Estrogen plays an essential physiologic role in reproduction and a pathologic one in breast cancer. The completion of the human genome has allowed the identification of the expressed regions of protein-coding genes; however, little is known concerning the organization of their cis-regulatory elements. We have mapped the association of the estrogen receptor (ER) with the complete nonrepetitive sequence of human chromosomes 21 and 22 by combining chromatin immunoprecipitation (ChIP) with tiled microarrays. ER binds selectively to a limited number of sites, the majority of which are distant from the transcription start sites of regulated genes. The unbiased sequence interrogation of the genuine chromatin binding sites suggests that direct ER binding requires the presence of Forkhead factor binding in close proximity. Furthermore, knockdown of FoxA1 expression blocks the association of ER with chromatin and estrogen-induced gene expression demonstrating the necessity of FoxA1 in mediating an estrogen response in breast cancer cells.

The aryl hydrocarbon receptor (AhR) is a receptor/transcription factor which regulates cytochrome P450 (CYP) gene transcription and which is activated by environmental carcinogens, some of which are associated with increased breast cancer risk. Here, we show that the AhR is over-expressed and constitutively active in human and rodent mammary tumors, suggesting its ongoing contribution to tumorigenesis regardless of tumor etiology. AhR regulation of CYP1A1 and CYP1B1 was studied to determine if constitutively active AhR effects the same transcriptional outcomes as environmental chemical-activated AhR. Elevated AhR and CYP1B1 but not CYP1A1 before tumor formation in a rat model of mammary tumorigenesis suggested differential CYP1B1 regulation by a constitutively active AhR. This hypothesis was tested with human mammary gland cell lines which hyper-express AhR and CYP1B1 but which express little or no CYP1A1. CYP1B1 expression was diminished by repression of AhR activity or by AhR knockdown, demonstrating AhR control of basal CYP1B1 levels. ChIP assays demonstrated constitutive AhR binding to both CYP1A1 and CYP1B1 promoters, demonstrating that differential CYP1A1 and CYP1B1 regulation by constitutively active AhR does not result from different amounts of promoter-bound AhR. While increasing AhR binding to both CYP1A1 and CYP1B1, 2,3,7,8-tetrachlorodibenzo-p-dioxin induced CYP1A1 mRNA in both a malignant and non-malignant line but increased only CYP1B1 mRNA in the malignant line, again demonstrating that the level of promoter binding does not necessarily correlate with gene mRNA levels. These studies suggest that constitutively active AhR mediates different molecular outcomes than environmental chemical-activated AhR, and further implicate the AhR in mammary tumorigenesis.

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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...