The subdomains of the transactivation domain of the aryl hydrocarbon receptor (AhR) inhibit AhR and estrogen receptor transcriptional activity q Rashmeet K. Reen, Adam Cadwallader, and Gary H. Perdew * Center for Molecular Toxicology and Carcinogenesis and Department of Veterinary Science, Pennsylvania State University, 226, Fenske Lab, University Park, PA 16802, USA Received 8 July 2002, and in revised form 27 August 2002 Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) activates the aryl hydrocarbon receptor (AhR) to mediate transcriptional activity of dioxin-responsive genes. The transactivation domain (TAD) of human AhR (hAhR) has potentially distinct acidic, glutamine- rich, and proline/serine/threonine-rich subdomains. Cotransfection of exogenous hAhR into BP8 cells with isolated subdomains of hAhR TAD fused to glutathione S-transferase exhibited squelching of TCDD-dependent dioxin-response element (DRE)-driven luciferasereporter-geneactivitywitheachsubdomain.TostudythepotentialcrosstalkbetweenAhR-andestrogenreceptor(ER)- mediated activities, BP8 cells were cotransfected with hAhR TAD subdomain constructs and ERa. The three hAhR TAD sub- domains inhibited the 17b-estradiol-induced estrogen-response element-mediated reporter-gene transactivation. Cotransfection of hAhR with the ligand-binding domain (LBD) of ERa also squelched TCDD-dependent DRE-driven reporter-gene activity in the presenceof17b-estradiol.SimilarresultswereobservedinT47DcellsthatexpressfunctionalAhRandERa.Theseresultsindicate thattheisolatedsubdomainsofhAhRÕsTADandLBDofERa arecapableofsquelchingligand-dependenttransactivationofeither the AhR or the ER, by titrating crucial proteins from an existing common pool of cofactors. Ó 2002 Published by Elsevier Science (USA). Keywords: Aryl hydrocarbon receptor; Estrogen receptor; 17b-Estradiol; 2,3,7,8-Tetrachlorodibenzo-p-dioxin; Dioxin response element; Estrogen response element; Transactivation subdomains; Ah receptor The aryl hydrocarbon receptor is a ligand-activated transcriptionfactorthathasbeenidentifiedasaprimary cellular target for 2,3,7,8-tetrachlorodibenzo-p-dioxin andrelatedcompounds.TheAhR 1 mediatesmost,ifnot all, toxic responses to TCDD, which include carcino- genicity, teratogenicity, hepatotoxicity, immune sup- pression, and reproductive and developmental toxicity. Biochemical responses to AhR activation include in- ductionofdrug-metabolizingenzymessuchasCYP1A1, CYP1B1, CYP1A2, and GSTYa [1,2]. The AhR is a member of the basic helix–loop–helix/PAS family of nuclear transcription factors [3] and is located in the cytoplasm as a heterotetrameric 9S complex in associa- tionwithHsp90andXAP2[4–6].Ligandbindingresults in translocation of the entire complex into the nucleus and subsequent shedding of Hsp90 and XAP2. AhR heterodimerizeswithitsnuclearpartner,Arnt[7,8],and interacts with DRE of target genes like CYP1A1, lead- ing to altered transcriptional rates [9]. The complex re- cruits other nuclear proteins, such as SRC-1, RIP140 Archives of Biochemistry and Biophysics 408 (2002) 93–102 www.academicpress.com ABB q This work was supported by the National Institute of Environ- mental Health Science, Grant ES04869, awarded to G.H. Perdew. * Corresponding author. Fax: 1-814-863-1696. E-mail address: ghp2@psu.edu (G.H. Perdew). 1 Abbreviations used: AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; ER, estrogen receptor; Hsp90, 90-kDa heat-shock protein; PAS, Per-Arnt-Sim; XAP2, hepatitis B virus X-associated protein; E2, 17b-estradiol; TCDD, 2,3,7,8-tetrachlo- rodibenzo-p-dioxin; DRE, dioxin-response element; ERE, estrogen- response element; TAD, transactivation domain; GST, glutathione S-transferase; Q-rich, glutamine-rich; PST, proline/serine/threonine; Mops, 4-morpholinepropanesulfonic acid; NLS, nuclear localization signal;RLU,relativelightunits;LBD,ligand-bindingdomain;MEM, minimal essential medium; FBS, fetal bovine serum; NP-40, Nonidet P-40; AAD, acidic activation domain. 0003-9861/02/$ - see front matter Ó 2002 Published by Elsevier Science (USA). PII:S0003-9861(02)00518-0