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• W2065956908 abstract "The aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) are DNA binding transcription factors with basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domains. These two proteins form a heterodimer that mediates the toxic and biological effects of the environmental contaminant and AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin. The coiled-coil protein coiled-coil coactivator (Co-CoA) is a secondary coactivator for nuclear receptors and enhances nuclear receptor function by interacting with the bHLH-PAS domain of p160 coactivators. We report here that CoCoA also binds the bHLH-PAS domains of AHR and ARNT and functions as a potent primary coactivator for them; i.e. CoCoA does not require p160 coactivators for binding to and serving as a coactivator for AHR and ARNT. Endogenous CoCoA was recruited to a natural AHR target gene promoter in a 2,3,7,8-tetrachlorodibenzo-p-dioxin -dependent manner. Moreover, reduction of CoCoA mRNA levels by small interfering RNA inhibited the transcriptional activation by AHR and ARNT. Our data support a physiological role for CoCoA as a transcriptional coactivator in AHR/ARNT-mediated transcription. The aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) are DNA binding transcription factors with basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domains. These two proteins form a heterodimer that mediates the toxic and biological effects of the environmental contaminant and AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin. The coiled-coil protein coiled-coil coactivator (Co-CoA) is a secondary coactivator for nuclear receptors and enhances nuclear receptor function by interacting with the bHLH-PAS domain of p160 coactivators. We report here that CoCoA also binds the bHLH-PAS domains of AHR and ARNT and functions as a potent primary coactivator for them; i.e. CoCoA does not require p160 coactivators for binding to and serving as a coactivator for AHR and ARNT. Endogenous CoCoA was recruited to a natural AHR target gene promoter in a 2,3,7,8-tetrachlorodibenzo-p-dioxin -dependent manner. Moreover, reduction of CoCoA mRNA levels by small interfering RNA inhibited the transcriptional activation by AHR and ARNT. Our data support a physiological role for CoCoA as a transcriptional coactivator in AHR/ARNT-mediated transcription. Polycyclic or halogenated aromatic hydrocarbons and other related planar organochlorinated compounds elicit many adverse biological effects, including immunosuppression, teratogenesis, tumor promotion, hormonal disregulation, and cardiovascular disease. All of these biological effects are believed to be mediated by the sustained activation of the aryl hydrocarbon receptor (AHR) 1The abbreviations used are: AHR, aryl hydrocarbon receptor; ARNT, AHR nuclear translocator; CoCoA, coiled-coil coactivator; NR, nuclear receptor; bHLH/PAS, basic helix-loop-helix/Per-Arnt-Sim; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XRE, xenobiotic response element; ChIP, chromatin immunoprecipitation; CBP, cAMP-response element-binding protein (CREB)-binding protein; HA, hemagglutinin; GST, glutathione S-transferase; HEK cells, human embryonic kidney cells; siRNA, small interfering RNA; ER, estrogen receptor; DBD, DNA-binding domain.1The abbreviations used are: AHR, aryl hydrocarbon receptor; ARNT, AHR nuclear translocator; CoCoA, coiled-coil coactivator; NR, nuclear receptor; bHLH/PAS, basic helix-loop-helix/Per-Arnt-Sim; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XRE, xenobiotic response element; ChIP, chromatin immunoprecipitation; CBP, cAMP-response element-binding protein (CREB)-binding protein; HA, hemagglutinin; GST, glutathione S-transferase; HEK cells, human embryonic kidney cells; siRNA, small interfering RNA; ER, estrogen receptor; DBD, DNA-binding domain. (1Hankinson O. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 307-340Crossref PubMed Scopus (1415) Google Scholar, 2Denison M.S. Pandini A. Nagy S.R. Baldwin E.P. Bonati L. Chem. Biol. Interact. 2002; 141: 3-24Crossref PubMed Scopus (365) Google Scholar). AHR is a ligand-dependent transcription factor belonging to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) gene family (3Gu Y.Z. Hogenesch J.B. Bradfield C.A. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 519-561Crossref PubMed Scopus (841) Google Scholar). bHLH-PAS proteins share a conserved N-terminal structural motif. The bHLH domain of most (but not all) bHLH-PAS proteins is used for specific DNA binding and/or heterodimerization with other bHLH-PAS proteins. The PAS domain located immediately after the bHLH domain harbors two conserved hydrophobic repeats termed A and B and functions as a sensor of specific environmental signals, a ligand binding surface, and a protein-protein interaction surface in various bHLH-PAS transcription factors, such as AHR and hypoxia-inducible factors. Another member of this family, AHR nuclear translocator (ARNT) is an indispensable heterodimer partner for AHR and hypoxia-inducible factors. In addition to the heterodimerization with AHR or hypoxia-inducible factor 1α, ARNT homodimers are likely to play physiological roles by binding to the E-box core sequence found in some types of enhancer elements (4Huffman J.L. Mokashi A. Bachinger H.P. Brennan R.G. J. Biol. Chem. 2001; 276: 40537-40544Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). AHR is found in the cytosol in a heterotetrameric complex with two Hsp90 molecules and one X-associated protein 2 (XAP2) molecule (5Meyer B.K. Pray-Grant M.G. Vanden Heuvel J.P. Perdew G.H. Mol. Cell. Biol. 1998; 18: 978-988Crossref PubMed Scopus (305) Google Scholar, 6Petrulis J.R. Perdew G.H. Chem. Biol. Interact. 2002; 141: 25-40Crossref PubMed Scopus (224) Google Scholar). Upon binding of xenobiotic compounds such as TCDD, the complexes translocate to the nucleus, where the AHR heterodimerizes with ARNT after Hsp90 dissociation (2Denison M.S. Pandini A. Nagy S.R. Baldwin E.P. Bonati L. Chem. Biol. Interact. 2002; 141: 3-24Crossref PubMed Scopus (365) Google Scholar, 6Petrulis J.R. Perdew G.H. Chem. Biol. Interact. 2002; 141: 25-40Crossref PubMed Scopus (224) Google Scholar). Heterodimeric AHR·ARNT complexes bind to specific enhancer elements called xenobiotic response elements (XREs) found in the regulatory domains of numerous genes (7Probst M.R. Reisz-Porszasz S. Agbunag R.V. Ong M.S. Hankinson O. Mol. Pharmacol. 1993; 44: 511-518PubMed Google Scholar). Genes transcriptionally activated by AHR·ARNT encode several enzymes that metabolize xenobiotic compounds (e.g. cytochrome P-450 enzymes such as CYP1A1) and proto-oncogenes c-jun and c-fos (8Hoffer A. Chang C.Y. Puga A. Toxicol. Appl. Pharmacol. 1996; 141: 238-247Crossref PubMed Google Scholar, 9Whitlock Jr., J.P. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 103-125Crossref PubMed Scopus (986) Google Scholar). AHR is also apparently involved in hepatic growth and development of the immune system, based on the phenotype of AHR knock-out mice, and may play a role in the cell cycle (10Gonzalez F.J. Fernandez-Salguero P. Drug Metab. Dispos. 1998; 26: 1194-1198PubMed Google Scholar, 11Ma Q. Whitlock Jr., J.P. Mol. Cell. Biol. 1996; 16: 2144-2150Crossref PubMed Scopus (242) Google Scholar). Despite the structural and functional differences between AHR·ARNT and nuclear receptors (NRs), both are ligand-regulated transcription factors and share common coregulators to mediate their transcription-enhancing activities. For example, the NR corepressor SMRT binds to AHR·ARNT and inhibits AHR·ARNT activity (12Nguyen T.A. Hoivik D. Lee J.E. Safe S. Arch. Biochem. Biophys. 1999; 367: 250-257Crossref PubMed Scopus (123) Google Scholar). The AHR·ARNT heterodimer also interacts with NR coactivators such as the p160 coactivators SRC-1, GRIP-1, and p/CIP (13Beischlag T.V. Wang S. Rose D.W. Torchia J. Reisz-Porszasz S. Muhammad K. Nelson W.E. Probst M.R. Rosenfeld M.G. Hankinson O. Mol. Cell. Biol. 2002; 22: 4319-4333Crossref PubMed Scopus (171) Google Scholar, 14Kumar M.B. Perdew G.H. Gene Expr. 1999; 8: 273-286PubMed Google Scholar), which enhance AHR/ARNT-mediated transcription in transient reporter gene assays. Although they have not been shown to bind DNA, the p160 coactivators also contain N-terminal bHLH-PAS domains (3Gu Y.Z. Hogenesch J.B. Bradfield C.A. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 519-561Crossref PubMed Scopus (841) Google Scholar, 15Xu J. Li Q. Mol. Endocrinol. 2003; 17: 1681-1692Crossref PubMed Scopus (389) Google Scholar). Furthermore, p160 coactivators are recruited to the CYP1A1 enhancer region in a TCDD-dependent fashion (13Beischlag T.V. Wang S. Rose D.W. Torchia J. Reisz-Porszasz S. Muhammad K. Nelson W.E. Probst M.R. Rosenfeld M.G. Hankinson O. Mol. Cell. Biol. 2002; 22: 4319-4333Crossref PubMed Scopus (171) Google Scholar). We previously isolated a novel NR coactivator, CoCoA, using the bHLH-PAS domain of GRIP1 as bait in a yeast two-hybrid screen (16Stallcup M.R. Kim J.H. Teyssier C. Lee Y.H. Ma H. Chen D. J. Steroid Biochem. Mol. Biol. 2003; 85: 139-145Crossref PubMed Scopus (94) Google Scholar, 17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). CoCoA is a new type of NR coactivator with a potent C-terminal activation domain and a central coiled-coil domain that binds the bHLH-PAS motif of p160 coactivators. Thus, CoCoA acts as a secondary coactivator for NRs, interacting with them indirectly through the primary p160 coactivators. Given the shared bHLH-PAS domains among p160 coactivators and bHLH-PAS transcription factors, we tested whether CoCoA could physically and functionally interact with AHR and ARNT as a coactivator in TCDD-dependent gene activation. Construction of Plasmids—The hemagglutinin (HA)-tagged mouse AHR (pACTAG-2.mAHR) and ARNT (pACTAG-2.mARNT) expression plasmids, Gal4 DBD-fused ARNT expression plasmid, and a CYP1A1-luciferase reporter plasmid (pGL-CYP1A1-LUC) containing 2.6 kilo-bases of the regulatory sequence of rat CYP1A1 gene are the kind gifts of Oliver Hankinson and Timothy Beischlag (University of California, Los Angeles, CA). PCR amplification and subcloning into the indicated restriction sites were performed to create plasmids encoding the following AHR and ARNT fragments (amino acid numbers in parentheses): AHR N (1–374), AHR C (375–805), ARNT N (1–458), and ARNT C (459–791) fragments digested with EcoRI and XhoI were inserted into EcoRI and XhoI sites of pSG5.HA, and ARNT N was inserted into EcoRI and SalI sites of pM. pSG5.HA-CoCoA, pSG5.HA-CoCoA (1–500), pcDNA3.1-CoCoA/V5-His, pGEX-5x-1-Co-CoA, pSG5.HA-GRIP1, pSG5.HA-GRIP1 (563–1462), and GK1-LUC were described previously (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Coimmunoprecipitation and Immunoblotting—COS-7 cell transfection, coimmunoprecipitation, and immunoblotting were performed as described previously (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). For coimmunoprecipitation, cell lysate containing 1 mg of protein was incubated with 1 μg of the anti-V5 antibody R960–25 (Invitrogen), mouse normal IgG (Santa Cruz Biotechnology), 10 μl of an equal mixture of the two rabbit antisera against CoCoA (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) or preimmune sera, and 20 μl of protein A/G-agarose suspension (Santa Cruz Biotechnology) overnight at 4 °C. Immunoblots were then performed on the precipitated proteins with anti-HA antibody 3F10 (Roche Applied Science). Subsequently, the membrane was stripped of bound antibody and re-probed with the anti-V5 antibody. GST Pull-down Assay—HA epitope-tagged AHR, ARNT, and their fragments were synthesized in vitro by using TNT-Quick coupled transcription/translation system (Promega) according to the manufacturer's protocol. GST pull-down assays were performed as described previously (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Bound proteins were analyzed by immunoblot with anti-HA antibody. Cell Culture and Transient Transfection—Hepa1c1c7 cells, hereafter referred to as Hepa-1 cells, were maintained in α minimal essential medium (Irvine Scientific) supplemented with 10% fetal bovine serum. HEK 293T cells and COS-7 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. For reporter gene assays, HEK 293T and Hepa-1 cells were plated at 2 × 105 cells/well in 12-well plates. HEK 293T cells were transiently transfected using Lipofectamine 2000 (Invitrogen). 48 h after transfection, cell extracts were prepared and assayed for luciferase activity as described previously (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Hepa-1 cells were transiently transfected by Superfect transfection reagent (Qiagen) according to the manufacturer's protocol. The total amount of plasmid DNA added to each well was adjusted to 1.0 μg by adding the necessary amount of pSG5.HA empty vector. Three hours after transfection cells were treated with either 10 nm TCDD or 0.1% dimethyl sulfoxide (Me2SO) vehicle and incubated for a further 18 h before luciferase assays. The results shown are the means and S.D. of triplicate points. Chromatin Immunoprecipitation (ChIP)—Hepa-1 cells were treated with 10 nm TCDD for 60 min. ChIP assays were performed largely as described previously (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The cross-linked, sheared chromatin solution was used for immunoprecipitation with 5 μl of anti-AHR antibody MA1–513 (Affinity BioReagents), 2 μg of anti-GRIP1 antibody A300–025A (Bethyl Laboratories), or 10 μl of an equal mixture of the two rabbit antisera against CoCoA (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The immunoprecipitated DNAs were purified by phenol-chloroform extraction, precipitated by ethanol, and amplified by PCR using primers flanking the murine CYP1A1 enhancer region (–1141 to –784 from the transcription initiation site) or β-actin promoter region (–527 to –205): CYP1A1 (–1141/–784), 5′-CTATCTCTTAAACCCCACCCCAA-3′ (forward) and 5′-CTAAGTATGGTGGAGGAAAGGGTG-3′ (reverse); β-actin (–527 to –205), 5′-ATTGCTAGCAATTGCTAGCAAGGGGGAGT-3′ (forward) and 5′-GAGAGAAAGCGAGATTGAGGAAGAGGATGA-3′ (reverse). RNA Interference and Reverse Transcription-PCR—COS-7 transfection with siRNAs, reverse transcription-PCR, and luciferase assays were performed as described previously (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Hepa-1 cells were plated into 12-well plates, grown until 70–80% confluence, and transfected with 40 or 80 pmol of scrambled sequence or CoCoA-specific siRNA duplex using Lipofectamine 2000 after the manufacturer's instructions. Total RNA was extracted with Trizol reagent (Invitrogen). The reverse transcriptase-PCR analysis was performed with 1–50 ng of total RNA using the Access reverse transcription-PCR system (Promega). The primers used in reverse transcriptase-PCR reactions were as follows: CoCoA, 5′-CACCTACATCCCCAACACCAA-3′ (forward) and 5′-CTTCATCAGCACTTTGTCACT-3′ (reverse); CYP1A1, 5′-CCAGACCTCTACAGCTTCACACTTATCACT-3′ (forward) and 5′-CCTGTCCTGACAATGCTCAATGAGGCTGTC-3′ (reverse); β-actin, 5′-TGTGATGGTGGGAATGGGTCAGAAGGACTC-3′ (forward) and 5′-TCATCGTACTCCTGCTTGCTGATCCACATC-3′ (reverse). The relative quantity of the PCR products was determined by densitometric analysis using the UN-SC-AN-IT gel software (Silk Scientific, Inc.). CoCoA Interacts with AHR and ARNT—Because the bHLH-PAS domain of the p160 coactivators is also present in AHR, ARNT, and other bHLH-PAS transcription factors, we tested whether CoCoA has the ability to interact with AHR and ARNT. In an in vitro GST pull-down assay GST-CoCoA efficiently bound full-length AHR and ARNT synthesized in vitro (Fig. 1, A and B). The interaction with AHR was TCDD-independent. To study in vivo interactions by co-immunoprecipitation, COS-7 cells were transfected with expression plasmids for V5-tagged CoCoA and HA-tagged AHR or ARNT. Anti-V5 antibodies efficiently and specifically precipitated AHR and ARNT (Fig. 1, C and D). Again, the interaction between CoCoA and AHR was TCDD-independent (data not shown). Other AHR coactivators can also bind efficiently to AHR without its ligand (13Beischlag T.V. Wang S. Rose D.W. Torchia J. Reisz-Porszasz S. Muhammad K. Nelson W.E. Probst M.R. Rosenfeld M.G. Hankinson O. Mol. Cell. Biol. 2002; 22: 4319-4333Crossref PubMed Scopus (171) Google Scholar, 18Ge N.L. Elferink C.J. J. Biol. Chem. 1998; 273: 22708-22713Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 19Kumar M.B. Tarpey R.W. Perdew G.H. J. Biol. Chem. 1999; 274: 22155-22164Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 20Jones L.C. Okino S.T. Gonda T.J. Whitlock Jr., J.P. J. Biol. Chem. 2002; 277: 22515-22519Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). This is in contrast to the ligand-dependent interactions of NRs with many coactivators. To characterize the CoCoA interaction domains in AHR and ARNT, the N-terminal (AHR 1–374 and ARNT 1–458) and C-terminal (AHR 375–805 and ARNT 459–799) portions (Fig. 2A) were tested for binding to CoCoA. In GST pull-down assays full-length AHR and its N-terminal and C-terminal domains interacted with CoCoA (Fig. 2B). The N-terminal bHLH-PAS domain of AHR (which contains the ligand binding function) interacted with CoCoA in the absence and the presence of TCDD (data not shown). Because ARNT fragments were not generated efficiently by in vitro translation, ARNT deletion mutants were tested in co-immunoprecipitation assays. CoCoA interacted with the N-terminal bHLH-PAS region but not with the C-terminal activation domain of ARNT (Fig. 2C). CoCoA Potentiates Dioxin-dependent AHR/ARNT-mediated Transcription—CoCoA functions as a secondary coactivator to enhance transactivation by NRs; i.e. CoCoA binds to NRs indirectly through p160 coactivators, and the ability of CoCoA to enhance NR function depends on the presence of p160 coactivators (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In contrast, CoCoA appears to interact directly with AHR and ARNT (Figs. 1 and 2). To investigate whether CoCoA can function as a primary coactivator for AHR·ARNT, we transfected AHR/ARNT-positive Hepa-1 cells with a luciferase reporter plasmid controlled by the XRE-containing CYP1A1 promoter together with expression vectors for GRIP1, CoCoA, or both. TCDD treatment alone enhanced CYP1A1 promoter-driven luciferase activity nearly 28-fold (Fig. 3A, assay 1). As expected (13Beischlag T.V. Wang S. Rose D.W. Torchia J. Reisz-Porszasz S. Muhammad K. Nelson W.E. Probst M.R. Rosenfeld M.G. Hankinson O. Mol. Cell. Biol. 2002; 22: 4319-4333Crossref PubMed Scopus (171) Google Scholar), overexpression of GRIP1 enhanced AHR function in a dose-dependent and TCDD-dependent manner (assays 2–5). CoCoA expression increased TCDD-dependent reporter gene activity up to 5-fold (assays 6–9). This stimulation of AHR·ARNT activity was dependent on the amount of CoCoA, and the magnitude of stimulation was similar to that produced by GRIP1 under the same conditions. Despite the fact that CoCoA binds to AHR in a ligand-independent manner (Fig. 1A), the effect of CoCoA on AHR-mediated transcription was TCDD-dependent. When CoCoA was co-expressed with GRIP1, an additive induction of AHR/ARNT-mediated transactivation was observed (assays 10–11). These results suggest that GRIP1 and CoCoA both interact directly with AHR·ARNT, function as primary coactivators, and make distinct contributions to AHR/ARNT-mediated transcriptional activation. Although p160 coactivators contain an N-terminal bHLH-PAS domain, they do not utilize this domain to interact with AHR and ARNT; instead a C-terminal region of p160 proteins, just proximal to the p300/CBP binding AD1 domain, binds to the bHLH-PAS domains of AHR and ARNT (13Beischlag T.V. Wang S. Rose D.W. Torchia J. Reisz-Porszasz S. Muhammad K. Nelson W.E. Probst M.R. Rosenfeld M.G. Hankinson O. Mol. Cell. Biol. 2002; 22: 4319-4333Crossref PubMed Scopus (171) Google Scholar). Therefore, we tested whether a GRIP1ΔN mutant lacking the bHLH-PAS domain could enhance AHR·ARNT function and cooperate with CoCoA. The GRIP1ΔN mutant enhanced AHR·ARNT function in a dose-dependent manner (Fig. 3B, assays 4 and data not shown). Furthermore, coexpression of CoCoA with the GRIP1ΔN mutant further potentiated the activity of AHR (Fig. 3B, assays 8–9). Thus, the bHLH-PAS domain of GRIP1 is not required for the coactivator function of CoCoA with AHR. These data are consistent with the conclusion that CoCoA interacts directly as a primary coactivator with AHR and ARNT. This result contrasts starkly with the secondary coactivator function of CoCoA with NRs, which depends entirely on the presence of a p160 coactivator with an intact N-terminal region (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The C-terminal segment of CoCoA (amino acids 501–691) possesses very strong autonomous transcriptional activation activity when fused to the GAL4 DNA binding domain and is necessary for the coactivator function of CoCoA with NRs (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The CoCoA mutant lacking the C-terminal activation domain failed to enhance AHR function (Fig. 3B, assay 5). CoCoA Enhances the Transcriptional Activity of ARNT—To further investigate a functional interaction between ARNT and CoCoA, we tested the effect of CoCoA expression on ARNT-mediated transactivation in a mammalian one-hybrid system. In transiently transfected HEK 293T cells the strong autonomous transactivation activity of a Gal4 DBD-ARNT fusion protein was further enhanced (up to 4-fold) by co-expression of CoCoA (Fig. 4, assays 4–6). Because the bHLH-PAS domain of ARNT mediates protein interaction with CoCoA (Fig. 2C), we tested the effect of CoCoA overexpression on the activity of Gal4-ARNT-N, which harbors the bHLH-PAS motif. Gal4-ARNT-N possesses very little autonomous transcriptional activation activity on a transiently transfected Gal4-responsive reporter plasmid in HEK 293T cells (Fig. 4, assays 7 and 9), consistent with studies indicating the requirement for the ARNT C-terminal activation domain for fully activated transcription (21Corton J.C. Moreno E.S. Hovis S.M. Leonard L.S. Gaido K.W. Joyce M.M. Kennett S.B. Toxicol. Appl. Pharmacol. 1996; 139: 272-280Crossref PubMed Scopus (8) Google Scholar). Nevertheless, co-expression of CoCoA increased luciferase activity by up to 7-fold (assays 9–11). Thus, CoCoA interacts with the bHLH-PAS domain of ARNT and is a potent coactivator for ARNT. CoCoA Is Specifically Targeted to the CYP1A1 XRE Region— ChIP assays were used to test whether CoCoA is recruited to the XREs of known AHR target genes. The CYP1A1 gene from mouse Hepa-1 cells has a well characterized regulatory region that responds to TCDD. Hepa-1 cells were treated either with 10 nm TCDD in Me2SO or with Me2SO vehicle. The cross-linked, sheared chromatin preparations were subjected to immunoprecipitation with various antibodies, and the precipitated DNA was analyzed by PCR amplification of the XRE region of the CYP1A1 promoter. TCDD treatment caused recruitment of AHR and GRIP1 to the CYP1A1 promoter region (Fig. 5), as shown previously (13Beischlag T.V. Wang S. Rose D.W. Torchia J. Reisz-Porszasz S. Muhammad K. Nelson W.E. Probst M.R. Rosenfeld M.G. Hankinson O. Mol. Cell. Biol. 2002; 22: 4319-4333Crossref PubMed Scopus (171) Google Scholar, 22Wang S. Hankinson O. J. Biol. Chem. 2002; 277: 11821-11827Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In addition, antibodies against CoCoA efficiently immunoprecipitated the XRE region of the CYP1A1 promoter from the TCDD-treated but not from the control cells, indicating the dioxin-dependent recruitment of CoCoA to the CYP1A1 promoter. Normal IgG failed to precipitate the CYP1A1 promoter. In contrast to the CYP1A1 promoter, the β-actin promoter region was not detected in association with AHR, GRIP1, or CoCoA. Thus, endogenous CoCoA was recruited to a native AHR-regulated promoter in a TCDD-dependent fashion, demonstrating a functional interaction between AHR and CoCoA occurring in an in vivo setting. Requirement for Endogenous CoCoA for Transcriptional Activation by AHR and ARNT—If CoCoA is a coactivator for AHR·ARNT, reducing the endogenous level of CoCoA should decrease the transcriptional activity of endogenous AHR and ARNT. In RNA interference experiments using a CoCoA-specific siRNA that was previously demonstrated to be effective in reducing CoCoA expression level (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), transfection of the Co-CoA-directed siRNA, but not a control scrambled-sequence siRNA, into Hepa-1 cells reduced the level of endogenous Co-CoA mRNA but had no effect on the β-actin mRNA level (Fig. 6A). TCDD-induced expression of the endogenous CYP1A1 gene was inhibited (more than 40%) by the CoCoA-directed siRNA, but the scrambled-sequence siRNA had no effect. To our surprise we also found that dioxin up-regulated the CoCoA mRNA level greater than 2-fold (Fig. 6A, lanes 1 and 2). Analysis of the mouse CoCoA gene (LocusLink locus ID 67488) for core AHR/ARNT binding sequences using the web-based TRANSFAC data base (23Wingender E. Chen X. Fricke E. Geffers R. Hehl R. Liebich I. Krull M. Matys V. Michael H. Ohnhauser R. Pruss M. Schacherer F. Thiele S. Urbach S. Nucleic Acids Res. 2001; 29: 281-283Crossref PubMed Scopus (553) Google Scholar) revealed one such sequence (tgcCACGCtgagtcca) and two putative ARNT binding sequences (CACGTG) within 2 kilobases upstream of the Co-CoA transcription start site. Thus, the CoCoA gene is apparently dioxin-responsive. The physiological significance of CoCoA in ARNT function was further tested by RNA interference in COS-7 cells. The siRNA against CoCoA, but not the scrambled-sequence siRNA, specifically reduced the level of endogenous CoCoA mRNA but not β-actin mRNA (Fig. 6B, inset). Reduction of CoCoA levels in COS-7 cells also reduced activity of a Gal4 DBD-ARNT fusion protein by more than 65% but had little or no effect on the activity of Gal4 DBD (Fig. 6B) Thus, endogenous CoCoA is required for efficient transcriptional activation by AHR and ARNT. The results from ChIP and RNAi assays strongly support a physiological role for CoCoA in AHR/ARNT-dependent transcription. CoCoA Is a Primary Coactivator for AHR·ARNT—CoCoA was initially identified as a novel type of NR coactivator with a coiled-coil domain (17Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). CoCoA binds to the bHLH-PAS domain of p160 coactivators but not directly to NRs, and the ability of CoCoA to enhance the activity of steroid receptors is highly dependent on the presence of a p160 coactivator with an intact N-terminal bHLH-PAS domain. By these characteristics, Co-CoA is classified as a secondary coactivator for nuclear receptors (16Stallcup M.R. Kim J.H. Teyssier C. Lee Y.H. Ma H. Chen D. J. Steroid Biochem. Mol. Biol. 2003; 85: 139-145Crossref PubMed Scopus (94) Google Scholar). In contrast, CoCoA bound to AHR and ARNT in vitro and in vivo and functioned as a coactivator for AHR and ARNT without co-expression of p160 coactivators or any other exogenously expressed proteins (Figs. 1 and 3). Thus, CoCoA binds indirectly to NRs and functions as a secondary coactivator for them but appears to bind directly to AHR·ARNT and serves as a primary coactivator for AHR·ARNT. The transient transfection data indicate that CoCoA has the activity of a coactivator for AHR·ARNT under those conditions. Under more physiologically relevant conditions endogenous CoCoA was efficiently recruited together with AHR to the endogenous CYP1A1 promoter in a TCDD-dependent fashion (Fig. 5) and was required for efficient TCDD induction of the endogenous CYP1A1 gene by AHR and for the autonomous transcriptional activation activity of ARNT (Fig. 6). These findings demonstrate that CoCoA is a physiologically relevant part of transcriptional activation by AHR·ARNT. Interestingly, we observed that the CoCoA mRNA level was up-regulated more than 2-fold by TCDD. Obviously, the resulting increase in CoCoA may further enhance AHR·ARNT activity. The regulation of CoCoA by ligand-activated AHR·ARNT is an additional physiological link between CoCoA and AHR·ARNT. The mechanism by which AHR·ARNT regulates CoCoA levels may involve direct binding of AHR·ARNT to the CoCoA gene promoter, since a putative AHR·ARNT binding sequence occurs within the proximal CoCoA promoter. Thus CoCoA and AHR·ARNT mutually regulate each other; AHR·ARNT regulates CoCoA levels, and Co-CoA serves as a coactivator for AHR·ARNT. Interaction of CoCoA with Mulitple Classes of bHLH-PAS Proteins—The interaction between CoCoA and AHR appears to be direct and involves two distinct regions within AHR, the N-terminal bHLH-PAS domain and a second site within the C-terminal activation domain (Fig. 2B). AHR bound CoCoA in a ligand-independent manner, suggesting that CoCoA may exist in a complex with AHR before ligand activation and after ligand binding travels with AHR to its target promoter. CoCoA and AHR are both recruited to the CYP1A1 promoter in a TCDD-dependent manner (Fig. 5), and CoCoA fails to enhance the activity of the CYP1A1 promoter in the absence of TCDD (Fig. 3). The ligand-independent association of AHR with Co-CoA and other coactivators (13Beischlag T.V. Wang S. Rose D.W. Torchia J. Reisz-Porszasz S. Muhammad K. Nelson W.E. Probst M.R. Rosenfeld M.G. Hankinson O. Mol. Cell. Biol. 2002; 22: 4319-4333Crossref PubMed Scopus (171) Google Scholar, 18Ge N.L. Elferink C.J. J. Biol. Chem. 1998; 273: 22708-22713Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 19Kumar M.B. Tarpey R.W. Perdew G.H. J. Biol. Chem. 1999; 274: 22155-22164Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 20Jones L.C. Okino S.T. Gonda T.J. Whitlock Jr., J.P. J. Biol. Chem. 2002; 277: 22515-22519Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) is in contrast to the interactions of many coactivators with NRs and may result from different structural organizations of AHR and NR proteins. Unlike the steroid receptors, where the ligand binding pocket and the important AF-2 activation function are part of the same structural domain, the major C-terminal activation domain of AHR is well separated from the N-terminal ligand binding domain; this may allow ligand-independent interactions between activation domain and coactivators. In contrast to the situation with AHR, CoCoA interacts with the bHLH-PAS domain but not with the activation domain of ARNT (Fig. 2C). These observations are consistent with previous reports which indicate that deletion of the C-terminal activation domain of ARNT failed to have a significant effect on activation of a reporter gene under control of the CYP1A1 enhancer and promoter, leading to the conclusion that the activation domain of AHR is dominant over that of ARNT during transcriptional activation (24Reisz-Porszasz S. Probst M.R. Fukunaga B.N. Hankinson O. Mol. Cell. Biol. 1994; 14: 6075-6086Crossref PubMed Scopus (210) Google Scholar, 25Ko H.P. Okino S.T. Ma Q. Whitlock Jr., J.P. Mol. Cell. Biol. 1996; 16: 430-436Crossref PubMed Scopus (144) Google Scholar). In general, the bHLH-PAS superfamily can be divided into three subgroups; members of one group are regulated in their expression or activity by binding of ligands or by cellular conditions (AHR, hypoxia-inducible factor, Single-minded (SIM), and Trachealess (TRH)); the second subgroup is ARNT, which does not need activation and has a central role in dimerization with members of group 1; the third subgroup is the p160 coactivators, which are not known to bind DNA directly but rather function as coactivators for diverse types of DNA binding transcription factors. CoCoA interacts with members of all three subgroups and enhances their activities as transcription factors or coactivators. These findings suggest that CoCoA may function as a general coactivator for all the bHLH-PAS transcriptional activators and may help us to understand the transactivation mechanisms of the bHLH-PAS protein family. Future studies of the interaction between CoCoA and the bHLH-PAS proteins may generate insights into regulation of genes involved in responses to hypoxia, circadian rhythms, development, and other pathways. Implications of CoCoA as a Common Coactivator for NRs and for AHR·ARNT—The fact that CoCoA serves as a common coactivator for two different classes of DNA binding transcription factors suggests a novel avenue for cross-talk between these two signaling pathways. It is interesting to note that TCDD inhibits several estrogen-dependent biological responses, decreases cellular estrogen receptor (ER) and progesterone receptor levels (26Ahlborg U.G. Lipworth L. Titus-Ernstoff L. Hsieh C.C. Hanberg A. Baron J. Trichopoulos D. Adami H.O. Crit. Rev. Toxicol. 1995; 25: 463-531Crossref PubMed Scopus (263) Google Scholar), and inhibits transcription of estrogen-regulated genes (27Porter W. Wang F. Duan R. Qin C. Castro-Rivera E. Kim K. Safe S. J. Mol. Endocrinol. 2001; 26: 31-42Crossref PubMed Scopus (59) Google Scholar). Conversely, 17β-estradiol (E2) can inhibit TCDD-induced reporter gene activity (28Kharat I. Saatcioglu F. J. Biol. Chem. 1996; 271: 10533-10537Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The possible mechanisms proposed for inhibitory AHR-ER cross-talk include induction of inhibitory factors, enhanced E2 metabolism, proteasome-dependent degradation of ERα by non-genomic inhibitory effects of AHR, and competition for common nuclear coactivators (29Safe S. Wormke M. Chem. Res. Toxicol. 2003; 16: 807-816Crossref PubMed Scopus (286) Google Scholar). Recent studies report that TCDD-induced interactions between AHR and ERα enhance ubiquitylation of ERα and subsequent proteasome-dependent degradation of ERα (30Wormke M. Stoner M. Saville B. Walker K. Abdelrahim M. Burghardt R. Safe S. Mol. Cell. Biol. 2003; 23: 1843-1855Crossref PubMed Scopus (239) Google Scholar). NRs and AHR·ARNT have been reported to interact with many common coactivators and components of the transcription machinery, including CoCoA, p160 coactivators (13Beischlag T.V. Wang S. Rose D.W. Torchia J. Reisz-Porszasz S. Muhammad K. Nelson W.E. Probst M.R. Rosenfeld M.G. Hankinson O. Mol. Cell. Biol. 2002; 22: 4319-4333Crossref PubMed Scopus (171) Google Scholar, 14Kumar M.B. Perdew G.H. Gene Expr. 1999; 8: 273-286PubMed Google Scholar), p300/CBP (31Kobayashi A. Numayama-Tsuruta K. Sogawa K. Fujii-Kuriyama Y. J. Biochem. (Tokyo). 1997; 122: 703-710Crossref PubMed Scopus (153) Google Scholar), BRG-1 (22Wang S. Hankinson O. J. Biol. Chem. 2002; 277: 11821-11827Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), ERAP140 (12Nguyen T.A. Hoivik D. Lee J.E. Safe S. Arch. Biochem. Biophys. 1999; 367: 250-257Crossref PubMed Scopus (123) Google Scholar), RIP140 (19Kumar M.B. Tarpey R.W. Perdew G.H. J. Biol. Chem. 1999; 274: 22155-22164Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), basal transcription factors (32Rowlands J.C. McEwan I.J. Gustafsson J.A. Mol. Pharmacol. 1996; 50: 538-548PubMed Google Scholar), and mediator complex (33Wang S. Ge K. Roeder R.G. Hankinson O. J. Biol. Chem. 2004; 279: 13593-13600Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In addition, a recent study has shown that ARNT, the obligatory heterodimerization partner for AHR, also functions as a coactivator of ERα and ERβ (34Brunnberg S. Pettersson K. Rydin E. Matthews J. Hanberg A. Pongratz I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6517-6522Crossref PubMed Scopus (116) Google Scholar). Some transcription factors have been reported to compete with one another for binding with limiting coactivators, resulting in mutually antagonistic effects (35Meyer M.E. Gronemeyer H. Turcotte B. Bocquel M.T. Tasset D. Chambon P. Cell. 1989; 57: 433-442Abstract Full Text PDF PubMed Scopus (438) Google Scholar, 36Kamei 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 (1915) Google Scholar). Competition for binding and squelching of these limiting common coactivators could be one of the mechanisms for the anti-estrogenic activity of TCDD. Our results add an additional possibility that cellular availability of CoCoA may also be an important factor in regulating ER and AHR transcriptional activities. We thank Drs. Oliver Hankinson and Timothy Beischlag for plasmids expressing mouse AHR, ARNT, and GAL4-ARNT, for CYP1A1-luciferase reporter plasmid, and for Hepa-1 cells. We thank Dan Gerke for expert technical assistance." @default.
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