The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls the expression of a diverse set of genes. The toxicity of the potent AhR ligand 2,3,7,8-tetrachlorodibenzop-dioxin is almost exclusively mediated through this receptor. However, the key alterations in gene expression that mediate toxicity are poorly understood. It has been established through characterization of AhR-null mice that the AhR has a required physiological function, yet how endogenous mediators regulate this orphan receptor remains to be established. A picture as to how the AhR/ARNT heterodimer actually mediates gene transcription is starting to emerge. The AhR/ ARNT complex can alter transcription both by binding to its cognate response element and through tethering to other transcription factors. In addition, many of the coregulatory proteins necessary for AhR-mediated transcription have been identified. Cross talk between the estrogen receptor and the AhR at the promoter of target genes appears to be an important mode of regulation. Inflammatory signaling pathways and the AhR also appear to be another important site of cross talk at the level of transcription. A major focus of this review is to highlight experimental efforts to characterize nonclassical mechanisms of AhR-mediated modulation of gene transcription.

The aryl hydrocarbon receptor (AHR) and the aryl hydrocarbon receptor nuclear translocator (ARNT) form a heterodimeric transcription factor upon binding a wide variety of environmental pollutants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR target gene activation can be repressed by estrogen and estrogen-like compounds. In this study, we demonstrate that a significant component of TCDD-inducible Cyp1a1 transcription is the result of recruitment of estrogen receptor (ER)-␣ by AHR/ARNT as a transcriptional corepressor. Both AHR and ARNT were capable of interacting directly with ER␣, as ascertained by glutathione S-transferase pull-down. 17␤-estradiol repressed TCDDactivated Cyp1a1 and Cyp1b1 gene transcription in MCF-7 cells in the presence of cycloheximide, as determined by reverse transcription/real-time PCR. Furthermore, chromatin immunoprecipitation (ChIP) assays have shown that ER␣ is present at the Cyp1a1 enhancer only after co-treatment with E2 and TCDD, in MCF-7 cells. Sequential two-step ChIP assays were performed which demonstrate that AHR and ER␣ are present together at the same time on the Cyp1a1 enhancer during transrepression. Taken together these data support a role for ER-mediated transrepression of AHR-dependent gene regulation. AHR1 and ER are both ligand activated transcription factors that transduce extracellular signals through DNA-binding-dependent and -independent mechanisms (1-4). AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT) form a heterodimeric transcription factor, the aryl hydrocarbon receptor complex (AHRC), that binds a wide variety of environmental pollutants including polycyclic aromatic hydrocarbons (PAHs), and halogenated aromatic hydrocarbons (HAHs) (5), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD). The binding of these compounds and subsequent activation of target genes are part of an organism's adaptive response to environmental contaminants (6). Furthermore, studies in AHR knock-out mice have revealed an important role for AHR in development and physiological homeostasis (7-11).Unliganded AHR exists in the cytoplasm as part of a multimeric complex containing two molecules of HSP90, the HSP90 co-chaperone p23, and a 36-kDa protein termed hepatitis B virus X-associated protein 2 (XAP2) (12-16). Upon ligand binding, AHR translocates to the nucleus where it associates with ARNT to form a functional transcription factor complex, the AHRC. As an activated complex, the AHRC is capable of recruiting several classes of co-activators such as SRC-1 (steroid receptor co-activator-1), NCoA2 (nuclear coactivator-2)/GRIP1/TIF2, and p/CIP (17, 18), RIP140 (receptorinteracting protein 140) (19), BRG-1 and components of the mediator complex (20, 21), and CBP and TRIP230 (thyroid hormone receptor/retinoblastoma protein-interacting protein) (22-25). These proteins are incorporated into multimeric complexes, which interact with and modulate the activity of the core transcriptional machinery, as well as modifying local chromatin structure (26). Howeve...

We demonstrate by yeast two-hybrid, glutathione S-transferase pulldown, and mammalian reporter gene assays that ARNT requires its helix 2 domain but not its transactivation domain to interact with SRC-1. This indicates a novel mechanism of action for SRC-1. SRC-1 does not require its bHLH-PAS domain to interact with ARNT or AHR, but utilizes distinct domains proximal to its p300/CBP interaction domain. Taken together, these data support a role for the SRC family of transcriptional coactivators in TCDD-dependent gene regulation.

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The pleiotropic cytokine interleukin 6 (IL-6) is involved in immune cell homeostasis. Additionally, IL-6 expression and signaling in tumor cells have been shown to elicit both protumor and antitumor properties. There is a plethora of mechanistic knowledge regarding how IL-6 signal transduction translates to biological responses. However, there is little understanding as to what factors control IL-6 expression within a tumor cell environment. The studies presented herein show that, in MCF-7 breast and ECC-1 endocervical cancer cells, the stimulation of aryl hydrocarbon receptor (AHR) activity, in combination with IL-1B or phorbol 12-myristate 13-acetate (PMA) treatment, results in a marked synergistic induction of IL-6 levels over what is seen without AHR activation. Chromatin immunoprecipitation experiments suggest that the regulation of IL-6 mRNA expression occurs at the chromatin level, as AHR presence on the IL-6 promoter was observed in response to treatment with AHR ligand. Synergistic induction of IL-6 expression was sustained for 72 hours, with accumulation of IL-6 protein reaching levels 4.8-fold above IL-1B treatment alone. In addition, transcriptional regulation of the prototypic AHR responsive gene Cyp1a1 was negatively regulated by PMA and IL-1B treatment. Silencing of RELA expression alleviated IL-1B-mediated repression of AHR transcriptional activity, whereas PMA-mediated repression was maintained. Additionally, small interfering RNA studies reveal that AHR and RELA are necessary for synergistic induction of IL-6. The findings presented here reveal the AHR as a potential therapeutic target for selective modulation of IL-6 expression in some tumor cell types. The data also suggest a possible previously unrecognized mechanism of AHRmediated tumor promotion. [Cancer Res 2008;68(10):3609-17]

The aryl hydrocarbon receptor nuclear translocator/ hypoxia-inducible factor (ARNT/HIF-1␤) mediates an organism's response to various environmental cues, including those to chemical carcinogens, such as 2,3,7,8-tetrachlorodibenzo--dioxin (TCDD or dioxin), via its formation of a functional transcription factor with the ligand activated aryl hydrocarbon receptor (AHR). Similarly, tissue responses to hypoxia are largely mediated through the HIF-1 heterodimeric transcription factor, comprising hypoxia-inducible factor-1␣ (HIF-1␣) and ARNT. The latter response is essential for a metabolic switch from oxidative phosphorylation to glycolytic anaerobic metabolism as well as for angiogenesis and has been implicated as necessary for growth in many solid tumors. In this report, we demonstrate that the thyroid hormone receptor/retinoblastoma-interacting protein 230 (TRIP230) interacts directly with ARNT and is essential for both hypoxic and TCDD-mediated transcriptional responses. We initially identified TRIP230 as an ARNT-interacting protein in a yeast two-hybrid assay screen. This interaction was confirmed in mammalian cell systems using co-immunoprecipitation and in mammalian two-hybrid assays. Furthermore, TRIP230 could be recorded at sites of activated transcription of either TCDD-or hypoxia-inducible genes in a stimulus-dependent fashion by chromatin immunoprecipitation analysis. Finally, using single-cell microinjection and RNA interference assays, we demonstrate that TRIP230 is indispensable for TCDD-and hypoxia-dependent gene transcription.

Upon hypoxia, the human erythropoietin (EPO) gene is transactivated by the heterodimeric hypoxia-inducible factor 1 (HIF-1). Mammalian SWI/SNF is a chromatin-remodeling complex involved in the modulation of gene expression. We demonstrate that Brahma (Brm) and Brahma/SWI2-related gene 1 (Brg-1), alternative ATPase subunits of SWI/SNF, potentiate reporter gene activation mediated by HIF-1 in an ATPase-dependent manner. Brm was more potent than Brg-1 in the reporter gene assays. Simultaneous depletion of both Brm and Brg-1 by small interfering RNAs significantly compromised the transcription of the endogenous EPO gene triggered by hypoxia. Whereas knocking down Brm alone resulted in a moderate reduction in transcription of the EPO gene, depletion of Brg-1 resulted in an augmentation of transcription of both the EPO gene and the Brm gene, indicating that Brm can compensate for loss of Brg-1. Chromatin immunoprecipitation (ChIP) and sequential ChIP (re-ChIP) analysis showed that both Brm and Brg-1 associate with the enhancer region of the EPO gene in vivo in a hypoxia-dependent fashion and that each is present in a complex with HIF-1. Brm and Brg-1 were also recruited to the promoter of the vascular endothelial growth factor (VEGF) gene in a hypoxiadependent fashion, although hypoxic induction of VEGF transcription was not affected by depletions of either or both Brm and Brg-1. Together these studies reveal a novel role for SWI/SNF in the activation of transcription of the EPO gene, indicate an important communication and compensation between Brm and Brg-1, and suggest that the requirement for SWI/SNF during hypoxic induction is gene-specific.Tissue oxygen concentration is an important regulatory stimulus for many physiological and pathological processes (1). Adaptation to hypoxia depends in part on appropriate alterations in the expression of a number of physiologically relevant genes. Induction of the erythropoietin (EPO) 1 gene by hypoxia is central to the regulation of the oxygen-carrying capacity of the blood (2). Hypoxic induction of genes encoding angiogenic growth factors such as the vascular endothelial growth factor (VEGF) leads to new blood vessel formation during development, wound repair, and tumor growth (3-5). Most if not all mammalian cell types share a common mechanism of oxygen sensing and signal transduction (6), enabling hypoxia-induced activation of the transcription factor hypoxia-inducible factor 1 (HIF-1) composed of HIF-1␣ and Arnt (7). The HIF-1␣ subunit is regulated by hypoxia both at the level of transactivation and protein stability (8 -11).In general, gene expression is not determined by the simple additive influences of individual transcription factor binding sites. Adjacent sites interact with each other to produce effects that range from repressive to highly synergistic. Multiprotein complexes involving transcription factors, coactivator proteins, and other proteins are thought to integrate signals and create the specificity and control required for the precise regulation of gene tra...

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