The long term goals of this proposal are to understand the roles of histones and functionally related gene products in transcription and chromatin structure in yeast. Mutations in the HTA1 and HTB1 genes, which encode histones H2A and H2B, were isolated as suppressors of Ty insertion mutations and cause a large number of mutant phenotypes and transcriptional defects. Strains that have a reduced level of histones H2A and H2B will be studied for changes in chromatin structure at particular loci to determine if changes in chromatin structure cause changes in transcription. These experiments will include analysis of transcriptional activators that are apparently required to derepress chromatin. To identify other aspects of histone function in transcription, genetic and biochemical studies of new histone mutants will be carried out. A systematic mutant isolation and analysis for each histone gene will be performed, to identify mutations that alter expression of different genes. Analysis of the mutants will focus on the mutant phenotypes and on the specific effects on transcription and chromatin structure. Mutations in the SPT4, SPT5, and SPT6 genes (SPT = suppressor of Ty), also isolated as suppressors of insertion mutations, cause the same transcriptional defects and mutant phenotypes as do mutations in the histone H2A- and H2B-encoding genes. These SPT genes are candidates to encode products required for normal chromatin structure and function. Experiments will be done to determine if the SPT4, SPT5, and SPT6 proteins are required for normal chromatin structure, if they bind to DNA or chromatin, if they are required for nucleosome assembly, and if they physically interact with each other. Given the extremely high conservation of histone proteins in eukaryotes, analysis of the function of histones and these related SPT gene products by a combination of genetic and biochemical experiments in yeast will be relevant to understanding gene expression in humans.
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