The CYP1A1 gene product, cytochrome P450IA1, has been most closely associated with the metabolic activation of polycyclic aromatic hydrocarbon (PAH) procarcinogens and protoxins to their ultimate pathological form. Many of these PAH are found prevalently in our environment. Although, normally transcriptionally inactive, CYP1A1 is known to be included when organisms are exposed to many of these same compounds. A large variation in the expression of CYP1A1 has been demonstrated in the human population. Our laboratory has been involved in experiments to elucidate mechanisms controlling the expression of this gene. Studies completed during the first grant period established the involvement of multiple cis-regulatory elements in the expression of human CYP1A1, i.e. proximal and a distal domains responding to the Ah receptor, glucocorticoid-responsive elements that act synergistically with PAH, and a negative regulatory element (NRE). Focusing on the NRE, we have established that this is able to down-regulate heterologous promoter and enhancers in an orientation- and distance- independent manner. Further, specific protein interactions with the NRE have been localized to two GC-rich domains located at -748 and -792. We hypothesize that the NRE functions as a silencer element, interfering with the ability of positive regulatory elements to interact with the RNA polymerase II initiation complex. To test this hypothesis, the objectives of this proposal are to: First, characterize NRE/protein interactions by DNaseI footprinting and confirm the function of identified cis-elements by competition with double stranded phosphorothioate oligonucleotides; Second, analyze critical NRE/protein interactions by a binding site selection assay; Third, examine the interaction of the NRE/repressor complex with other CYP1A1 regulatory elements; Fourth, clone and characterize the repressor cDNA; Fifth, purify and characterize the repressor protein; and Sixth, characterize the expression of the repressor in cell lines known to have altered regulation of CYP1A1, and in HepG2 cells in response to exogenous agents. These studies will add greatly to our understanding of the mechanisms controlling the expression of CYP1A1 and will also contribute to our knowledge of eucaryotic gene regulation in general.
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