with these results, CBP/p300 can interact with the activation domain of STAT3 to regulate transcription (38, 39). Since NcoA/SRC1a has been found to be associated with p300/CBP, we made the hypothesis that it could be a coactivator of STAT3 proteins. In this study, we show that, following IL-6 stimulation, NcoA/SRC1a interacts with STAT3 and potentiates its transcriptional activity through its CBP/p300-interacting domain AD1. Moreover, using chromatin immunoprecipitation experiments, we found that STAT3, NcoA/SRC1a, and CBP/p300 are all rapidly recruited to the promoter of the p21waf1 gene. This suggests that NcoA/SRC1a and CBP/p300 function at the same step in the process of STAT3-mediated activation of gene transcription.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Stable Cell Lines-- Cell lines obtained from the American Type Culture Collection (Manassas, VA) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum.

Reagents and Plasmid Constructs-- Polyclonal STAT3 (C20), polyclonal anti-NcoA/SRC1 (M-341), polyclonal anti-CBP (A22), and phospho-STAT3 Tyr705 were obtained from Santa Cruz Biotechnology and New England Biolabs, respectively. The NcoA/SRC1a expression vector was used as a template for PCR amplification of the various domains used in pull-downs and luciferase experiments. Some of these plasmids have been described elsewhere (32). Constructs were subcloned in the pcDNA3 vector using specific oligonucleotides containing BamHI and EcoRI restriction sites. Details of constructs are available upon request.

Preparation of Nuclear Extracts-- Cells were plated at a density of 1.4 × 106/10-cm plate and serum-starved for 1-3 days, and after two washings with cold PBS, nuclear extracts were prepared according to the method of Lee et al. (40). Briefly, 1 ml of ice-cold extraction buffer was added to the plates (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin). After three cycles of freeze-thaw, cytoplasmic extracts were recovered by centrifugation at 12,000 rpm for 5 min, and pellets were resuspended in buffer C (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 420 mM KCl, 0.2 mM EDTA, 25% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin). Following a 30-min incubation at 4 °C, nuclear extracts were spun down at 12,000 rpm for 5 min.

Immunoprecipitation and Western Blot Analysis-- Immunoprecipitation reactions were performed with nuclear cell extracts (5 mg) precleared with 40 µl of protein A-Sepharose (10% slurry in PBS) for 2 h at 4 °C. Cleared extracts were immunoprecipitated with 2 µg of the indicated antibodies overnight at 4 °C followed by the addition of 40 µl of protein A-Sepharose for 1 h at 4 °C. Note that the NcoA/SRC1a immunoprecipitation was performed in the presence of 1% Nonidet P-40. Immunoprecipitates were washed two times in Buffer C (10 mM Tris, pH 8, 150 mM NaCl) and one time with Tris 20 mM (pH 8) prior to the addition of sample buffer. Following electrotransfer, membranes were analyzed by Western blot with the indicated antibodies diluted in TBS buffer (10 mM Tris, pH 8, 150 mM NaCl) supplemented for NcoA/SRC1a and STAT3 (C20) with bovine serum albumin (2.5%) and milk powder (5%), whereas only bovine serum albumin (6%) was added for phospho-STAT3 Tyr705. Note that the NcoA/SRC1a membranes were incubated overnight at 4 °C with the primary antibody. Proteins were visualized using the ECL system of Amersham Biosciences, Inc.

GST Pull-down Experiments and in Vitro Transcription/Translation-- Recombinant cDNAs were transcribed and translated in vitro in reticulocyte lysate in the presence of [35S]methionine, according to the manufacturer's instructions and using the TNT kit (Promega). Pull-down reactions were performed by incubating purified His-STAT3-(716-770) (100 ng) with 50 ng of GST or GST-SRC-(361-782) coupled to glutathione beads in binding buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 1% Brij 96). After a 30-min incubation at 4 °C, the beads were washed once with binding buffer, twice with binding buffer containing 0.5 M NaCl, and once with 20 mM Tris-HCl, pH 8. The reverse experiment was performed with His-STAT3-(716-770) coupled to nickel-agarose resin. When using in vitro NcoA/SRC1a translated proteins, 2 µg of His-STAT3-(716-770) fusion protein coupled to nickel-agarose resin where used. In this case, proteins were allowed to interact for 1 h at 4 °C in the absence of Brij 96.

RNA Extraction and Northern Blot Analysis-- Northern blot analysis was performed essentially as described previously (41). RNA was extracted using the TRIzol reagent (Invitrogen), and 8.5 µg of total RNA was then size-fractionated on a denaturing 6% formaldehyde, 1% agarose gel and transferred to Nitrocellulose (Amersham Biosciences). After 6 h of prehybridization, hybridization was carried out overnight at 42 °C in 5 ml of 50 mM Hepes (pH 7), 0.75 M NaCl, 50% formamide, 3.5% SDS, 5× Denhardt's solution, 2 mM EDTA, 0.1% SDS, and 200 µg/ml salmon sperm DNA. The full-length p21 human cDNA was labeled with [32P]dCTP using the random priming labeling kit from Amersham Biosciences (specific activity >109 cpm/µg) and was used as a probe. Following hybridization, filters were then washed four times in 0.1× SSC, 0.1% SDS, at room temperature for 20 min each. They were then exposed for 2 days to x-ray film with intensifying screens at -80 °C.

Chromatin Immunoprecipitation (CHIP) Assay-- CHIP experiments were performed according to the method of Shang et al. (42). HepG2 cells were grown to 60% confluence and serum-starved for at least 2 days. Following IL-6 (20 ng/ml) addition for various times, cells were washed twice with PBS and then cross-linked with 1% formaldehyde at room temperature for 10 min. Cells were recovered by centrifugation at 1000 rpm for 5 min; rinsed twice with ice-cold PBS, collected in 1 ml of 100 mM Tris-HCl (pH 9.7), 10 mM dithiothreitol; incubated for 15 min at 30 °C; and then recovered by centrifugation at 2000 rpm for 5 min. Cells were washed sequentially with 1 ml of ice-cold PBS, 1 ml of ice-cold buffer I (0.25% Triton X-100, 10 mM EDTA, 0,5 mM EGTA, 10 mM Hepes, pH 6.5), and 1 ml of ice-cold buffer II (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Hepes, pH 6.5). Cells were then resuspended in 0.5 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin) and sonicated three times for 10 s each at the maximum setting. Supernatants were then recovered by centrifugation at 12,000 rpm for 10 min at 4 °C, diluted 3-10 times in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1), and subjected to one round of immunoclearing for 2 h at 4 °C with 2 µg of sheared salmon sperm DNA, 2.5 µg of preimmune serum, and 45 µl of protein A-Sepharose (of 50% slurry). Immunoprecipitation was performed overnight with specific antibodies, and then 2 µg of sheared salmon sperm DNA and 45 µl of protein A-Sepharose (of 50% slurry) were further added for 1 h at 4 °C. Immunoprecipitates were washed sequentially for 10 min each in TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl) and buffer III (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Bead precipitates were then washed three times with TE buffer and eluted three times with 1% SDS, 0.1 M NaHCO3. Eluates were pooled and heated at 65 °C overnight to reverse the formaldehyde cross-linking. Supernatants were then incubated for 1 h at 45 °C with proteinase K (80 µg each), and DNA was precipitated using classical procedures. For PCR, 10 µl from a 50-µl DNA preparation were used for 30 cycles of amplifications. The following primers were used: region -879/-593 of the p21waf1 promoter, 5'-TTCAGGAGACAGACAACTCACTCG-3' (forward primer) and 5'-GACACCCCAACAAAGCATCTTG-3' (backward primer); region -2760/-2486 of the p21waf1 promoter, 5'-TTGTGCCACTGCTGACTTTGTC-3' (forward primer) and 5'-AGCCTGAAGAAGGAGGATGTGAGG-3' (backward primer).

Luciferase Assays-- Transient transfections were done using the calcium phosphate precipitation method and repeated at least five times. Cells were plated at a density of 8 × 104 in six-well plates 24 h prior to transfection. After 36-48 h post-transfection, cells were stimulated with IL-6 (20 ng/ml) for 6 h and washed twice with ice-cold PBS, and 300 µl of lysis buffer was added (0.1 M KHPO4, pH 7.8, 0.1% Triton). Luciferase activity was normalized based on protein concentrations and then measured using a Packard Topcount scintillation counter.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NcoA/SRC1 Interacts with STAT3 Proteins-- To determine whether NcoA/SRC1 might be involved in the regulation of STAT3 activation, we first asked whether it could bind specifically to STAT3 proteins. HepG2 hepatoma cells were stimulated with IL-6 for 15 min, nuclear extracts were recovered, and coimmunoprecipitations were performed alternatively with polyclonal antibodies directed against NcoA/SRC1 (Fig. 1A, lane 2), polyclonal antibodies directed against STAT3 (Fig. 1A, lane 4), or nonspecific antibodies (Fig. 1A, lanes 1 and 3). Proteins present in the immunoprecipitates were revealed by immunoblotting with the reciprocal antibodies. In both cases, NcoA/SRC1 and STAT3 were found to co-immunoprecipitate (Fig. 1A, compare lanes 1 and 2 and lanes 3 and 4). Similar effects were also observed in a different cell line that also expresses the IL-6 receptor, the murine M1 monocyte cell line (Fig. 1A, lanes 5 and 6). This interaction was dependent on the presence of IL-6, since a very weak interaction was detected between the two proteins when cells were serum-starved for 2 days (Fig. 1B). By contrast, NcoA/SRC1 and STAT3 coimmunoprecipitate in response to IL-6 stimulation (Fig. 1B, lanes 3 and 4). Whether the phosphorylation has a direct effect on the interaction or is only related to an increased quantity of STAT3 in the nucleus remains, however, to be determined. Importantly, these co-immunoprecipitations were carried out using nuclear extracts from nontransfected cells; therefore, the association between NcoA/SRC1 and STAT3 does not require that these proteins be overexpressed. Altogether, these results indicate that STAT3 transcription factors interact with NcoA/SRC1.


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  		Fig. 1.   NcoA/SRC1a interacts with STAT3 proteins. A, HepG2 cells were serum-starved for 48 h and then stimulated with IL-6 (20 ng/ml) for 15 min. Nuclear cell extracts were immunoprecipitated with polyclonal antibodies directed against NcoA/SRC1a proteins (lane 2) or a control serum (lane 1), separated by SDS-PAGE, transferred to a nitrocellulose filter, and probed with polyclonal antibodies directed against STAT3 proteins (lanes 1 and 2). Reciprocal immunoprecipitations were performed with polyclonal antibodies directed against STAT3 proteins (lane 4), a control serum (lane 3), followed by membrane blotting with polyclonal antibodies directed against NcoA/SRC1 proteins (lanes 3 and 4). The same experiment was repeated using the M1 cell line (lanes 5 and 6). B, HepG2 cells were serum-starved for 48 h and then stimulated for 15 min with IL-6 (20 ng/ml; lanes 2 and 4) or left untreated (lanes 1 and 3). Nuclear extracts were then directly analyzed by Western blot (lanes 1 and 2) or first immunoprecipitated with polyclonal antibodies directed against NcoA/SRC1 (lanes 3 and 4). Following SDS-PAGE, membranes were probed with polyclonal antibodies directed against STAT3 proteins (bottom panel) or directed only against its Tyr705-phosphorylated form (top panel).

NcoA/SRC1 Potentiates STAT3 Transcriptional Activity-- Recent results have shown that NcoA/SRC1 can potentiate the transcriptional activity of AP-1, SRF, NF-kappa B, and nuclear receptors. Having shown that NcoA/SRC1 and STAT3 could interact, we therefore hypothesized that this interaction might increase the transcriptional activity of STAT3. To verify this, HepG2 cells were cotransfected with a reporter construct containing two STAT3 consensus binding sites upstream of a thymidine kinase minimal promoter together with a vector expressing NcoA/SRC1a. Following transfection, cells were serum-starved and stimulated with IL-6 for 6 h, and luciferase activity was measured on cytoplasmic extracts. IL-6 stimulation induced a 3-fold increase in reporter gene activity, and this activation was further potentiated in the presence of an NcoA/SRC1a expression vector (Fig. 2A, compare lanes 1-3). This suggests that the co-activator is able to potentiate the activity of the endogeneous STAT3 proteins. Inclusion of a STAT3-expressing vector in the transfection mix led to a 10-fold increase in expression following cell stimulation. This activation was further increased 8-fold in the presence of NcoA/SRC1a (Fig. 2A, lanes 4-7). Importantly, NcoA/SRC1a had no effect on the basal expression of control reporter genes (Fig. 2A, compare lanes 4 and 5; see also Fig. 3).


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  		Fig. 2.   NcoA/SRC1a potentiates STAT3 transcriptional activity. A, HepG2 cells were co-transfected with a vector expressing a luciferase reporter gene (100 ng) containing two copies of a STAT3 consensus binding site linked to a minimal thymidine kinase promoter, in the presence (lane 3) or absence (lanes 1 and 2) of a vector encoding for NcoA/SRC1a (1 µg). The same experiment was repeated with a STAT3 expression vector (300 ng) included in the reaction mix (lanes 4-7). Following transfection, cells were serum-starved for 24 h and stimulated with IL-6 for 6 h (20 ng/ml, lanes 2 and 3 and lanes 6 and 7). Cytoplasmic extracts were then prepared and processed to measure luciferase activity. The mean of five transfections is shown. B, HepG2 cells were transfected as described, except that a vector encoding for the second isoform of NcoA, SRC1e, was used. Cells were stimulated as described above, and cytoplasmic extracts were prepared and processed to measure luciferase activity.


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  		Fig. 3.   NcoA/SRC1a interacts with the activation domain of STAT3. A, HepG2 cells were co-transfected with vectors expressing a Gal4 luciferase reporter gene (100 ng) together with vectors expressing a Gal4 fusion protein linked to the STAT3 activation domain (300 ng), in the presence (lane 3) or absence (lanes 1 and 2) of a plasmid encoding NcoA/SRC1a (1 µg). As a control, the same experiments were performed in parallel using a Gal4-p53 plasmid (300 ng; lanes 4-6). Following transfection, cells were serum-starved for 24 h and stimulated with IL-6 for 6 h, and cytoplasmic extracts were then prepared and processed to measure luciferase activity. The mean of five transfections is shown. B, representation of the carboxyl-terminal His-STAT3-(716-770) fusion protein used in the pull-down experiments. C, His-tagged STAT3 fusion proteins corresponding to the activation domain of STAT3 (His-STAT3-(716-770); 2 µg) were tested for binding to in vitro 35S-labeled full-length NcoA/SRC1 proteins. Bound proteins were analyzed by SDS-PAGE and autoradiography. Lane 1 contains 20% of the amount of the 35S-labeled NcoA/SRC1 proteins used in the pull-down reactions (20% Input).

NcoA/SRC1 exists as two functionally distinct isoforms, SRC1a and SRC1e, due to differential splicing of an exon at the 3'-end of the SRC1 mRNA. The unique C-terminal sequence in SRC1a contains an additional NR binding (LXXLL) motif and also acts to repress the activity of a CBP-independent activation domain (AD2) (43). To investigate whether the two isoforms differ in their ability to potentiate STAT3 mediated transcription, these experiments were repeated using an NcoA/SRC1e-expressing vector. As shown in Fig. 2B, NcoA/SRC1e potentiated the activity evoked by STAT3 to the same extent as the SRC1a isoform. Its effect on the IL-6-induced transactivation increased when increasing amounts of NcoA/SRC1e expression vectors were included in the transfection mix.

Thus, we concluded from these results that NcoA/SRC1a can potentiate the transcriptional activity of STAT3 proteins. Importantly, electrophoretic mobility shift assay experiments using stable cell lines overexpressing NcoA/SRC1a showed that NcoA/SRC1a had no effect on STAT3 DNA binding and nuclear expression following IL-6 stimulation (data not shown).

The Effect of NcoA/SRC1 Is Mediated through Its Interaction with the Activation Domain of STAT3-- Functioning as a coactivator, we then hypothesized that NcoA/SRC1a should potentiate the activity of a chimeric Gal4-STAT3 fusion protein corresponding to the activation domain of STAT3. Using a Gal4-dependent luciferase reporter gene, we found that NcoA/SRC1a was able to potentiate transactivation by Gal4-STAT3 (Fig. 3A, compare lanes 2 and 3). Importantly, NcoA/SRC1a had no effect on the transcriptional activity of control Gal4 fusion proteins such as Gal4-p53 (Fig. 3A, compare lanes 5 and 6). These results indicate that the effect of NcoA/SRC1a is specific and also suggest that NcoA/SRC1a interacts with the carboxyl-terminal activation domain of STAT3. To verify this, in vitro pull-down experiments were performed using bacterially produced 6× histidine-tagged STAT3 containing the 716-770 amino acids corresponding to the activation domain of STAT3 (Fig. 3B, His-STAT3-(716-770)) and [35S]methionine-labeled NcoA/SRC1 proteins via in vitro transcription/translation. We found that in vitro translated NcoA/SRC1a was retained by His-tagged STAT3-(716-770) immobilized on beads, whereas it was not retained by histidine beads alone (Fig. 3C, compare lanes 2 and 3). We concluded from these results that NcoA/SRC1 binds at least to the carboxyl-terminal activation domain of STAT3. Moreover, these pull-down experiments also suggest that the interaction between NcoA/SRC1 and STAT3 is probably direct, although we cannot rule out the possibility that NcoA/SRC1a functions via another partner that could be co-purified with His-tagged STAT3-(716-770) or NcoA/SRC1.

The Transactivation Domain of STAT3 Interacts with Amino Acids 361-567 of NcoA/SRC1a-- To further extend these results and investigate which domain(s) in NcoA/SRC1a mediated the interaction with STAT3, we tested various fragments of NcoA/SRC1a for interactions with STAT3 in pull-down experiments. These fragments (Fig. 4A) were labeled with [35S]methionine using in vitro transcription/translation and mixed with bacterially produced His-STAT3-(716-770). Whereas in vitro translated full-length NcoA/SRC1a was retained by His-tagged STAT3-(716-770) (see Fig. 3B and data not shown), the fusion protein did not interact with amino acids 781-1140 of NcoA/SRC1a or with the 567-1140 fragment (Fig. 4A, data not shown). This result suggests that the NcoA/SRC1a nuclear interaction domain is probably not involved in the interaction with STAT3 (44-46). We then used a GST-SRC1a fusion protein containing the 361-782 amino acids of NcoA/SRC1a (GST-SRC-(361-782)). Using bacterially produced proteins, we observed a strong interaction between His-STAT3-(716-770) and amino acids 361-782 of NcoA/SRC1a (Fig. 4B). Altogether, these results suggest that the activation domain of STAT3 binds to amino acids 361-567 of NcoA/SRC1a. To confirm these results, we then hypothesized that a truncated form of NcoA/SRC1a that does not contain the STAT3 interaction domain should not be able to potentiate the activity of the transcription factor. To this end, we used a truncated form of NcoA/SRC1a, NcoA/SRC1a-(567-1140), where the first 567 amino acids of wild type NcoA/SRC1a have been deleted. Importantly, NcoA/SRC1a-(567-1140) retained the CBP interaction domain AD1 that is sufficient to mediate the SRC1 enhancement of STAT3 activity (see Fig. 5). The ability of this mutant to function as a coactivator was investigated in transiently transfected HepG2 cells using a Gal4-STAT3 fusion protein. Whereas the full-length NcoA/SRC1a potentiated the activity of STAT3, we found that the truncation of the N-terminal residues in NcoA/SRC1a-(567-1140) resulted in a complete loss of STAT3 enhancement activity (Fig. 4C, compare lanes 4 and 6). Taken together, these results suggest a direct interaction between the activation domain of STAT3 and residues 361-567 of NcoA/SRC1a.


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  		Fig. 4.   STAT3 interacts with the N-terminal part of NcoA/SRC1a. A, schematic representation of the NcoA/SRC1a constructs and their interaction with His-STAT3-(716-770). The nuclear receptor interaction domain (LXXLL motifs), CBP interaction domain/AD1 and AD2 are shown (not to scale). B, His-tagged STAT3 fusion proteins corresponding to the activation domain of STAT3 (His-STAT3-(716-770); 100 ng) were tested for binding to GST or to GST-SRC-(361-782) (50 ng) immobilized on Sepharose beads (lanes 1 and 2). Samples were then washed four times and separated on 6% polyacrylamide gels, and STAT3 binding was detected by Western blot using anti-STAT3 polyclonal antibodies (C20; lanes 1 and 2). Purified GST-SRC-(361-782) proteins (50 ng) were incubated for 30 min at 4 °C with histidine or His-tagged STAT3-(716-770) immobilized on nickel-agarose beads (100 ng). Samples were then washed four times and separated on 6% polyacrylamide gels, and NcoA/SRC1a binding was detected by Western blot using anti-NcoA/SRC1 polyclonal antibodies (lanes 3 and 4). C, HepG2 cells were co-transfected with vectors expressing a Gal4 luciferase reporter gene (100 ng) together with vectors expressing a Gal4 fusion protein linked to the STAT3 activation domain (300 ng; lanes 2, 4, and 6), with a plasmid encoding the full-length NcoA/SRC1a (1 µg; lanes 3 and 4) or with a deletion construct corresponding to the residues 567-1140 of NcoA/SRC1a that retains the CBP-interacting domain AD1 (1 µg; lanes 5 and 6; note that AD1 that is sufficient to mediate the SRC1 enhancement of STAT3 activity; see Fig. 5). Following transfection, cells were serum-starved for 24 h and stimulated for 6 h with IL-6 (20 ng/ml). Cytoplasmic extracts were then prepared and processed to measure luciferase activity. The mean of five transfections is shown.


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  		Fig. 5.   NcoA/SRC1a Potentiates STAT3 transcriptional activity mainly through its CBP-interacting domain AD1. A, schematic representation of the NcoA/SRC1a deletion constructs. B, HepG2 cells were transfected as described above with full-length NcoA/SRC1a or NcoA/SRC1a with the deletion mutants as indicated. Following transfection, cells were serum-starved for 24 h and stimulated for 6 h with IL-6 (20 ng/ml). Cytoplasmic extracts were then prepared and processed to measure luciferase activity. The mean of five transfections is shown.

NcoA/SRC1a Enhancement of STAT3 Activity Requires the CBP Interaction Domain AD1-- Recent results have shown that NcoA/SRC1a could function as an adaptor to recruit secondary cofactors such as CBP/p300, P/CAF, or the methyltransferase CARM1 (32-34, 47). Since transcriptional activation by STAT3 proteins requires interactions with CBP/p300 (38, 39), we hypothesized that NcoA/SRC1a might potentiate the transcriptional activity of STAT3 through secondary recruitment of CBP/p300. Therefore, our next aim was to determine the importance of the AD1 and AD2 domains to the NcoA/SRC1a function as a STAT3 coactivator. To this end, we used two NcoA/SRC1a deletion mutants, Delta AD1 and Delta AD2, that retained the STAT3 interacting region but have their interacting domains with CBP/p300 and CARM1 deleted, respectively (Fig. 5A). Full-length NcoA/SRC1a potentiated the STAT3 transcriptional activity (Fig. 5B, compare lanes 2 and 4). A C-terminal deletion resulting in the loss of AD2 and part of the HAT domain (SRC1a Delta AD2 corresponding to amino acids 1-1240) retained strong coactivator function (Fig. 5B, compare lanes 4 and 6). By contrast, truncation of the CBP/p300 interaction domain in SRC1a Delta AD1 (corresponding to a deletion of amino acids 900-950) resulted in a significantly reduced enhancement of NcoA/SRC1a activity. Interestingly, SRC1a Delta AD1 was reproducibly found to potentiate STAT3, but only with a 2-fold enhancement. Further confirming the results presented in Fig. 4, the construct SRC1a-(567-1140), which retained AD1 but in which the N-terminal STAT3 interaction domain is deleted, showed a complete loss of transcriptional enhancement (Fig. 5B, lane 10). Altogether, these results suggest that NcoA/SRC1a recruits CBP/p300 to STAT3 to potentiate its transcriptional activity.

IL-6 Induces Occupancy of the p21waf1 Promoter by STAT3, NcoA/SRC1a, and CBP Coactivators-- Our next aim was to determine whether NcoA/SRC1a could function as a transcriptional coactivator of STAT3 under physiological conditions. STAT proteins can recognize a conserved response element (Fig. 6A) in the promoter of the gene encoding the cell cycle regulator p21waf1 and activate the induction of the p21waf1 mRNA (2, 3). In line with these previous results, Northern blot analysis showed that IL-6 induced a significant induction of the p21waf1 mRNA in HepG2 cells (Fig. 6B, lanes 1 and 2), confirming that activation of STAT3 proteins leads to the activation of the p21waf1 gene. Equal loading was verified by visualization of ribosomal RNA staining (Fig. 6B, lanes 3 and 4). To determine the role of NcoA/SRC1a on the regulation of the p21waf1 promoter, we examined the recruitment of the transcription factor and its cofactor to the STAT3-responsive region of the promoter using chromatin immunoprecipitation. Kinetic experiments following IL-6 stimulation of HepG2 cells showed that maximal Tyr705 phosphorylation of nuclear STAT3 was observed after 30 min of stimulation and that the signal then gradually decreased (Fig. 6C). In light of this result, chromatin was prepared using a formaldehyde cross-linking protocol (42), and occupancy of the promoter was analyzed using specific pairs of primers spanning the STAT3-responsive region (Fig. 6D, lanes 1-4). To ensure the specificity of the reaction, all immunoprecipitations were subjected to one round of preclearing with an excess of nonrelated IgG (see "Materials and Methods"). PCR analysis was also performed with a second set of primers spanning a region 2 kb upstream of the STAT3 binding site (Fig. 6D, lanes 5-8). Under these conditions, antibodies directed against STAT3 precipitated DNA encompassing the STAT3-responsive element of the p21waf1 promoter. The STAT3 occupancy of the p21waf1 promoter started as early as 5 min following IL-6 stimulation and then gradually increased (Fig. 6D, second panel). As a control of DNA sonication efficiency, PCR analysis did not detect any increase in the STAT3 occupancy of a region 2 kb upstream of the STAT3-responsive region of the p21waf1 promoter (Fig. 6D, second panel, lanes 5-8). We next determined the participation of the two coactivators NcoA/SRC1a and CBP/p300 in the formation of the STAT3 transcription complex. As was observed for STAT3, NcoA/SRC1a and CBP/p300 were all rapidly recruited to the promoter (Fig. 6D, third and fourth panels). Importantly, within the first 30 min of IL-6 stimulation, the two cofactors associate with the same timing, suggesting that they function at the same step in the process of STAT3-mediated activation. As above, PCR analysis did not detect any increase in the NcoA/SRC1a or CBP/p300 occupancy of the control region of the p21waf1 promoter (Fig. 6D, lanes 5-8). Taken together and in accordance with studies on other NcoA/SRC1a binding transcription factors (32-34, 47), our results suggest that the transcriptional activity of STAT3 is dependent on recruitment of both NcoA/SRC1a and CBP/p300 coactivators. However, it remains to be determined whether STAT3, NcoA/SRC1a, and CBP/p300 are present in the same complex on the p21waf1 promoter.


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  		Fig. 6.   Recruitment of STAT3, NcoA/SRC1a and CBP to the p21waf1 promoter after IL-6 stimulation. A, schematic representation of the STAT3 consensus binding sites in the p21waf1 promoter. B, HepG2 cells were serum-starved for 2 days and stimulated for 6 h with IL-6 (20 ng/ml). Total RNA was prepared, and 8.5 µg of RNA was subjected to Northern blot analysis using a human cDNA probe. Equal RNA loading was verified using ethidium bromide staining. C, HepG2 cells were serum-starved for 48 h and stimulated by adding fresh RPMI medium with IL-6 (20 ng/ml). The indicated times correspond to the time elapsed, since cytokine stimulation. Nuclear extracts were prepared, and proteins from the same extracts were separated by SDS-PAGE and blotted with polyclonal antibodies directed against STAT3 proteins (bottom panel) or directed only against its Tyr705-phosphorylated form (top panel). D, soluble chromatin was prepared from HepG2 cells treated with IL-6 (20 ng/ml) for various times as indicated and immunoprecipitated with antibodies directed against STAT3, NcoA/SRC1a, or CBP. The final DNA extractions were amplified using pairs of primers that cover the STAT3 binding sites (lanes 1-4) or a distal region of the promoter (lanes 5-8).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STAT3 proteins are important mediators of cell growth, they play an essential role during embryonic development, they regulate cell survival and proliferation, and their activation is often associated with cell transformation. At the molecular level, STAT3 proteins are believed to act as transcriptional activators, since it has been demonstrated that STAT3 can regulate different genes such as p21waf1, c-myc, cyclin D1, Bcl2, Bcl-xL, and beta 2-macroglobulin (4-8). Activation of transcription requires the recruitment of coactivators to facilitate access of the transcriptional machinery to the DNA template. Accordingly, the transcriptional coactivator CBP/p300 has been shown to interact with the activation domain of STAT3 to potentiate its activity. Initially discovered as a nuclear receptor-binding protein (25), the NcoA/SRC1a coactivator is thought to contribute to transcriptional activation by recruiting CBP/p300 to transcription factors. In this study, we describe a new pathway for regulating STAT3 activation, and we establish a new role for NcoA/SRC1a as a STAT3 transcriptional coactivator. We have shown that NcoA/SRC1a can interact with STAT3 proteins and potentiates their activity. NcoA/SRC1a binds to the STAT3 activation domain through a domain spanning amino acids 361-567. In line with this result, NcoA/SRC1a has been recently shown to bind to the activation domain of STAT6 to enhance its transcriptional activity (29). The region in NcoA/SRC1a responsible for the STAT6 interaction has been located to amino acids 213-462, suggesting that STAT6 and STAT3 both bind to the N-terminal part of NcoA/SRC1a but not exactly to the same region. The reason for this difference is currently under investigation in the laboratory. As for STAT6, the CBP/p300 interacting domain, AD1, is required for STAT3 coactivation, since deletion of this domain resulted in a significantly reduced enhancement of NcoA/SRC1a activity. Therefore, NcoA/SRC1 probably functions as a STAT3 coactivator through its interaction with AD1-binding proteins such as CBP/p300. Interestingly, deletion of the AD2 domain did not lead to a complete loss of transcriptional enhancement. This suggests that some AD2-binding proteins such as methyltransferases could also participate in STAT3 activity.

A few possibilities can be raised concerning the molecular mechanisms whereby NcoA/SRC1a interacts with STAT3. The pull-down experiments suggest that the interaction between the two proteins is direct; however, it remains to be determined if this interaction occurs as soon as STAT3 enters the nucleus or whether it is regulated through phosphorylation. Interestingly, phosphorylation of the STAT3 activation domain on Ser727 has been reported to be essential for maximal activation (48). The mechanism by which serine phosphorylation increases transcription remains to be determined, but we might speculate that a phosphorylated form of STAT3 interacts much better with co-activators such as NcoA/SRC1a. Therefore, one hypothesis would be that NcoA/SRC1a can interact only with the Ser727-phosphorylated form of STAT3. We consider this possibility unlikely, since pull-down experiments were conducted with nonphosphorylated proteins. Moreover, preliminary experiments using in vitro kinase assay indicate that Ser727 phosphorylation of STAT3 does not potentiate its interaction with NcoA/SRC1a. Therefore, its seems likely that this interaction does not depend on the phosphorylation of Ser727.

Chromatin immunoprecipitation assay is a powerful technique that offers the advantage of being able to detect proteins that are not directly bound to DNA but depend on other proteins for promoter binding. We found that NcoA/SRC1a probably plays an important role in the regulation of the p21waf1 promoter by activated STAT3 proteins. Using CHIP, we found that STAT3 was rapidly recruited to the promoter of the p21waf1 gene following IL-6 stimulation. NcoA/SRC1a and CBP/p300 were also recruited and with the same timing to the p21waf1 promoter after IL-6 stimulation. These results indicate that NcoA/SRC1a and CBP/p300 act as transcriptional coactivators of STAT3 under physiological conditions. According to the pull-down and CHIP results, we would propose a model by which NcoA/SRC1a binds to the activation domain of STAT3 and further recruit CBP/p300 to the promoter. Importantly, it remains to be determined whether NcoA/SRC1a could facilitate the binding of CBP/p300 to the activation domain of STAT3 and whether STAT3, NcoA/SRC1a, and CBP/p300 are present in the same complex on the p21waf1 promoter. Since it has been demonstrated that these cofactors can also be recruited sequentially to different promoters (42), it will be also interesting to determine whether NcoA/SRC1a and CBP/p300 are always recruited to the promoters of all STAT3-responsive genes and, if so, whether the two cofactors always associate with the same timing on different regulatory regions.

We have previously found that cyclin D1 can interact with the activation domain of STAT3 proteins to block their transcriptional activity (49). Interestingly, cyclin D1 has also been shown to interact with NcoA/SRC1a (50). In light of these results, we might speculate that cyclin D1 would block the interaction between STAT3 and NcoA/SRC1a or between NcoA/SRC1a and CBP/p300, so that steric hindrance would lead to transcriptional repression. CHIP experiments will determine whether cyclin D1 is present on the p21waf1 promoter. If this model is correct, one would expect that cyclin D1, acting as a transcriptional inhibitor, should be recruited at a late stage to allow the initial transcriptional activation by the STAT3-NcoA-CBP complex to occur.

In summary, these results point to a novel role for NcoA/SRC1a as a cofactor of STAT3 transcriptional activity. We propose a dynamic model by which IL-6 stimulation induces the binding of STAT3 proteins to DNA. This is immediately followed by the recruitment of a SRC1a-CBP complex that leads to the activation of STAT3-responsive genes, probably through histone acetylation.

    ACKNOWLEDGEMENTS

We thank Dr. M. G. Parker, Dr. J. Torchia, and Dr. R. Bernards for the gifts of NcoA/SRC1a, SRC1e, and GST-SRC expression vectors.

    FOOTNOTES

* This work was supported by a fellowship (to S. G.) from the Ministere de la Recherche et de la Technologie and by a fellowship (to F. B.) and a grant from the Ligue Nationale Pour la Recherche Sur le Cancer as an "Equipe Labelisée La Ligue Contre le Cancer."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.

Dagger To whom correspondence should be addressed. Tel.: 33-2-41-35-47- 33; Fax: 33-2-41-73-16-30; E-mail: olivier.coqueret@univ-angers.fr.

Published, JBC Papers in Press, December 31, 2001, DOI 10.1074/jbc.M111486200

    ABBREVIATIONS

The abbreviations used are: STAT, signal transducers and activators of transcription; CBP, CREB-binding protein; IL, interleukin; PBS, phosphate-buffered saline; GST, glutathione S-transferase; AD1 and AD2, activation domain 1 and 2, respectively.

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