The overall objective of the proposed research is to determine how chromosome structure affects gene expression and how the transcription machinery contends with this structure. Our general strategy is to use a combined biochemical and genetic approach to dissect the contributions of histone domains and nonhistone proteins to chromatin structure and function. Genetic studies in the yeast Saccharomyces cerevisiae have led to the identification of a HMG1-like protein, SIN1, and a domain of the histone octamer, comprised of residues from both histones H3 and H4, that play crucial roles in chromatin-mediated repression of transcription in vivo. In addition, biochemical studies in higher eukaryotic systems and genetic analyses in yeast have indicated a role for the histone N-termini in modulating transcription factor function on nucleosomal templates. Our working hypothesis is that SIN1, a domain of the histone octamer, and the histone N-termini regulate transcription factor binding to nucleosomes either by influencing compaction of nucleosomal arrays or the accessibility of DNA within nucleosome cores. This proposal exploits the powerful genetic and biochemical opportunities available in yeast to directly test this hypothesis. During the past year, my lab has made significant progress in developing the biochemical and genetic tools required for the proposed studies. Most importantly, we have succeeded in purifying large quantities of yeast histones that we competent for reconstitution onto DNA templates. We propose to reconstitute DNA templates with mutant histones that have been defined functionally in vivo, and thus these studies will provide an unique opportunity to address the role of histone domains in nucleosome structure and function.
The first aim of this proposal will investigate the functional role of an octamer domain that was identified by genetic analyses and plays a crucial role in chromatin-mediated transcriptional repression.
This aim i s addressed by nuclease digestion studies, analysis of the salt stability of histone-histone interactions, and quantitative agarose gel electrophoresis and analytical ultracentrifugation analyses of nucleosome arrays. The second objective uses various footprinting assays to test the hypothesis that this octamer domain plays a role in restricting transcription factor accessibility.
The third aim will investigate the ability of the SIN1 protein to bind to nucleosomal arrays and to influence array compaction as assayed by quantitative agarose gel electrophoresis and analytical ultracentrifugation. In this aim we will also use a genetic screen to identify proteins that are functionally related to SIN1. The fourth objective will define the role of the histone H4 N-terminal domain in compaction of nucleosome arrays. The results from the proposed studies will enrich our knowledge of the functions of nonhistone proteins and histone domains in chromatin structure and function as well as provide valuable insights into mechanisms by which transcription is regulated on nucleosomal templates.
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