Agonist and Chemopreventative Ligands Induce Differential Transcriptional Cofactor Recruitment by Aryl Hydrocarbon Receptor

Abstract

Aryl hydrocarbon receptor (AHR) is a transcription factor whose activity is regulated by environmental agents, including several carcinogenic agonists. We measured recruitment of AHR and associated proteins to the human cytochrome P4501A1 gene promoter in vivo. Upon treatment with the agonist ␤-naphthoflavone, AHR is rapidly associated with the promoter and recruits the three members of the p160 family of coactivators as well as the p300 histone acetyltransferase, leading to recruitment of RNA polymerase II (Pol II) and induction of gene transcription. AHR, coactivators, and Pol II cycle on and off the promoter, with a period of ϳ60 min. In contrast, the chemopreventative AHR ligand 3,3-diindolylmethane promotes AHR nuclear translocation and p160 coactivator recruitment but, remarkably, fails to recruit Pol II or cause histone acetylation. This novel mechanism of receptor antagonism may account for the antitumor properties of chemopreventative compounds targeting the AHR.The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor belonging to the basic helix-loop-helix/Per-ARNT-Sim family of proteins (12). The AHR mediates the toxic effects of several chemical carcinogens, including polycyclic and halogenated aromatic hydrocarbons. These are but examples of the diverse ligands for the AHR, which include dietary compounds, natural and synthetic flavonoids, natural products, and pharmaceuticals (6). Prior to ligand binding, AHR exists in the cytoplasm in a complex with heat shock protein 90 (24), the cochaperone p23 (14), and the immunophilin homolog XAP2 (3). Following ligand binding, AHR moves to the nucleus, dissociates from the chaperone complex, and forms a heterodimer with the basic helix-loop-helix/Per-ARNT-Sim protein ARNT. This heterodimer binds to xenobiotic response elements (XREs) in the promoter and enhancer regions of target genes to regulate their transcription. Induction of cytochrome P4501A1 (CYP1A1) expression has been studied extensively as a model of AHR action (35).Although structurally unrelated, AHR activity shares several features with members of the nuclear receptor superfamily. These transcription factors recruit a host of cofactor proteins to gene promoters in order to regulate transcription. Several nuclear receptor coactivators also interact with the AHR, including ERAP140 (22), RIP140 (20), p300, CBP (16), BRG-1 (34), and the three members of the p160 family of coactivators: NCoA1 (SRC-1), NCoA2 (GRIP-1 and TIF-2), and NCoA3 (AIB-1, p/CIP, and ACTR) (1). AHR interacts with these factors via its C-terminal transactivation domain (19), and ARNT may also be involved in recruiting cofactors to the promoter. The cofactors are involved in recruiting of additional proteins, ATP-dependent chromatin remodeling, and acetylation of promoter histones. The net effect of these activities is to relax chromatin, reposition nucleosomes, and facilitate recruitment of RNA polymerase II (Pol II).AHR and its agonists have been implicated in the initiation and progression of cancers in m...

Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells

“…The results illustrated in Figure 4A show that under normoxia, treatment with TCDD resulted in recruitment of both AhR and Arnt to the distal region of the CYP1A1 promoter which contains 4 DRE motifs. Pol II was not significantly associated with this complex as previously reported (Hestermann et al, 2003). However, in cells treated with 500 μM CoCl 2 for 2 or 6 hr, we also observed comparable recruitment of both AhR and Arnt to the CYP1A1 promoter, suggesting that hypoxia does not directly affect association of this complex with the promoter.…”

The aryl hydrocarbon receptor constitutively represses c-myc transcription in human mammary tumor cells

“…Several protein chaperones, coactivators, and corepressors can modulate AhR function (Meyer et al, 1998;Nguyen et al, 1999;Tohkin et al, 2000;Rushing and Denison, 2002;Hestermann and Brown, 2003;Fallone et al, 2004;Wang et al, 2004a;Beischlag and Perdew, 2005). Even other receptors, such as ER-a, can associate with and inhibit gene transactivation (Beischlag and Perdew, 2005).…”

“…ChIP and DNA quantification were performed as described previously (Hestermann and Brown, 2003). For gel electrophoresis, the region of the c-myc promoter 95-245 bp upstream of the transcription start site was amplified using the primers 5 0 -AGGCGCGCGTAGTTAATTCAT-3 0 and 5 0 -CGCCCT CTGCTTTGGGA-3 0 for 43 cycles (951C for 15 s, 581C for 60 s).…”

“…For gel electrophoresis, the region of the c-myc promoter 95-245 bp upstream of the transcription start site was amplified using the primers 5 0 -AGGCGCGCGTAGTTAATTCAT-3 0 and 5 0 -CGCCCT CTGCTTTGGGA-3 0 for 43 cycles (951C for 15 s, 581C for 60 s). The region of the CYP1A1 promoter 784-1156 bp upstream of the start site was amplified using the primers 5 0 -CACCCGCCACCCTTCGACAGTTCT-3 0 and 5 0 -CTCC CGGGGTGGCTAGTGCTTTGA-3 0 for 40 cycles (951C for 45 s, 581C for 45 s, and 701C for 60 s) as we described previously (Hestermann and Brown, 2003). In our hands, DNA is sheared within approximately 1 kb of the amplified fragment.…”

2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole is a ligand and shows species-specific partial agonism of the aryl hydrocarbon receptor

“…This represents an important advance, insofar as a great deal of existing literature describes antagonism of the AhR in a qualitative manner, and quantitative measurements will enable insight into the molecular mechanisms underlying ligand-specificity of response. So for example, where there is differential recruitment of cofactors by ligands of AhR (Boronat, 2007) (Hestermann and Brown, 2003) (Zhang et al, 2008), it would be of interest to quantitate the agonistic and antagonistic activity of the ligands.…”