Chaperones, chromatin, and transcriptional control by PR

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DESCRIPTION (provided by applicant): Understanding the regulation of gene expression is essential to a comprehension of development and differentiation, and thus, cancer and other disorders that may arise when these processes go awry. Biological regulatory systems have mechanisms to ensure the timely onset and dissipation of a signal. In the case of steroid receptors, binding of the hormonal ligand initiates an elaborate sequence that culminates in the regulation of target gene expression. This regulation is often imposed at the transcription initiation step but the mechanistic basis remains poorly understood. Even less well understood are the biological mechanisms that mediate the cessation of the activation signal and the resetting of the biological response system. These studies propose an in vitro approach to elucidate the mechanisms that govern the activity cycle of the progesterone receptor. We have developed a progesterone receptor-dependent in vitro transcription system that uses chromatin templates and have incorporated chaperone-mediated receptor recycling as an integral part of the system. Not only is chaperone activity required for restoration of hormone binding by purified receptors, it is required for an additional step as well. A first aim is to elucidate the roles of chaperones in the progesterone receptor activity cycle in vitro. With this hormone- and receptor dependent cell-free transcription system, we will also explore the roles of chromatin modification and remodeling in turning on and turning off receptor action at target genes. Since different steroid receptor coactivators have been shown to possess histone methyltransferase and histone acetyltransferase activity, we will evaluate the contribution of these coactivators to hormonal activation of receptor. Our previous studies have demonstrated an absolute requirement for acetyl CoA for transcription from chromatin templates. The acetyl CoA is required for preinitiation complex assembly at all promoters, including steroid receptor-dependent promoters, indicating that the presumptive acetylation step is a fundamental one. We will use a purified transcription system to identify the chromatin proteins that are being acetylated and investigate which of these are playing a central role in the process of transcription. Together, the studies proposed will provide fundamental knowledge that will advance our understanding of steroid receptor action in human health and disease.