The cytochrome P450IA1 (IA1) gene product, aryl hydrocarbon hydroxylase, 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, the IA1 gene is known to be induced when organisms are exposed to many of these same compounds. A large variation in the expression of the IA1 gene 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 two years of this proposal have established the involvement of four distinct cis-regulatory elements in the expression of the human IA1 gene, i.e. a proximal and a distal positive acting xenobiotic- responsive element (XRE), a glucocorticoid-responsive element that acts synergistically with the two XREs, and a negative regulatory element (NRE). Although several groups have described specific trans-regulatory proteins and DNA binding sites involved in the positive regulation of the IA1 gene, very little is understood about the negative regulatory components. This proposal is designed to further characterize the NRE and to extend these studies to the isolation and characterization of the postulated trans negative regulatory factor (NRF) whose action is mediated by these sequences. First, experiments will be conducted to determine the orientation-, position-, enhancer-, and promoter-dependence of the NRE. The tissue- and species-specificity of the NRE, if any, will also be examined. Second, the specific interaction of trans- acting proteins with the NRE will be analyzed. Third, the effect of specific mutations on the identified protein binding sites will be determined. Fourth, we will purify and characterize the trans- acting NRF. Finally, a cDNA clone for the NRF will be isolated, characterized, and employed to examine the expression of the NRF and obtain an initial estimate of its genetic complexity. These studies will add greatly to our understanding of the mechanisms controlling the activity of the IA1 enzyme and will also contribute to our knowledge of eucaryotic gene regulation in general.
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