The coordinated regulation of gene expression in response to hormonal signals is a fundamental process in biology. The lipophilic hormones (e.g., estrogens, retinoic acid, thyroid hormone) comprise a structurally diverse array of compounds that play important roles in many physiological processes including growth, development, reproduction, and metabolism. Aberrations in the signaling pathways controlled by these hormones lead to disease states, including developmental abnormalities and cancers. The actions of the lipophilic hormones are mediated through nuclear receptor proteins that function as ligand- regulated, DNA-binding transcription factors. The receptors function with coregulator proteins (e.g., corepressors and coactivators) to alter patterns of gene expression from arrays of hormone-regulated genes. The long-term objective of these studies is to elucidate of the mechanisms of ligand-regulated gene transcription by nuclear hormone receptors and their coregulator proteins in the chromatin environment of the nucleus. Analysis of the molecular and biochemical mechanisms of gene regulation by nuclear receptors and coregulators has been limited by the difficulty of recreating in vitro the known biological activities of the receptor and coregulator proteins with chromatin. We have developed a biochemical (""""""""cell-free"""""""") assay that (1) includes all components of the hormone signaling pathways (e.g., ligand, receptor, coregulator proteins, chromatin, the basal transcriptional machinery), (2) can be easily modified by the addition, subtraction, or modification of the components, and (3) accurately recreates ligand-, receptor-, and coactivator- dependent transcriptional events with chromatin templates assembled and transcribed in vitro. We will use this assay, along with complementary cell-based and molecular techniques, to test the broad hypothesis that transcriptional outcomes in nuclear receptor signaling pathways are ultimately determined by significant physical and functional interactions among the receptors, coregulators, transcriptional machinery, and chromatin. In our studies, we will focus on the activity of a coactivator (i.e., p300) and a corepressor (i.e., repressor of estrogen receptor activity, REA) with representative nuclear receptors (e.g., receptors for estrogens, retinoic acid, and thyroid hormone). These studies should lead to significant advances in the way hormone-regulated gene expression is analyzed and should increase our understanding of how these processes occur in normal and disease states.
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