The goal of this proposal is to discover how genetic regulatory proteins assemble onto specific sites in mammalian chromatin, perturbing nucleosomes and forming local higher-order structures that activate gene expression. The tools are now in hand to address this question with regard to the developmentally regulated transcriptional enhancer of the serum albumin gene: Essential sequences of the enhancer have been defined; trans-acting binding proteins have been cloned; and basic information about the enhancer's nucleosome structure in liver cell has been obtained. The investigator proposes the following aims to reveal the underlying mechanisms of control: 1. Provide a detailed molecular characterization of the nucleosomal structure and proteins binding to the albumin enhancer in native liver chromatin and control tissues. 2. Determine the order with which nucleosomal rearrangements occur at the albumin enhancer during hepatocyte development. 3. Reconstitute the albumin enhancer into nucleosomal segments in vitro and test relevant regulatory proteins for their ability to bind, perturb nucleosome structure, and to permit the binding of other factors that cannot bind on their own. 4. Fractionate nuclear extracts and screen for non-DNA binding activities that facilitate the reorganization of enhancer chromatin fragments in vitro. The proposal is designed to reveal hierarchies by which regulatory factors bind to chromatin and activate gene transcription; these hierarchies govern basic processes that may be essential for normal growth and development in humans.
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