The Aryl Hydrocarbon Receptor/ARNT dinner mediates carcinogenesis by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and certain polycyclic aromatic hydrocarbons by activating gene transcription. The overarching goal of this proposal is to more fully understand the mechanism(s) of AHR/ARNT-mediated gene activation. The first specific aim will analyze induction of the human CYP1A1 gene, determining (i) which factors and coactivators are required for its transcriptional activation, (ii) whether there is a nucleosome located in a fixed position over the CYP1A1 promoter that is displaced by treating the cells with ligand, (iii) which of the above proteins are required for displacement of this nucleosome, (iv) which are recruited to the enhancer, and which to the promoter, (v) what covalent modifications occur on histones at the enhancer and promoter, (vi) what is the order in which the proteins are recruited and the histones modified after ligand treatment, (vii) which proteins are required for specific subsequent protein recruitments and histone modifications, and (viii) what are the compositions of the different multi-subunit complexes recruited to the enhancer and promoter.
Specific aim 2 will address the hypotheses that induction of the human CYP1B1 gene requires different proteins and histone modifications than CYP1A1, and that this, at least in part, may explain the different cell-specific distribution of CYP1A1 and CYP1B1 inducibility.
Specific aim 3 will investigate two novel coactivators (SPAG5, SUG2) of AHR/ARNT transcriptional activation, and a TCDD- inducible protein (B94) that appears to be a repressor. Data confirming physical interactions of SPAG5 with ARNT and SUG2 with AHR will be sought, and the protein-protein interaction sites on each protein identified. The roles of SPAG5 and SUG2 in TCDD-dependent transcriptional activation of the CYP1A1 and CYP1B1 genes will be probed as in aims 1 and 2. Experiments will be directed towards confirming that B94 acts as a repressor of AHR/ARNT-mediated transcription and determining whether repression depends upon B94 binding to AHR and/or ARNT, and upon its recruitment to the CYP1A1 enhancer or promoter along with histone deacetylases. Overall, these studies should provide fundamental insights into the process of carcinogenesis by a variety of chemicals involved in the causation of a significant portion of human cancers, and may eventually lead to the development of strategies for reducing the frequencies of these cancers. ? ? ?
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