MOLECULAR MECHANISMS OF GLUCOCORTICOID HORMONE ACTION

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Understanding the mechanisms of regulation of gene expression is essential to a comprehension of development and differentiation, and thus, cancer and the other disorders that may arise when these processes go awry. Steroid hormone regulated gene expression provides an excellent model system with which to investigate regulatory mechanisms. The mouse mammary tumor virus (MMTV) promoter has served as the prototype for studies developing the conceptual framework of the molecular mechanisms of glucocorticoid hormone action. Although glucocorticoid regulatory element (GRE) consensus sequences have been proposed, functional response elements cannot be reliably predicted from sequence data. In addition, more response elements, especially those mediating negative regulation, are being mapped which do not conform to the consensus. Our studies have focused on a systematic mutagenesis approach to defining the glucocorticoid response element of the MMTV promoter. During the previous grant period we developed and characterized a library of GRE point mutants. The quantitation of the activity of more than 60 of the mutants has allowed us to refine our conception of the GRE. We propose to complement the biological activity data with in vitro analyses of receptor binding to the mutant HREs. The results of the binding studies will be combined with the functional activity analyses to assemble a map of residues critical for receptor binding and for hormone response. These mapping data will yield insights as to molecular details of the receptor-DNA interaction particularly to identify the likely sites where receptors make hydrogen bond contacts that allow it to discriminate HRE sequences. Because the MMTV promoter has now been shown to respond to at least 4 different classes of steroids, the mutant library can be further exploited by extending these analyses to the other steroid-receptor systems that regulate this promoter. Such comparative analyses will reveal how the different receptors distinguish different features of the recognition sequence. The studies focusing on the prototypical positive GRE will be complemented by similar analyses on the negative GRE of the proliferin gene a receptor recognition site that bears only weak homology to the MMTV GRE. Finally, approaches are proposed to investigate receptor DNA interaction in vivo, not only with GRE mutants, but also under circumstances associated with steroid non-responsiveness e.g. steroid antagonists or steroid resistance associated with cell transformation.