Reciprocal Recruitment of DRIP/Mediator and p160 Coactivator Complexes in Vivo by Estrogen Receptor

Abstract

Two functionally distinct classes of coactivators are recruited by liganded estrogen receptor, the DRIP/Mediator complex and p160 proteins, although the relative dynamics of recruitment is unclear. Previously, we have shown a direct, estradiol-dependent interaction between the DRIP205 subunit of the DRIP complex and the estrogen receptor (ER) AF2 domain. Here we demonstrate the in vivo recruitment of other endogenous DRIP subunits to ER in response to estradiol treatment in MCF-7 cells. To explore the relationship between DRIP and p160 coactivators, we examined the kinetics of coactivator recruitment to the ER target promoter, pS2, by chromatin immunoprecipitation. We observed a cyclic association and dissociation of coactivators with the promoter, with recruitment of p160s and DRIPs occurring in opposite phases, suggesting an exchange between these coactivator complexes at the target promoter. Two functionally distinct classes of coactivators are recruited by liganded estrogen receptor, the DRIP/Mediator complex and p160 proteins, although the relative dynamics of recruitment is unclear. Previously, we have shown a direct, estradiol-dependent interaction between the DRIP205 subunit of the DRIP complex and the estrogen receptor (ER) AF2 domain. Here we demonstrate the in vivo recruitment of other endogenous DRIP subunits to ER in response to estradiol treatment in MCF-7 cells. To explore the relationship between DRIP and p160 coactivators, we examined the kinetics of coactivator recruitment to the ER target promoter, pS2, by chromatin immunoprecipitation. We observed a cyclic association and dissociation of coactivators with the promoter, with recruitment of p160s and DRIPs occurring in opposite phases, suggesting an exchange between these coactivator complexes at the target promoter. The actions of estradiol are mediated by two isoforms of the estrogen receptor (ER), 1The abbreviations used are: ERestrogen receptorE217β-estradiolChIPchromatin immunoprecipitationRTreverse transcriptasePMSFphenylmethylsulfonyl fluorideCATDcathepsin DCBPCREB-binding protein (where CREB is cAMP-response element-binding protein)pol IIRNA polymerase IITBPTATA-binding protein ERα and ERβ, which function as ligand-regulated transcription factors. The liganded ER homodimer binds the promoters of target genes and interacts with coactivators to facilitate transcriptional activation (1.Gamble M.J. Freedman L.P. Trends Biochem. Sci. 2002; 27: 165-167Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). A number of coactivators interact with the C-terminal activation domain (AF2) in a ligand-dependent manner and have been implicated in ER-mediated transcription. One class of coactivators, collectively termed the p160 family, includes SRC1/NCoA-1, TIF2/GRIP1/NCoA-2, and pCIP/ACTR/AIB1 (reviewed in Refs.16.Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar and 23.Freedman L.P. Cell. 1999; 97: 5-8Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). The p160 coactivators not only possess weak histone acetyltransferase activity but also recruit CBP/p300 (2.Demarest S.J. Martinez-Yamout M. Chung J. Chen H. Xu M. Dyson H.J. Evans R.M. Wright P.E. Nature. 2002; 415: 549-553Crossref PubMed Scopus (369) Google Scholar), presumably leading to the generation of an open chromatin structure at the promoter (3.Lemon B.D. Freedman L.P. Curr. Opin. Genet. Dev. 1999; 9: 499-504Crossref PubMed Scopus (90) Google Scholar). A second distinct class of coactivators, alternatively called DRIP, ARC, or TRAP (4.Malik S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 5.Rachez C. Freedman L.P. Curr. Opin. Cell Biol. 2001; 13: 274-280Crossref PubMed Scopus (236) Google Scholar), comprises a multi-protein complex that interacts with liganded nuclear receptors, including ERα and ERβ, via the DRIP205/TRAP220 subunit (6.Burakov D. Wong C.W. Rachez C. Cheskis B.J. Freedman L.P. J. Biol. Chem. 2000; 275: 20928-20934Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 7.Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Naar A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Crossref PubMed Scopus (638) Google Scholar, 8.Warnmark A. Almlof T. Leers J. Gustafsson J.A. Treuter E. J. Biol. Chem. 2001; 276: 23397-23404Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 9.Yuan C.X. Ito M. Fondell J.D. Fu Z.Y. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7939-7944Crossref PubMed Scopus (391) Google Scholar, 10.Zhu Y. Qi C. Jain S. Le Beau M.M. Espinosa III, R. Atkins G.B. Lazar M.A. Yeldandi A.V. Rao M.S. Reddy J.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10848-10853Crossref PubMed Scopus (150) Google Scholar). The DRIP complex shares several subunits with the mammalian Mediator complex, suggesting that it functions in the direct recruitment of RNA polymerase II to the promoter (11.Chiba N. Suldan Z. Freedman L.P. Parvin J.D. J. Biol. Chem. 2000; 275: 10719-10722Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). estrogen receptor 17β-estradiol chromatin immunoprecipitation reverse transcriptase phenylmethylsulfonyl fluoride cathepsin D CREB-binding protein (where CREB is cAMP-response element-binding protein) RNA polymerase II TATA-binding protein It is evident that both p160s and DRIP205 play a key role in ER-mediated transcription (12.Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1931) Google Scholar, 13.Kim M.Y. Hsiao S.J. Kraus W.L. EMBO J. 2001; 20: 6084-6094Crossref PubMed Scopus (91) Google Scholar, 14.Llopis J. Westin S. Ricote M. Wang Z. Cho C.Y. Kurokawa R. Mullen T.M. Rose D.W. Rosenfeld M.G. Tsien R.Y. Glass C.K. Wang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4363-4368Crossref PubMed Scopus (131) Google Scholar, 15.Mak H.Y. Parker M.G. Mol. Cell. Biol. 2001; 21: 4379-4390Crossref PubMed Scopus (12) Google Scholar), and despite the functional distinction between the p160 coactivators and the DRIP complex, the molecular determinants of the interactions between these coactivators and ER appear to be very similar. Both classes of coactivators interact with the receptor AF2 via LXXLL signature motifs (16.Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar), and the same residues in the ERα-AF2 are critical for interactions with both p160s and DRIP205 (6.Burakov D. Wong C.W. Rachez C. Cheskis B.J. Freedman L.P. J. Biol. Chem. 2000; 275: 20928-20934Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), raising the question of whether these complexes are utilized by the receptor dimer simultaneously or sequentially. It has been reported that ACTR can be acetylated by CBP/p300, leading to the dissociation of p160 coactivator complexes from the promoter-bound ER (17.Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar), suggesting a mechanism whereby coactivator exchange may take place. Recently, spectroscopic methods have been used to demonstrate that the stoichiometry of the SRC1/ERα/E2 complex is one coactivator molecule per ERα dimer, supporting a sequential model of coactivator recruitment (18.Margeat E. Poujol N. Boulahtouf A. Chen Y. Muller J.D. Gratton E. Cavailles V. Royer C.A. J. Mol. Biol. 2001; 306: 433-442Crossref PubMed Scopus (61) Google Scholar). Conversely, chromatin immunoprecipitation (ChIP) experiments reported by Shang et al. (19.Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1469) Google Scholar) favor a combinatorial model. Here we show the recruitment of multiple DRIP complex subunits by liganded ER both in vitro and in vivo, suggesting that the entire DRIP complex is utilized by ER. Using ChIP, we have examined the kinetics of coactivator recruitment to an endogenous estrogen-regulated promoter. We observe a cycling of coactivators on and off the promoter, and strikingly, there is an inverse relationship between DRIP205 and p160 promoter occupancy, suggesting an exchange between the two coactivator complexes at the promoter and supporting a sequential model of coactivator recruitment. BV-FLAG-hER (α or β) was purified from SF9 cells (20.Kraus W.L. Kadonaga J.T. Genes Dev. 1998; 12: 331-342Crossref PubMed Scopus (290) Google Scholar) and pull-downs performed as described (21.Rachez C. Suldan Z. Ward J. Chang C.P. Burakov D. Erdjument-Bromage H. Tempst P. Freedman L.P. Genes Dev. 1998; 12: 1787-1800Crossref PubMed Scopus (334) Google Scholar). Immobilized proteins were extracted with SDS buffer, separated, and Western blotted with the specified antibodies. MCF-7 cells, grown in phenol red-free Dulbecco's modified Eagle's medium with 10% dextran-charcoal stripped fetal bovine serum, were treated with 100 nm E2 as indicated. For RT-PCR, 5 μg of total RNA (Trizol, Invitrogen) was reverse transcribed (Superscript™ Preamplification System, Invitrogen), and 10% of the RT product was PCR-amplified (pS2 (+871 to +1256) and β-actin (+16 to +696)). For ChIP, cells were cross-linked with 1% formaldehyde at 37 °C for 30 min then quenched with glycine to 125 mm. The cells were washed with phosphate-buffered saline and collected into 100 mm Tris·Cl (pH 9.4), 10 mm dithiothreitol. The cell pellet was resuspended (10 mm Tris·Cl (pH 8.0), 0.25% Triton X-100, 0.5% Nonidet P-40, 10 mm EDTA, 0.5 mm EGTA, 1 mm PMSF) and incubated on ice for 10 min. Nuclei were collected by centrifugation, washed (10 mm Tris·Cl (pH 8.0), 0.2 m NaCl, 1 mm EDTA, 0.5 mmEGTA, 1 mm PMSF) and resuspended in the same buffer without NaCl. Samples were sonicated (10 × 10 s), centrifuged, and 0.10 volume of 10× radioimmune precipitation buffer (10% Triton X-100, 1% sodium deoxycholate, 1.4 m NaCl) was added to the supernatants. Immunoprecipitations and subsequent washes were as described (19.Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1469) Google Scholar). The washed resin was resuspended in 100 μl of elution buffer (1% SDS, 0.1 m NaHCO3) and reverse-cross-linked at 65 °C, 6 h. DNA fragments were purified (QIAquick Spin Kit, Qiagen) and PCR-amplified (pS2 promoter region (−484 to −103), pS2 coding region (+871 to +1256)) (Fig. 3 A). Amplified products were analyzed by agarose gel electrophoresis with ethidium bromide staining and quantitated (Quantity One® Gel Doc System, Bio-Rad). We reported previously that the DRIP205 subunit of the DRIP coactivator complex interacts with ERα and ERβ in a ligand-dependent manner (6.Burakov D. Wong C.W. Rachez C. Cheskis B.J. Freedman L.P. J. Biol. Chem. 2000; 275: 20928-20934Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar); we failed, however, to observe the corecruitment of other DRIP subunits from nuclear extracts by ER. We have re-addressed this question using a modified assay. Full-length baculovirus-expressed FLAG-tagged ERα and ERβ were affinity-purified (Fig. 1 A) and used to pull down ER-interacting proteins from nuclear extracts of the human breast-derived cell line, HBL100. In addition to DRIP205 we were able to detect several DRIP subunits, including DRIP150, DRIP130 (Fig. 1 B), and DRIP240 (data not shown), as well as two members of the p160 family, SRC1 and ACTR. These factors all interacted with ER in an agonist-dependent manner, and this interaction was abolished in the presence of an antagonist, 4-Hydroxytamoxifen (Fig. 1 B). Importantly, these interactions were also detected among endogenous proteins. DRIP205, DRIP150, DRIP130 and SRC1 were coimmunoprecipitated from MCF-7 cell lysates with both ERα and ERβ in a ligand-dependent manner (Fig. 1 C). These results suggest that both ER isoforms recruit the whole DRIP complex in response to ligand. A series of ChIP experiments were performed to examine thein vivo recruitment of DRIPs and p160 coactivators by liganded ER to the endogenous pS2 promoter in MCF-7 cells. Initially, sequential dilutions of the coprecipitated DNA template and input DNA were PCR-amplified to determine the linear range of the assay (data not shown); all subsequent analyses used 5 μl of DNA template, which lies within the linear range. To establish an appropriate time course of ligand treatment, we examined the kinetics of E2-induced pS2 gene expression by RT-PCR (Fig. 2 A). pS2 mRNA increased above control levels within 30 min of treatment, reaching a maximum at 45 min, after which it remained elevated through to the last time point at 165 min. The recruitment of several classes of transcription factors to the pS2 promoter was examined over a 165-min time course of E2treatment (Fig. 2 B). Within 30 min of E2treatment we observed a significant increase in ER occupancy at a region of the pS2 promoter containing a single, well characterized estrogen response element (22.Jeltsch J.M. Roberts M. Schatz C. Garnier J.M. Brown A.M. Chambon P. Nucleic Acids Res. 1987; 15: 1401-1414Crossref PubMed Scopus (94) Google Scholar) (panel i). In accordance with a previous report (19.Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1469) Google Scholar), we observed a cycling of cofactors on and off the promoter over the time course of estradiol addition to cells, although with some minor differences in the absolute timing of peaks of recruitment. This might be explained in part by the different promoters used in each study; pS2 here compared with cathepsin D (CATD) in the previous report. One significant difference was in the kinetics of CBP/p300 recruitment to the promoter. The first (45 min) and last (150 min) peaks of CBP recruitment in our study coincide with those observed by Shang et al. (19.Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1469) Google Scholar); however, we detected an additional peak of CBP binding at 105 min (panel ii). Moreover, we consistently observed p300 throughout the time course of E2 treatment, unlike the transient appearance of p300 at 30 min reported previously. This might reflect the use of different anti-p300 antibodies in each study. The appearance of histone H4 acetylation within 30 min is consistent with the kinetics of ER and CBP/p300 recruitment to the promoter (panel ii). The recruitment of two members of the p160 family of coactivators, SRC1 and ACTR, is shown in panel iii. The level of SRC1 at the promoter increased over 30–45 min, reaching a peak at 60 min, with a second less pronounced peak at 150 min. This kinetics parallels that of CBP/p300, consistent with the fact that CBP/p300 is recruited to nuclear receptors through its interaction with p160 coactivators (23.Freedman L.P. Cell. 1999; 97: 5-8Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). ACTR was detected at 30 min, after which we observed reduced levels of this protein at the promoter until 165 min, when there was a second pronounced peak. We cannot distinguish whether the difference in the kinetics of SRC1 and ACTR is due to functional differences between these related coactivators or because of intrinsic differences in the antibodies used in the assays. Using ChIP we found that at least three subunits of the DRIP complex, DRIP205, DRIP150, and DRIP130, are brought to the pS2 promoter following ligand addition to MCF-7 cells (panel iv). These three DRIP subunits are also recruited to the E2-regulated c-Myc promoter in this cell line (data not shown). For DRIP205, using two different antibodies, we detected two distinct peaks of recruitment, a transient peak at 45 min and a broader peak at 120 min after E2 addition. Interestingly, in the context of both the pS2 and c-Myc promoters, DRIP130 and DRIP150 were corecruited with DRIP205 only during the second peak, suggesting a stabilization of DRIP205 at the promoter in the presence of other subunits. It is possible that an intact DRIP complex that includes DRIP205, DRIP150, and DRIP130 may act at a later stage in E2-dependent gene activation and that free DRIP205, or an alternate DRIP205-containing subcomplex, might be recruited at earlier time points. Finally, we examined the recruitment of components of the basal transcription machinery to the pS2 promoter (panel v). The level of RNA pol II remained steady over the time course of treatment. TBP was also detected on the promoter throughout the time course, reaching a maximal level at 45 min, which correlates with the induction of pS2 gene expression. From these experiments it is evident that both DRIPs and p160 coactivators are involved in ER-mediated transcription in vivo. Strikingly, however, there appears to be an inverse relationship between DRIP205 and p160 coactivator recruitment to the pS2 promoter. When we compared the promoter occupancy of DRIP205 with either ACTR or SRC1 over the time course of hormone treatment, we observed a clear reciprocal relationship (Fig. 2 C). This was also true at the c-Myc promoter (data not shown). It should be noted that this reciprocal pattern of recruitment of DRIP205 and p160 coactivators is in contrast to the study of Shang et al. (19.Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1469) Google Scholar), where AIB1 (ACTR) and PBP (DRIP205) were detected simultaneously at the CATD promoter. As our ChIP data indicated that ER and most transcriptional cofactors are already recruited to the pS2 promoter at the first time point following estradiol addition to cells (i.e. 30 min), we looked at earlier time points in the hope that we might elucidate the order of recruitment of coactivator complexes. MCF-7 cells were treated with E2 for a short time course (2.5–30 min), and ChIP was performed using a subset of the antibodies used previously. The results, presented in Fig. 3 B, show that ERα is recruited to the promoter by the earliest time point (2.5 min) after E2 stimulation. We also observed the recruitment of CBP and p300 at 2.5 min, followed by the appearance of acetylated histone H4. Two different antibodies, anti-ACTR and anti-SRC1, were used to assess the recruitment of p160 coactivators to the promoter within 2.5 min after E2 addition. Remarkably, three peaks (2.5, 10, and 30 min) were detected with the anti-SRC1 antibody, although this cycling was not as apparent when the anti-ACTR antibody was used. Using three independent antibodies to DRIP205, we also observed its recruitment to the promoter within 2.5 min following addition of ligand, with peaks at 5 and 15 min. As with the longer time course, this pattern is the reciprocal of that seen for the p160 coactivators (Fig. 3 C), suggesting an exchange between the two coactivator complexes when recruited to ER. We believe that the early time points shown in Fig. 3 represent the limit of resolution of the ChIP technique. However, the use of fluorescently tagged receptors and stably integrated hormone response element arrays have enabled the visualization of receptor-cofactor dynamics at the scale of seconds in living cells. Using this approach, it appears that ligand-occupied glucocorticoid and estrogen receptors undergo rapid (within seconds) exchange with DNA targets; moreover, rapid coregulator exchange also occurs (24.McNally J.G. Muller W.G. Walker D. Wolford R. Hager G.L. Science. 2000; 287: 1262-1265Crossref PubMed Scopus (659) Google Scholar, 25.Stenoien D.L. Nye A.C. Mancini M.G. Patel K. Dutertre M. O'Malley B.W. Smith C.L. Belmont A.S. Mancini M.A. Mol. Cell. Biol. 2001; 21: 4404-4412Crossref PubMed Scopus (129) Google Scholar). The peaks of coactivator-chromatin association that we observed by ChIP may represent the average of multiple peaks of oscillation that take within a time point the of the ChIP assay it to determine the order of coactivator recruitment in vivo. the most from our ChIP was a clear reciprocal relationship between DRIP205 and p160 coactivator recruitment to the pS2 promoter following ligand addition 2 and 3 C), suggesting that the receptor not both coactivator complexes at the same but one complex for We believe that this is consistent with a sequential model of coactivator whereby one coactivator complex, as is recruited to ER and at one level of transcription This complex then from the receptor recruitment of a distinct complex, as which in facilitate a functional interaction with RNA pol II (11.Chiba N. Suldan Z. Freedman L.P. Parvin J.D. J. Biol. Chem. 2000; 275: 10719-10722Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). this was in the recruitment of the whole DRIP complex by liganded ER was reported M. C. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar), supporting the results presented We are to M. Brown for very of antibody and C. Rachez and M. Gamble for and anti-SRC1 antibodies, We also D. and T. for on the ChIP as well as W. L. Kraus for