Mechanistic Problems in Eukaryotic Gene Regulation

Northwestern University, Evanston IL

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Abstract

DESCRIPTION (provided by applicant): The proper regulation of genes is essential for the development and health of all organisms, yet many fundamental aspects of gene regulation in eukaryotes are not understood. We are addressing the question of how gene regulatory proteins gain access to DNA target sites in chromatin. In the past we focused our studies on spontaneous DNA target site accessibility in chromatin. This work led to the development of the "site exposure" model for dynamic properties of nucleosomes that makes possible the binding of sequence-specific gene regulatory proteins to DNA target sites buried inside nucleosomes. Subsequent studies have distinguished between different mechanisms for site exposure, and have investigated the position-dependence of site exposure, and roles of specific DNA sequences, histone tail domains, and histone acetylation. Studies proposed for the next project period extend this work in two new directions. In Aim 1 we characterize the nucleosome structural changes, and their kinetics, that accompany uncatalyzed binding to buried DNA target sites. We will study both individual nucleosomes and test nucleosomes embedded in long chains. In vivo, access to specific DNA target sites is facilitated by ATP-dependent chromatin "remodeling" factors. In Aim 2 we study the mechanism of action of the simplest of these systems, the protein ISWI and a two protein complex comprising ISWI and Acf1. These studies will be carried out both from the perspective of the remodeling factors viewed as enzymes, and also from the perspective of the nucleosome, to elucidate what happens to the nucleosome during catalyzed translocation. We will fully characterize the substrate and product binding properties and associated enzyme kinetics, interdependences among these quantities, and the mechanism through which ATP energy is harnessed to drive the movement of nucleosomes.