Role of Mediator in Transcriptional Activation by the Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AHR) binds many aromatic hydrocarbon compounds and mediates their carcinogenesis. We demonstrate that the endogenous AHR physically associates with the endogenous TRAP/DRIP/ARC/Mediator complex in a ligand-dependent manner. The Med220 subunit, which is known to interact with several nuclear hormone receptors through its LXXLL motifs, potentiates AHR-dependent reporter gene activity in an LXXLL-independent manner. Depletion of Med220 substantially reduces endogenous AHR-mediated transcription from the mouse cytochrome P4501A1 gene (CYP1A1). Both Med220 and CDK8 (another subunit of TRAP/DRIP/ARC/Mediator) are recruited to the CYP1A1 enhancer in a TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-dependent fashion in vivo, and Med220 LXXLL motifs are not required. Med220 rapidly and persistently associates with the enhancer but not the promoter of the CYP1A1 gene after TCDD treatment with similar kinetics as AHR and the coactivators p300 and p/CIP. Our findings demonstrate a novel role for Med220 in AHR-regulated transcription that differs mechanistically from its role in transcriptional regulation by other previously studied transcription factors. The aryl hydrocarbon receptor (AHR) binds many aromatic hydrocarbon compounds and mediates their carcinogenesis. We demonstrate that the endogenous AHR physically associates with the endogenous TRAP/DRIP/ARC/Mediator complex in a ligand-dependent manner. The Med220 subunit, which is known to interact with several nuclear hormone receptors through its LXXLL motifs, potentiates AHR-dependent reporter gene activity in an LXXLL-independent manner. Depletion of Med220 substantially reduces endogenous AHR-mediated transcription from the mouse cytochrome P4501A1 gene (CYP1A1). Both Med220 and CDK8 (another subunit of TRAP/DRIP/ARC/Mediator) are recruited to the CYP1A1 enhancer in a TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-dependent fashion in vivo, and Med220 LXXLL motifs are not required. Med220 rapidly and persistently associates with the enhancer but not the promoter of the CYP1A1 gene after TCDD treatment with similar kinetics as AHR and the coactivators p300 and p/CIP. Our findings demonstrate a novel role for Med220 in AHR-regulated transcription that differs mechanistically from its role in transcriptional regulation by other previously studied transcription factors. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) 1The abbreviations used are: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AHR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; NR, nuclear receptors; TR, thyroid hormone receptor; ER, estrogen receptor; MEF, mouse embryo fibroblast; SC, scrambled control oligo; pol II, polymerase II; ChIP, chromatin immunoprecipitation. and certain polycyclic aromatic hydrocarbons are widespread environmental pollutants that are designated human carcinogens and have been shown to be potent toxicants and carcinogens in experimental animals (1DeVito M. Birnbaum L. Schecter A. Dioxins and Health. Plenum Press, New York1994: 139-162Google Scholar). The aryl hydrocarbon receptor (AHR), a ligand activated transcription factor, is the intracellular protein that mediates most of the toxic effects of these compounds. Additionally, AHR may also play an important role in normal developmental and physiological setting possibly via an endogenous ligand(s). The evidence in support of this is largely derived from AHR knock-out mice. These mice are viable but exhibit many physiological changes and developmental abnormalities, including reduced liver weight, a depressed immune system, and reproductive defects such as small litter size (2Fernandez-Salguero P. Pineau T. Hilbert D.M. McPhail T. Lee S.S. Kimura S. Nebert D.W. Rudikoff S. Ward J.M. Gonzalez F.J. Science. 1995; 268: 722-726Google Scholar). Unliganded AHR forms a complex with the chaperone protein hsp90, a co-chaperone protein called p23 (3Kazlauskas A. Poellinger L. Pongratz I. J. Biol. Chem. 1999; 274: 13519-13524Google Scholar), and the hepatitis B virus X-associated protein 2 (XAP2) (4Meyer B.K. Pray-Grant M.G. Vanden Heuvel J.P. Perdew G.H. Mol. Cell. Biol. 1998; 18: 978-988Google Scholar). This complex resides in the cytoplasm. Upon ligand binding, the AHR undergoes a conformation change and dissociates from the complex. The resulting exposure of its nuclear localization sequence triggers the nuclear translocation of AHR (5Pollenz R.S. Sattler C.A. Poland A. Mol. Pharmacol. 1994; 45: 428-438Google Scholar). Inside the nucleus, AHR dimerizes with a related nuclear protein called the aryl hydrocarbon receptor nuclear translocator (ARNT). This heterodimer can then recognize its cognate DNA binding site called the xenobiotic response element and activates genes involved in xenobiotic metabolism and cell proliferation (6Hankinson O. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 307-340Google Scholar, 7Schmidt J. Bradfield C. Annu. Rev. Cell Dev. Biol. 1996; 12: 55-89Google Scholar). AHR-regulated transcription is under intense scrutiny since the biological activities of AHR are largely due to its ability to alter gene expression. A model system frequently used to study AHR-regulated transcription is the murine cytochrome P4501A1 gene (CYP1A1). Early studies established that AHR is capable of initiating enhancer-promoter communication during CYP1A1 transcriptional activation (8Ko H. Okino S.T. Ma Q. Whitlock J.P. Mol. Cell. Biol. 1996; 16: 430-436Google Scholar, 9Morgan J. Whitlock J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11622-11626Google Scholar); occupation of the enhancer region by liganded AHR/ARNT elicits chromatin remodeling over the promoter and assembly of the general transcription machinery. The mechanistic details of these events are, however, not fully elucidated. Direct interactions between transcriptional activators such as AHR/ARNT and general transcription factors are not sufficient for nucleation of the general transcription machinery. For example, activators are unable to render productive and robust transcription in vitro with transcription reactions reconstituted with basal factors alone, indicating that additional cofactors are required (10Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Google Scholar, 11Meisterernst M. Roy A.L. Lieu H.M. Roeder R.G. Cell. 1991; 66: 981-993Google Scholar). Biochemical isolation and studies of the related or identical multisubunit complexes, murine Mediator (12Jiang Y.W. Veschambre P. Erdjument-Bromage H. Tempst P. Conaway J.W. Conaway R.C. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8538-8543Google Scholar), TRAP/DRIP/ARC/human Mediator (13Boyer T.G. Martin M.E. Lees E. Ricciardi R.P. Berk A.J. Nature. 1999; 399: 276-279Google Scholar, 14Fondell J.D. Ge H. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8329-8333Google Scholar, 15Näär A.M. Beaurang P.A. Zhou S. Abraham S. Solomon W. Tjian R. Nature. 1999; 398: 828-832Google Scholar, 16Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. Naar A.M. Erdjument-Bromage H. Tempst P. Freedman L.P. Nature. 1999; 398: 824-828Google Scholar) (hereafter referred to as the Mediator complex), suggest that these complexes can stimulate in vitro transcription mediated by several activators. Certain subunits of the Mediator complex can directly interact with transcription activators, whereas other subunits associate with the general transcription apparatus (17Malik S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Google Scholar). Thus, Mediator may function as an adaptor to convey regulatory signals from enhancers to promoters. We demonstrate here a role for Mediator in AHR/ARNT-dependent transcription of CYP1A1. The mechanism of Mediator action differs for AHR/ARNT from other previously described transcription factors. Our observations shed light on the enhancer-promoter communication invoked by AHR. Furthermore, potential differences in the structural composition of the Mediator in different tissue and promoter contexts could help explain differential regulation of gene expression mediated by AHR/ARNT and impact our understanding of receptor-mediated chemical toxicity and carcinogenesis. Antibodies and Cell Culture—Affinity-purified rabbit polyclonal antibodies to AHR and ARNT were described previously (18Probst M.R. Reisz-Porszasz S. Agbunag R.V. Ong M.S. Hankinson O. Mol. Pharmacol. 1993; 44: 511-518Google Scholar). Antibodies to p/CIP were generated against amino acids 691–851 of mouse p/CIP in this laboratory. Med220 (M-255), CDK8 (C-19), p300 (N-15), and pol II (N-20) antibodies were purchased from Santa Cruz Biotechnology. The Med130 antibody was purchased from Rockland Inc. Hepa-1 and human hepatocarcinoma cell line HepG2 were maintained in α-minimum Eagle's medium supplemented with 10% fetal bovine serum. Med220+/+ and Med220-/- mouse embryonic fibroblasts (19Ge K. Guermah M. Yuan C.X. Ito M. Wallberg A.E. Spiegelman B.M. Roeder R.G. Nature. 2002; 417: 563-567Google Scholar) were grown in Dulbecco's modified Eagle's medium high glucose with 10% fetal bovine serum. Med220-/- mouse embryonic fibroblasts stably infected with wild type Med220, and LXXLL-mutated Med220 (19Ge K. Guermah M. Yuan C.X. Ito M. Wallberg A.E. Spiegelman B.M. Roeder R.G. Nature. 2002; 417: 563-567Google Scholar) were maintained in Dulbecco's modified Eagle's medium high glucose with 10% fetal bovine serum and 150 μg/ml hygromycin. Plasmid Constructs—Expression constructs of wild type Med220 and LXXLL-mutated Med220 were derived from pWZLhyg-FH220 (19Ge K. Guermah M. Yuan C.X. Ito M. Wallberg A.E. Spiegelman B.M. Roeder R.G. Nature. 2002; 417: 563-567Google Scholar). Coimmunoprecipitation—Hepa-1 cells were treated with Me2SO or TCDD at a final concentration of 10-8m for 60 min and then washed extensively with ice-cold phosphate-buffered saline. Nuclear extracts were as described R. K. in New York1994: Scholar). The final concentration in the nuclear was the of the nuclear was to and of nuclear was with of protein at for and were at with AHR ARNT or were of protein bovine serum The were washed with and were with and in by and with Med220, AHR, or ARNT and of expression were the For Hepa-1 and HepG2 a system was DNA of the of Med220 or constructs were with of reporter by the virus promoter and of reporter by of the regulatory region of the CYP1A1 including its promoter and xenobiotic response The final DNA were expression to that of DNA were used in cells were treated with TCDD or for were then and in activities were the The activity was used to the activity of was in in HepG2 Med220 small a scrambled with similar and their by were and the For reporter or were with a of of reporter and of reporter or a of expression of and of were at cell with to the were with medium cells with the reporter were treated with Me2SO or TCDD for an additional whereas the cells with the reporter were Cell were and reporter activities were as described For the of Med220 on endogenous CYP1A1 or were at cells were with the for an additional and cells were treated with Me2SO or TCDD for was then and for AHR, and by For of Med220 protein cells were in 10% and and of cell was then on by with the Med220 antibody or AHR and were with transcription of in a 10% of the was used with to a that was and on an were for then of at and min at 60 A of the after an a at the of the were and for human Med220, and for human and for murine and for human and for murine Med220 and CYP1A1 were with the of were as described previously S. Hankinson O. J. Biol. Chem. 2002; Scholar). cells were treated with TCDD for 60 min for the The of the of chromatin were reduced to by The used to the murine CYP1A1 promoter region were and a similar that the with the p300 antibody was by with at for The was with 150 2 and with the Med220 AHR/ARNT with the Mediator in the potential of Mediator in transcription to the AHR/ARNT can interact with Hepa-1 cells were treated with TCDD or for 60 Nuclear extracts from these cells were and were an AHR of the Mediator Med220 and were with AHR from cells treated with TCDD but not from cells not treated with this AHR Med220 may be a subunit, since of Med220 not to the of the Mediator complex (19Ge K. Guermah M. Yuan C.X. Ito M. Wallberg A.E. Spiegelman B.M. Roeder R.G. Nature. 2002; 417: 563-567Google Scholar), Med130 is an of the the of Med130 the of and with the Mediator complex J. A. A. Berk A.J. Science. 2002; Scholar). this that AHR/ARNT with the Mediator complex. We also ARNT from a nuclear of Hepa-1 cells with We were unable to between Med220, and ARNT These ARNT, at not with AHR, not interact with the Mediator complex. Med220 and Med220 with LXXLL in Hepa-1 and HepG2 the potential of subunits of the Mediator complex to expression constructs for Mediator including and Med220 were Hepa-1 cells with a reporter of the regulatory region of the CYP1A1 including its promoter and xenobiotic response cells were treated with Me2SO or TCDD for an additional and reporter activities were then the constructs Med220 a of reporter activity B and and not the potential of other Mediator subunits protein and the composition of the Mediator complex could also impact the of the a role for Med220 is the subunit the Mediator complex for several nuclear receptors including thyroid hormone receptor and estrogen receptor J.D. Ge H. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8329-8333Google Scholar, 16Rachez C. Lemon B.D. Suldan Z. Bromleigh V. Gamble M. 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Perdew 1999; Scholar). our study Med220 with LXXLL We that Med220 with or of its LXXLL motifs reporter activity with as wild type Med220 in Hepa-1 and HepG2 cells B and Depletion of Med220 AHR-regulated CYP1A1 used to endogenous Med220 in to the of Med220 to AHR-regulated We were from Med220 wild type and Med220 mouse embryo fibroblasts in this study CYP1A1 expression is known to be in K. R. Poellinger L. A. J. Biol. Chem. 1999; 274: Scholar) and also the receptor to be in Med220-/- in Med220+/+ not HepG2 cells were with a small against human Med220 or a scrambled control for by Me2SO or TCDD treatment for was then and by to Med220 cell extracts of the HepG2 cells were also for Med220 protein shown in A and the reduced Med220 and protein but not AHR protein For the Med220 on reporter gene or was with the reporter HepG2 after cells were treated with Me2SO or TCDD for an additional and reporter activities were then in treatment reduced reporter but the were HepG2 cells with expression and reporter in reporter activities were between and cells in with studies that the subunit in the Mediator complex and that reporter activities are by the of Med220 M. Yuan C.X. Roeder R.G. Mol. Cell. 2000; Scholar). study the effects of small on the endogenous CYP1A1 HepG2 cells were with the or the control for by Me2SO or TCDD treatment for was then and by TCDD CYP1A1 transcription was in cells We that the reporter gene activity and the endogenous CYP1A1 in the of This is due to transcription of CYP1A1 in the of TCDD, also on AHR. of evidence support this of genes and in AHR mice as with wild type mice (2Fernandez-Salguero P. Pineau T. Hilbert D.M. McPhail T. Lee S.S. Kimura S. Nebert D.W. Rudikoff S. Ward J.M. Gonzalez F.J. Science. 1995; 268: 722-726Google Scholar), and a of basal transcription is also in with P. Gonzalez J. Cell Sci. 1998; Scholar). additional support for this used to CYP1A1 in Hepa-1 cells with in a of Hepa-1 that a substantially of AHR W. J. Hankinson O. J. Biol. Chem. Scholar). We that in the of TCDD, CYP1A1 Hepa-1 The of CYP1A1 transcription in the of the ligand is due to the of an endogenous ligand in Hepa-1 cells O. R.D. M. Nebert D.W. J. Biol. Chem. Scholar) or to the of a small of AHR the at resulting from of the receptor between the and T. H. T. Y. K. J. Biol. Chem. 1998; Scholar). Mediator with the CYP1A1 in in a to the Mediator complex is recruited to the endogenous CYP1A1 gene its transcription Hepa-1 cells were treated with Me2SO or TCDD for 60 min by were in and that the size of the of chromatin was reduced Antibodies against Med220 and CDK8 were then used to their chromatin of and were then DNA were and by with the enhancer region We enhancer for Med220 and CDK8 indicating that the Mediator was recruited to the CYP1A1 enhancer region via studies from several different that the of subunits with the Mediator complexes of certain such as and A.M. Beaurang P.A. Zhou S. Abraham S. Solomon W. Tjian R. Nature. 1999; 398: 828-832Google Scholar, Berk A.J. Mol. Cell. Biol. Scholar). The of the Mediator may an additional of to different transcription activators and promoters. Our that in the of CYP1A1 the complex of Mediator is in Med220+/+ binding of Med220 and AHR to the CYP1A1 enhancer in these cells Med220 with the enhancer was in Med220-/- binding of AHR to the enhancer binding of CDK8 to the enhancer could be in Med220+/+ This that Med220 is for the of the Mediator complex by with the that CYP1A1 expression is in in these cells that receptor binding and Med220 to the enhancer were 60 min of pol II over the promoter could be after 2 of TCDD treatment that AHR is capable of Mediator in the of a complex over the study the role of the LXXLL motifs of Med220 in the of in Med220 stably infected with a Med220 in which of LXXLL motifs are shown in Med220 with LXXLL motifs can be recruited to the enhancer in response to these that Med220 is recruited to the murine CYP1A1 enhancer after TCDD that the LXXLL motifs in Med220 are not required for and that the complex of the Mediator CDK8 is of of AHR, Med220, and at the CYP1A1 after TCDD transcription activators different complexes in to their as is from a studied nuclear receptor The Med220 and the p/CIP of the of coactivators to the regulatory and these to associate and from the regulatory of the and the genes in a the with receptor DNA binding and pol II over the regulatory region Freedman L.P. J. Biol. Chem. 2002; Scholar, Y. J. M. Cell. 2000; Scholar). the of TR, the receptor-mediated of of coactivators Med220 J. Proc. Natl. Acad. Sci. U. S. A. 2002; Scholar). We studies on the murine CYP1A1 regulatory antibodies against AHR, Med220, and other cofactors in Hepa-1 The of that reduced the of the of chromatin to to binding to the promoter from binding to the enhancer from the We AHR binding to the enhancer as as min after the TCDD and in min in different A and binding for min Med220, and p300 were recruited to the enhancer rapidly after TCDD with kinetics similar to other and AHR with AHR, were in binding of these coactivators to the enhancer over a after TCDD AHR Med220 was at the promoter that the Med220 is to the enhancer to the promoter in the protein complex that is with the CYP1A1 gene during transcriptional TCDD treatment of pol II with the promoter as not with the enhancer The kinetics of binding of pol II to the promoter were similar to the kinetics of AHR, Med220, and p/CIP binding to the enhancer A p300 and Med220 antibodies was chromatin from Hepa-1 cells were with the p300 The was and to with the Med220 Our that p300 and Med220 can the enhancer These suggest that a similar of AHR to chromatin with a different from nuclear receptors that have been studied Freedman L.P. J. Biol. Chem. 2002; Scholar, Y. J. M. Cell. 2000; Scholar, J. Proc. Natl. Acad. Sci. U. S. A. 2002; Scholar). transcriptional activation is in the that from different final by to be to promoter to be to the general transcription and in to binding of transcription factors to their cognate DNA in chromatin remodeling activities such as complexes as as such as V. V. Y. Cell. 1996; Scholar), J. S. J. H. Y. K. Dev. 1998; 12: Scholar), and the of cofactors H. A. L. Y. Cell. Scholar, H. W. Cell. 1999; Scholar, T. Zhou J. C.A. Nature. Scholar) have been shown to or remodeling of chromatin transcription can be in vitro on a DNA general transcription factors and pol II, additional cofactors are to to by activators (10Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Google Scholar, 11Meisterernst M. Roy A.L. Lieu H.M. Roeder R.G. Cell. 1991; 66: 981-993Google Scholar). The Mediator complex from (10Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Google Scholar) and from cells this Our study that AHR can interact with the Mediator complex and that the Med220 subunit is directly involved in AHR-dependent mediated transcriptional activation of the CYP1A1 is that AHR to the of transcription factors the nuclear receptor these different of transcription factors similar of transcription studies that several known nuclear receptor cofactors including and are also by AHR. this with is important to that in to the nuclear the AHR ligand binding transcription activation and that its complex transcription activation is in the of the AHR transcription activation can on transcription and also J. Mol. Pharmacol. 1996; Scholar, K. K. H. Y. J. 1995; Scholar, M.R. Reisz-Porszasz S. Hankinson O. J. Biol. Chem. 1995; Scholar). between endogenous AHR and the endogenous Mediator subunits Med220 and Med130 that the of Med220 with AHR in the of an Mediator complex. the of the Mediator complex was to demonstrate of Med220 and CDK8 with the CYP1A1 enhancer in with the of for the LXXLL motifs for the of AHR-dependent transcription by Med220, the LXXLL motifs were also not required for of Med220 with the endogenous CYP1A1 The is different from the of with AHR, with the of AHR, and is a on the LXXLL of M. Perdew 1999; Scholar). also with the that the LXXLL motifs of Med220 are required for its activity with several nuclear Our observations and of other on the including the suggest that transcriptional between AHR and nuclear receptors could at is a known of TCDD and is mediated through AHR. TCDD been shown to of the estrogen receptor S. W. R. A. Toxicol. 1998; Scholar), receptor S. M. J. C. H. Biochem. 1999; Scholar), receptor C. A. J. Biol. Chem. 1998; Scholar), P. Gonzalez J. Cell Sci. 1998; Scholar), and possibly receptor J. M. J. Cell. Biochem. 1994; Scholar). Med220 mediates between the Mediator complex and to help the complex. between AHR/ARNT and at this of transcription activation could to by TCDD in to other such as ligand metabolism J. 1993; Scholar), of nuclear receptor T. M. S. Biochem. Pharmacol. 1991; Scholar), and hormone D.W. Proc. Natl. Acad. Sci. U. S. A. Scholar). the of Med220 to AHR-regulated CYP1A1 after of endogenous Med220 of Med220 not to study the in cell to TCDD but also or effects after of Med220 in Our that of Med220 in HepG2 cells AHR-regulated CYP1A1 CYP1A1 was not could be several for this including the effects of of Med220 or the of between Mediator and AHR via additional subunits other A was with TR, that a small of in Med220-/- with Med220 wild type M. Yuan C.X. Roeder R.G. Mol. Cell. 2000; Scholar). of CYP1A1 in the is at the promoter TCDD treatment chromatin remodeling over the promoter and activation of The transcription activation of AHR is for this The of chromatin at the promoter is after J. Whitlock J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11622-11626Google Scholar). Our studies on a by which AHR-mediated complex assembly can be during The kinetics of of Med220 to the enhancer could not be from that of or AHR. of these with the enhancer was as as min after TCDD a after and pol II over the promoter the of these Our observations are with nuclear indicating that a transcription is min after TCDD treatment of Hepa-1 cells and is maintained for several M.G. Whitlock J.P. J. Biol. Chem. Scholar, Whitlock J.P. J. Biol. Chem. Scholar), and a study (8Ko H. Okino S.T. Ma Q. Whitlock J.P. Mol. Cell. Biol. 1996; 16: 430-436Google Scholar, 9Morgan J. Whitlock J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11622-11626Google Scholar), indicating a chromatin and transcription after TCDD treatment of Hepa-1 Our that the of cofactors with the CYP1A1 enhancer are different from of estrogen receptor and Med220 and a associate and in on estrogen in a similar to the estrogen whereas the of p300 is the of TR, the and p300 are but not to the basal after 2 of ligand Med220 a The of p300 in the of AHR may be a of different of p300 in and Our study an role for Med220 in the composition of the Mediator complex and potential and of its this complex may play a role in tissue and developmental in response to of AHR. We Freedman for expression constructs for several Mediator