Challenge and hypotheses. Regulation of transcription is central to all organisms and viruses. Specificity of metazoan transcription is determined combinatorially: different combinations of broadly expressed regulatory factors assemble as cell- and gene-specific complexes at genomic response elements. However, what determines unique assemblages of common factors remains a fundamental problem. This project will test the idea that key regulatory factors receive and integrate multiple signals as allosteric effectors, producing novel patterns of functional surfaces that nucleate distinct assemblies. The work will additionally explore whether regulatory mechanisms correlate with physiological processes. Project objectives. This study will define signal-induced allosteric effects on glucocorticoid receptor (GR) structure, the consequences of those effects on the combinatorial assembly, structure, dynamics and selectivity of GR regulatory complexes, and the mechanisms by which these complexes modulate transcription. This information will suggest design principles that determine context specificity, and may link molecular features of regulation to physiology.
Three specific aims are envisioned: 1. Identify and analyze poised and causative primary GREs. Approach. Identify genes that are glucocorticoid-activated in one cell line and repressed in another, and linked to GR-occupied glucocorticoid response elements (GREs) that are thus poised to switch regulatory modes and mechanisms. In parallel, identify causative primary GR-regulated genes (CPRGs) as GRE-linked genes essential for physiologic process, or, when misregulated, responsible for disease such as glucocorticoid- induced osteoporosis, or acute lymphoblastic leukemia. Analyze features of GREs that generate specificity, and determine which transcriptional processes/events are regulated by a given GRE in a given context. 2. Determine GRE-specific allosteric effects on structure of GR and an associated co-regulator. Approach. Carry out structural and biophysical analyses to determine allosteric paths extending from the signaling actions of GR binding sequences and non-GR regulatory factors at GREs through the GR DNA binding domain (DBD) and into its flanking regulatory domains and bound cofactors. Define potentially functional GR surfaces that are differentially displayed in different GRE contexts. 3. Quantitatively analyze and reconstitute regulatory complexes. Approach. Measure context effects on interactions of regulatory complex components, and determine conditions for reconstitution in vitro of functional regulatory complexes. Characterize and analyze biochemically the regulatory complexes at selected GREs. Exploit enzymatic activities resident within certain regulatory complexes as partial reactions that enable quantitative determination of regulatory mechanisms.
Complex organisms such as humans have evolved exquisitely specific ways to regulate which genes are switched on or off in a given tissue, or at different times during development, or in response to physiologic signals or environmental cues. While it is clear that anomalous gene regulation is responsible for many birth defects and diseases, most of the precise mechanisms and the exact genes that are regulated have not been determined or identified. This research project will define in molecular detail how one gene regulatory protein is able to control different networks of genes under different conditions, how misregulation leads to specific diseases, and how elucidation of these control mechanisms may yield new ways to find disease treatments and cures.
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