Human cytochrome P450 CYP1A1 is not normally expressed in adult tissues. However, transcript of this gene is induced several-fold upon an individual's exposure to a variety of environmental xenobiotics, including halogenated aromatic hydrocarbons (HAH) and polyaromatic hydrocarbons (PAH). This process is mediated by the Ah receptor (AhR). The physiological function of CYP1A1 is unknown. However, the enzyme can activate PAH and HAH procarcinogens and protoxicants to their ultimate pathological forms and, therefore, plays an important role in chemical carcinogenesis and toxicity. Previous studies in this laboratory identified a mechanism for the negative regulation of the CYP1A1 gene. This regulatory system seems to be important in maintaining CYP1A1 in an inactive state and, more important, modulates the AhR-mediated induction response. The negative regulatory element (NRE) and its cognate binding protein are postulated to contribute to the large interindividual variation in human CYP1A1 inducibility and possibly to an increased incidence of bronchogenic carcinoma and poor prognosis in human breast cancer. The applicant proposes to characterize the trans-acting factor mediating CYP1A1 repression. Based on previous studies, he hypothesizes the repressor is a heteromeric transcription factor that acts to stabilize nucleosome structure within the -YPiAi regulatory domain, thereby antagonizing the action of the AhR. Proposed research will focus on the following specific aims to test this hypothesis: (i) Purify and characterize the human CYP1A1 NRE binding proteins employing DNA affinity chromatography and1or immunopurification and microsequence analysis; (2) Clone and characterize cDNAs representing the human CYPIAI NRE binding proteins using probes based on the purified proteins; (3) Complete the characterization of the NRE using a binding site selection assay; (4) Examine the molecular mechanism whereby the CYPIAI transcriptional repressor functions by: (a) searching for associated proteins using a yeast two-hybrid system; (b) mapping functional domains on the repressor using deletion mutagenesis, in vitro DNA binding assays, co- immunoprecipitation, and transient transfection assays, and testing for nucleosome1repressor interactions employing in vitro nucleosome binding and transcription assays.
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