Transcriptional Activation by STAT6 Requires the Direct Interaction with NCoA-1

Signal transducer and activator of transcription 6 (STAT6) is a transcription factor that is activated by interleukin-4 (IL-4)-induced tyrosine phosphorylation and mediates most of the IL-4-induced gene expression. Transcriptional activation by STAT6 requires the interaction with coactivators like p300 and the CREB-binding protein (CBP). In this study we have investigated the function of the CBP-associated members of the p160/steroid receptor coactivator family in the transcriptional activation by STAT6. We found that only one of them, NCoA-1, acts as a coactivator for STAT6 and interacts directly with the transactivation domain of STAT6. The N-terminal part of NCoA-1 interacts with the far C-terminal part of the STAT6 transactivation domain but does not interact with the other members of the STAT family. This domain of NCoA-1 has a strong inhibitory effect on STAT6-mediated transactivation when overexpressed in cells, illustrating the importance of NCoA-1 for STAT6-mediated transactivation. In addition, we showed that both coactivators CBP and NCoA-1 bind independently to specific regions within the STAT6 transactivation domain. Our results suggest that multiple contacts between NCoA-1, CBP, and STAT6 are required for transcriptional activation. These findings provide new mechanistic insights into how STAT6 can recruit coactivators required for IL-4-dependent transactivation. Signal transducer and activator of transcription 6 (STAT6) is a transcription factor that is activated by interleukin-4 (IL-4)-induced tyrosine phosphorylation and mediates most of the IL-4-induced gene expression. Transcriptional activation by STAT6 requires the interaction with coactivators like p300 and the CREB-binding protein (CBP). In this study we have investigated the function of the CBP-associated members of the p160/steroid receptor coactivator family in the transcriptional activation by STAT6. We found that only one of them, NCoA-1, acts as a coactivator for STAT6 and interacts directly with the transactivation domain of STAT6. The N-terminal part of NCoA-1 interacts with the far C-terminal part of the STAT6 transactivation domain but does not interact with the other members of the STAT family. This domain of NCoA-1 has a strong inhibitory effect on STAT6-mediated transactivation when overexpressed in cells, illustrating the importance of NCoA-1 for STAT6-mediated transactivation. In addition, we showed that both coactivators CBP and NCoA-1 bind independently to specific regions within the STAT6 transactivation domain. Our results suggest that multiple contacts between NCoA-1, CBP, and STAT6 are required for transcriptional activation. These findings provide new mechanistic insights into how STAT6 can recruit coactivators required for IL-4-dependent transactivation. signal transducer and activator of transcription interleukin germline cAMP responsive element-binding protein CREB-binding protein, HAT, histone acetyltransferase steroid receptor coactivator glutathione S-transferase polyacrylamide gel electrophoresis transactivation domain Per-Arnt-Sim nuclear receptor interaction domain basic helix loop helix activation domain DNA-binding domain, NCoA, nuclear receptor coactivator STAT1 proteins are transcription factors that transmit signals from activated cytokine receptors to the nucleus. Following their obligatory tyrosine phosphorylation exerted by JAK kinases, STATs dimerize and move to the nucleus where they modulate transcription through specific DNA sequence elements (1Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5025) Google Scholar, 2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3380) Google Scholar, 3Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1265) Google Scholar). Thus far, seven mammalian STATs have been identified. They share the same structure and functional domains. The N-terminal portion mediates cooperative binding to multiple DNA sites (4Xu X. Sun Y.L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (407) Google Scholar, 5Vinkemeier U. Cohen S.L. Moarefi I. Chait B.T. Kuriyan J. Darnell J.E. EMBO J. 1996; 15: 5616-5626Crossref PubMed Scopus (249) Google Scholar). The region that determines the DNA-binding site specificity is located between amino acids 400 and 500 (6Horvath C.M. Wen Z. Darnell J.E. Genes Dev. 1995; 9: 984-994Crossref PubMed Scopus (452) Google Scholar). The STAT-SH2 domain mediates association with the activated receptor (7Greenlund A.C. Farrar M.A. Viviano B.L. Schreiber R.D. 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Although STAT3 and STAT5 are expressed in most cell types and activated by a variety of cytokines and growth factors, other STAT proteins play specific roles in host defenses (2Darnell J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3380) Google Scholar). In the present study we focused on STAT6, which is activated in response to IL-4 and IL-13, another cytokine that binds to the α chain of the IL-4 receptor (12Smerz-Bertling C. Duschl A. J. Biol. Chem. 1995; 270: 966-970Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). IL-4 regulates immune and anti-inflammatory responses. It promotes the differentiation of T helper precursors toward the Th2 lineage while inhibiting Th1 development. Furthermore, IL-4 stimulation of B-cells triggers Ig class switching to IgE isotype. This recombination is thought to be initiated following the transcriptional activation of the germline (GL) ε promoter, which leads to the generation of the sterile ε transcript (13Jung S. 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They are best characterized in the Ig GL ε promoter, which contains a composite binding element for STAT6 and the CAAT/enhancer-binding protein (17Delphin S. Stavnezer J. J. Exp. Med. 1995; 181: 181-192Crossref PubMed Scopus (188) Google Scholar). The transactivation domain of STAT6 was characterized as a modular, proline-rich region in the C terminus of the protein. The structure of this domain is quite different from that of the other members of the STAT family (18Lu B. Reichel M. Fisher D.A. Smith J.F. Rothman P. J. Immunol. 1997; 159: 1255-1264PubMed Google Scholar, 19Moriggl R. Berchtold S. Friedrich K. Standke G.J. Kammer W. Heim M. Wissler M. Stocklin E. Gouilleux F. Groner B. Mol. Cell. Biol. 1997; 17: 3663-3678Crossref PubMed Scopus (111) Google Scholar). Recently studies have mapped two distinct transactivation functions in this domain that cooperate in transcriptional activation (20Goenka S. Youn J. Dzurek L.M. Schindler U., Yu- Lee L.Y. Boothby M. J. Immunol. 1999; 163: 4663-4672PubMed Google Scholar). Activation of transcription in general requires the recruitment of transcriptional coactivators that are part of the chromatin modifying complexes possessing histone acetyltransferase activities and serve as a bridge to the basal transcriptional apparatus (21Hampsey M. Reinberg D. Curr. Opin. Genet. Dev. 1999; 9: 132-139Crossref PubMed Scopus (138) Google Scholar). In previous studies we demonstrated that the functionally conserved coactivators p300 and CREB-binding protein (CBP) are recruited by STAT6 and are required for transcriptional activation by IL-4 (22Gingras S. Simard J. Groner B. Pfitzner E. Nucleic Acids Res. 1999; 27: 2722-2729Crossref PubMed Scopus (109) Google Scholar). p300/CBP are also recruited by different classes of transcription factors, including nuclear receptors, AP-1, p53, p65 subunit of NFκB, and STAT1, STAT2, and STAT5 (23Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar, 24Pfitzner E. Jahne R. Wissler M. Stoecklin E. Groner B. Mol. Endocrinol. 1998; 12: 1582-1593Crossref PubMed Google Scholar). p300/CBP possesses intrinsic histone acetyltransferase activity and associates with other histone acetyltransferases (HATs) like p/CAF and members of the p160/steroid receptor coactivator (SRC) family (23Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar). The p160/SRC coactivator family, also called the NCoA coactivator family, was identified as nuclear receptor-binding proteins, which enhance transcriptional activation, by these ligand-induced transcription factors (25Xu L. Glass C.K. Rosenfeld M.G. Curr. Opin. Genet. Dev. 1999; 9: 140-147Crossref PubMed Scopus (814) Google Scholar). Three homologous factors, termed NCoA-1, also called SRC-1 (26Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2057) Google Scholar, 27Kamei Y. Xu L. 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Science. 1998; 279: 703-707Crossref PubMed Scopus (560) Google Scholar, 43Kurokawa R. Kalafus D. Ogliastro M.H. Kioussi C. Xu L. Torchia J. Rosenfeld M.G. Glass C.K. Science. 1998; 279: 700-703Crossref PubMed Scopus (199) Google Scholar). One important question concerning the function of NCoA coactivators focuses on whether or not the different NCoA cofactors fulfill redundant functions. All three family members possess similar properties in terms of interaction with nuclear receptors and enhancement of nuclear receptor transcriptional activation. However, several reports suggest that their activities are not completely redundant (30Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1107) Google Scholar, 35Lee S.K. Kim H.J. Kim J.W. Lee J.W. Mol. Endocrinol. 1999; 13: 1924-1933Crossref PubMed Scopus (68) Google Scholar). Because NCoA coactivators are associated with p300/CBP, which in turn coactivates STAT6, we investigated their influence on STAT6 transactivation. We tested whether STAT6-mediated transactivation requires the activity of specific coactivators. In this paper we demonstrate that NCoA-1, but not the other members of the NCoA coactivator family, acts as a coactivator of STAT6. We found a direct interaction of STAT6 and NCoA-1 in cells and in vitro. Overexpression of the STAT6-interacting domain of NCoA-1 inhibits transactivation by STAT6 in a transdominant manner, demonstrating the importance of NCoA-1 for STAT6 transactivation. Additionally, we showed that CBP and NCoA-1 bind independently to specific parts of the STAT6 transactivation domain. The analysis of NCoA-1 mutants in which different functional domains were deleted demonstrates that coactivation by NCoA-1 requires its activation domain 1 and the STAT6 interaction region. Our results show that STAT6 enhances transcription by directly contacting at least two different coactivators (CBP and NCoA-1) with its modular transactivation domain. HepG2 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mm l-glutamine, and penicillin/streptomycin. They were transfected with Superfect transfection reagent (Qiagen) according to the manufacturer's instructions. In a typical transfection experiment 1.5 × 105 cells were transfected with 2 μg of luciferase reporter plasmid, the indicated amounts of expression vectors, and 0.025 μg of SV40 promoter-drivenLacZ expression vector to control the transfection efficiency. After 1 day the cells were induced with IL-4 (10 ng/ml) and lysed after a further 16 h of incubation. The murine pre-B-cell line Ba/F3-IL-4R, stably transfected with the human IL-4R α and γ chains (44Lischke A. Kammer W. Friedrich K. Eur. J. Biochem. 1995; 234: 100-107Crossref PubMed Scopus (21) Google Scholar), was cultured in RPMI medium containing 10% fetal calf serum, 2 mm l-glutamine, penicillin/streptomycin, and 5% supernatant of murine IL-3-overproducing WEHI cells as previously described (44Lischke A. Kammer W. Friedrich K. Eur. J. Biochem. 1995; 234: 100-107Crossref PubMed Scopus (21) Google Scholar). These cells (5 × 106) were transfected by electroporation with a Bio-Rad gene pulser at 350 V/960 microfarads. In a typical transfection experiment 5 μg of luciferase reporter construct, the indicated amount of expression vectors, and 1 μg of SV40-LacZcontrol plasmid was used. The total amount of DNA was adjusted to 20 μg with pBSK. 4 h later the cells were aliquoted into two fractions; one was treated with murine IL-4 (10 ng/ml), and the other was left untreated. 20 h later the cells were harvested. 293T cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mm l-glutamine, and penicillin/streptomycin. These cells were transfected by the calcium phosphate precipitation method. Luciferase and β-galactosidase activities were assayed as recommended by the manufacturer (Promega). Luciferase activities were normalized to the LacZexpression. At least three independent experiments were performed. The reporter genes (GAL4-RE)3TK LUC, N4(STAT-RE)3 LUC, and LacZ expression plasmid (pCH110) have been described previously (19Moriggl R. Berchtold S. Friedrich K. Standke G.J. Kammer W. Heim M. Wissler M. Stocklin E. Gouilleux F. Groner B. Mol. Cell. Biol. 1997; 17: 3663-3678Crossref PubMed Scopus (111) Google Scholar). The reporter gene Igε-TK LUC, containing nucleotides −111 to −62 of the human C ε promotor, was kindly provided by Andre Zimmer (Albert-Ludwigs-University, Freiburg, Germany). The expression vectors for hSTAT6 (pXM-Stat6), hSTAT6 residues 1–792 in the pXM vector, GAL4-DBD, and the derivates containing residues 677–792, 677–847, and 792–846 of STAT6 have been previously described (19Moriggl R. Berchtold S. Friedrich K. Standke G.J. Kammer W. Heim M. Wissler M. Stocklin E. Gouilleux F. Groner B. Mol. Cell. Biol. 1997; 17: 3663-3678Crossref PubMed Scopus (111) Google Scholar). pcDNA3-STAT1 and pBSK-STAT2 were kindly provided by Markus Heim (University Hospital, Basel, Switzerland). pSG5-hSTAT3 and pXMmSTAT4 have been described previously (45Caldenhoven E. van Dijk T.B. Solari R. Armstrong J. Raaijmakers J.A. Lammers J.W. Koenderman L. de Groot R.P. J. Biol. Chem. 1996; 271: 13221-13227Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 46Yamamoto K. Quelle F.W. Thierfelder W.E. Kreider B.L. Gilbert D.J. Jenkins N.A. Copeland N.G. Silvennoinen O. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4342-4349Crossref PubMed Scopus (204) Google Scholar). pSG6-mSTAT4 was generated by insertion of the KpnI fragment of pXM-mSTAT4 into the KpnI sites of pSG6. pBSK-hSTAT5A and pBSK-mSTAT5B were kindly provided by Fabrice Gouillieux (University of Picardie-Jules Verne, Amiens, France). pSG6-hSTAT6 was generated by insertion of theEcoRI/NotI fragment of pXM-hSTAT6 into theEcoRI/NotI sites of pSG6. The expression vectors for murine NCoA-1, NCoA-2, and NCoA-3 (pCMV-NCoA-1/SRC-1, pCMV-NCoA-2, and pCMV-NCoA-3/p/CIP) were kindly provided by Joe Torchia (University of Western Ontario, London, Canada). The expression vectors for full-length rat SRC-1/NCoA-1 (pSG6-SRC-1a) and rat SRC-1/NCoA-1 residues 569–804 fused to six Myc tags were provided by Ludger Klein-Hitpass (Universitätsklinikum, Essen, Germany). The series of expression vectors for murine NCoA-1 residues 1–462, 100–462, and 213–462 fused to six Myc tags were generated by inserting each corresponding region (generated by digestion or polymerase chain reaction) in frame into the StuI/XbaI andXhoI/XbaI sites, respectively, of pCS2+. The fragments containing the NCoA-1 sequence residues 1–571 and 1–787 of pSG6-SRC-1/NCoA-1 were subcloned into theXbaI/EcoRI and XbaI/BamHIsites of pSG6. The constructs containing the NCoA-1 sequence residues 361–571 and 313–462 were obtained by insertion of theEcoRI and PvuII/XbaI fragments of pCMV-SRC-1/NCoA-1 into the EcoRI andEcoRV/XbaI sites of pcDNA3.1/His (Invitrogen). The construct containing the NCoA-1 sequence residues 1–361 was obtained by deletion of the EcoRI fragment of pSG6-SRC-1/NCoA-1. The expression vectors for GST fusion proteins pGEX-AHK and pGEX-KGK were kindly provided by Thorsten Heinzel (Georg-Speyer-Haus, Frankfurt, Germany). The GST fusion construct containing residues 1–787 of NCoA-1 was generated by insertion of the corresponding BamHI fragment of pCMV-SRC-1/NCoA-1 into theBamHI site of pGEX-KGK. The GST fusion constructs containing residues 677–792, 677–847, and 792–847 of human STAT6 were generated by subcloning of the corresponding insert from the series of GAL4-DBD-STAT6 vectors, generated by digestion withEcoRI/NheI, in frame in theEcoRI/XbaI sites of pGEX-AHK. Insertion of all constructs was verified by digestion. Inserts generated by polymerase chain reaction were additionally verified by sequencing analysis. The SRC-1/NCoA-1 deletion constructs pSG5-SRC1e-ΔAD1, pSG5-SRC1e-ΔAD2 (Δ 1053–1123), pSG5-SRC1e-1–1240, and pSG5-SRC1eΔPAS (381–1399) have been described previously (47Bevan C.L. Hoare S. Claessens F. Heery D.M. Parker M.G. Mol. Cell. Biol. 1999; 19: 8383-8392Crossref PubMed Scopus (334) Google Scholar, 48Belandia B. Parker M.G. J. Biol. Chem. 2000; 275: 30801-30805Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The full-length pSG5-SRC1e was obtained by insertion of the NheI/BglII fragment of pSG5-SRC1eΔPAS (381–1399) into theNheI/BglII sites of pSG5-SRC1e(Δ 1053–1123). Recombinant cDNAs in the pSG6, pBSK, pCMXPL2, or pCS2+ expression vectors were transcribed and translated in vitro in reticulocyte lysates (Promega) in the presence of [35S]methionine according to the manufacturer's instructions. GST or GST fusion proteins were expressed in Escherichia coli and purified with glutathione-Sepharose beads (Amersham Pharmacia Biotech). For binding assays, GST fusions or GST alone (1.5–5 μg) bound to glutathione-Sepharose beads were incubated with labeled proteins in 200 μl of binding buffer as described previously (24Pfitzner E. Jahne R. Wissler M. Stoecklin E. Groner B. Mol. Endocrinol. 1998; 12: 1582-1593Crossref PubMed Google Scholar). After extensive washing bound proteins were eluted and separated on SDS-polyacrylamide gels. Radiolabeled proteins were visualized by fluorography. Amounts and integrity of bound proteins were estimated on SDS-PAGE by Coomassie staining. 293T cells were transfected with 8 μg of pXM-Stat6 or pXM-STAT6Δ792 and 8 μg of pSG6-SRC-1/NCoA-1 per 10-cm dish. 2 days after transfection the cells were lysed in NETN buffer (20 mm Tris/HCl, pH 8, 100 mm NaCl, 1 mm EDTA, 10% glycerol, 0.2% Nonidet P-40, and complete protease inhibitor; Roche Molecular Biochemicals). Cleared cell lysates were incubated with 2 μg of STAT6 antibody (Zymed Laboratories Inc.), SRC-1 antibody (M341, Santa Cruz) or 5 μl of rabbit serum for 2 h and for a further hour with protein A/G-agarose beads as described previously (24Pfitzner E. Jahne R. Wissler M. Stoecklin E. Groner B. Mol. Endocrinol. 1998; 12: 1582-1593Crossref PubMed Google Scholar). The immunoprecipitates were separated by SDS-PAGE, and Western blots were analyzed with SRC-1 antibody (C20, Santa Cruz) and STAT6 antibody (Transduction Laboratories). Previous studies characterized the transactivation domain (TAD) of STAT6 (18Lu B. Reichel M. Fisher D.A. Smith J.F. Rothman P. J. Immunol. 1997; 159: 1255-1264PubMed Google Scholar, 19Moriggl R. Berchtold S. Friedrich K. Standke G.J. Kammer W. Heim M. Wissler M. Stocklin E. Gouilleux F. Groner B. Mol. Cell. Biol. 1997; 17: 3663-3678Crossref PubMed Scopus (111) Google Scholar, 20Goenka S. Youn J. Dzurek L.M. Schindler U., Yu- Lee L.Y. Boothby M. J. Immunol. 1999; 163: 4663-4672PubMed Google Scholar) and identified autonomously transactivating elements. We have recently demonstrated that the activity of the STAT6 TAD is enhanced by the coactivators p300 and CBP and observed an interaction of STAT6 with p300 and CBP in vivo (22Gingras S. Simard J. Groner B. Pfitzner E. Nucleic Acids Res. 1999; 27: 2722-2729Crossref PubMed Scopus (109) Google Scholar). The region between amino acids 1850–2176 was characterized as the STAT6-interacting domain of CBP. This domain also mediates the interaction of p300/CBP with the NCoA family of nuclear receptor coactivators (33Yao T.P. Ku G. Zhou N. Scully R. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10626-10631Crossref PubMed Scopus (395) Google Scholar), which together form a large coactivator complex. To investigate whether the NCoA coactivators are involved in the transactivation by STAT6, transient transfection assays in the IL-4-responsive liver cell line HepG2 were carried out. The cells were transfected with a luciferase reporter construct containing multimerized STAT6 response elements and expression vectors encoding STAT6 and the coactivators NCoA-1, NCoA-2, and NCoA-3, respectively. After transfection, the cells were treated with IL-4 or left untreated. Induction with IL-4 led to a 6-fold enhancement of basal reporter gene expression (Fig. 1, lanes 1and 2). This induction was further enhanced up to 20-fold when NCoA-1 was cotransfected (lanes 3 and 4), whereas cotransfection of NCoA-2 or NCoA-3, respectively, had no effect (lanes 5–8). These results indicate that NCoA-1 is a coactivator of STAT6, whereas the other members of the NCoA family, NCoA-2 and NCoA-3, do not seem to be involved in transactivation by STAT6. The results from our transfection experiments suggested that NCoA-1 has, in contrast to the other CBP-associated NCoA-family members, a specific function in STAT6-mediated transcriptional activation. To analyze whether NCoA-1 can directly interact with the transactivation domain of STAT6, GST pull-down experiments were performed with NCoA-1 and the two other related members of the NCoA family. Equal amounts of GST and GST-STAT6-TAD containing the STAT6 transactivation domain fused to GST were incubated with in vitro synthesized,35S-labeled coactivators. NCoA-1 strongly bound to GST-STAT6 TAD (Fig. 2, lane 7). In contrast, NCoA-2 and NCoA-3 failed to interact with STAT6 TAD (lanes 8 and 9). No binding was observed with GST alone (lanes 4–6). These findings are consistent with the transient transfection experiments shown in Fig. 1, where NCoA-1, but not NCoA-2 and NCoA-3, enhanced STAT6 transactivation potential. Taken together, only NCoA-1 interacts with STAT6 and serves as a specific coactivator for STAT6. Previous studies have characterized the transactivation domain of STAT6 as a modular domain with different transactivation functions that mediates transcriptional activation when fused to the heterologous GAL4-DNA-binding domain (18Lu B. Reichel M. Fisher D.A. Smith J.F. Rothman P. J. Immunol. 1997; 159: 1255-1264PubMed Google Scholar, 19Moriggl R. Berchtold S. Friedrich K. Standke G.J. Kammer W. Heim M. Wissler M. Stocklin E. Gouilleux F. Groner B. Mol. Cell. Biol. 1997; 17: 3663-3678Crossref PubMed Scopus (111) Google Scholar, 20Goenka S. Youn J. Dzurek L.M. Schindler U., Yu- Lee L.Y. Boothby M. J. Immunol. 1999; 163: 4663-4672PubMed Google Scholar). We investigated whether NCoA-1 enhances the transactivation potential of STAT6 by contacting a specific part of the STAT6 transactivation domain. Two GAL4 fusion proteins, possessing either the N-terminal part of the STAT6 transactivation domain (amino acids 677–791) or the far C-terminal part of the STAT6 transactivation domain (amino acids 792–847) (Fig.3 A) were used to analyze whether NCoA-1 is recruited to these domains. The GAL4-STAT6-TAD constructs and a luciferase reporter construct containing three GAL4 response elements in its promoter region were transiently transfected into HepG2, with or without cotransfection of the coactivator NCoA-1. Expression of the isolated GAL4-DBD did not result in a significant enhancement of the luciferase reporter gene activity, which was also not affected by cotransfection of NCoA-1 (Fig. 3 B,lanes 1 and 2). GAL4-STAT6 (677) strongly induced the reporter gene expression (lane 3). Cotransfection of NCoA-1 led to a slight enhancement (lane 4). GAL4-STAT6 (792) did not significantly induce the reporter gene activity, indicating that this domain has only a minor transactivation potential when fused to the GAL4 DBD. Surprisingly, cotransfection of NCoA-1 strongly enhanced the transactivation function of this far C-terminal part of the STAT6 TAD to a level as high as the N-terminal transactivation domain of STAT6 (compare lanes 6and 4). To validate this finding and to exclude the possibility that the strong effect of NCoA-1 on the transactivation activity of GAL4-STAT6 (792) was cell type-specific, we repeated the experiments in the pre-B-cell line Ba/F3-IL-4R (Fig. 2 C). Again, GAL4-DBD alone had no significant transa