UNIVERSITY OF CALIFORNIA, SANTA CRUZ
DESCRIPTION (provided by applicant): Transcription is the first step of gene expression and a key point of biological regulation, which requires specific changes in chromatin structure and chemistry. The long-term objective of this proposal is to understand the structural underpinnings, functions and mechanisms of chromatin dynamics in the regulation of eukaryotic genes. Recent work on the inducible PHO5 promoter of Saccharomyces cerevisiae provided the first example of signal-induced promoter nucleosome disassembly, and has led to the following working hypotheses concerning the nature and function of chromatin dynamics in gene regulation: first, nucleosome disassembly is a general mechanism of promoter activation; second, the activated promoter state is characterized by a dynamic equilibrium of nucleosome dis- and reassembly (dynamic equilibrium hypothesis); third, nucleosome disassembly at PHO5 is a fast reaction governed by a slow stochastic rate constant (stochastic model of nucleosome disassembly), and fourth, PHO5 expression is controlled by the rate of promoter nucleosome disassembly. To test the dynamic equilibrium hypothesis, we will use chromatin immunoprecipitation to determine if newly-synthesized histones associate with promoter DNA under activating conditions. The stochastic model of nucleosome disassembly will be tested by analyzing the diversity of promoter chromatin structure in the transition from low to high levels of expression by gel chromatographic fractionation of small promoter circles formed in vivo. To test the generality of nucleosome disassembly as a mechanism of promoter activation, we will investigate the question of whether nucleosome disassembly occurs at other promoters. The investigation will employ chromatin circles formed in vivo, limit digestion and DNA topology analyses, sedimentation and in vivo cross-linking studies. The factors catalyzing nucleosome disassembly are unknown. We will use genetic and biochemical tools to identify nucleosome disassembly factors. Transcriptional control underlies many biological phenomena from pattern formation in development and the regulation of cell proliferation to long term memory and animal behavior. Investigations of the molecular mechanisms of transcriptional control, as suggested here, will thus eventually contribute the understanding and treatment of disease. Not surprisingly, factors regulating chromatin structure and chemisty have been implicated in oncogenesis.