Carcinogen metabolizing Cytochrome P450 Genes: Regulation of Expression The aryl hydrocarbon (Ah)-inducible P450s catalyze both the detoxification of carcinogens, and their activation to highly carcinogenic forms. Differences between individuals in the expression of specific P450s may influence the balance between these pathways, and in turn, the susceptibility of an individual to specific forms of cancer. The source of interindividual differences in specific P450s, and possibly in cancer susceptibility, could reside in either P450 structural genes or in regulatory genes that control P450 expression. However, susceptibility to chemical carcinogens has been correlated primarily with the inducibility of aryl hydrocarbon hydroxylase (primarily P450IAI). This means that there may be interindividual differences in the regulatory mechanisms of this gene that relate to susceptibility to carcinogens. Thus, understanding the regulation of this gene may be critical to understanding chemical carcinogenesis. The proposed research will obtain in-depth, basic knowledge concerning the molecular mechanisms by which expression of the P450IAI gene (CYP1A1) is regulated, focusing on understanding the response of this gene to polycyclic aromatic carcinogens. This research should be useful in developing the molecular- genetic tools needed to directly assess the role of this P450 in chemical carcinogenesis. We will investigate two mechanisms that may be central to modu1ating the response to Ahs: I.Transcription regulatory factors that are distinct from the A"""""""" receptor and that recognize sites distinct from XREs, may act cooperatively with XRE-bound Ah receptor to modulate the ability of the receptor to activate CYP1A1 transcription. Investigating this mechanism will focus on six DXEs, averse sequence :xenobiotic response elements, which are diverse in sequence and distinct from the XREs at which the Ah receptor binds. Constitutive nuclear factors bind to DXEs in response to Ahs and we have shown that one DXE cooperates with XREs to activate transcription in response to Ahs. We will carry out transfection experiments to study DXE function and LMPCR-amplified in vivo footprinting to study the regulation of DXE function. We will clone the cDNAs encoding mRNAs for selected DXE-specific nuclear factors and study the regulation of those mRNAs. 2.Alterations in chromatin organization, induced by binding of the Ah receptor to XREs, may modulate the response of CYP1A1 to Ahs by controlling access of other regulatory factors to their recognition sites. To follow up preliminary evidence supporting this mechanism, we will investigate the organization and properties of chromatin on CYP1A1 regulatory sequences and measure directly whether Ahs induce chromatin alterations. We will examine nucleosome positioning by low and high resolution methods and measure histone acetylation and phosphorylation. We will immunoprecipitate and quantitate crosslinked DNA-protein complexes, to measure in parallel histone HI and transcription factor occupation of CYP1A1 regulatory sequences in response to Ahs.
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