DESCRIPTION: X-chromosome inactivation; epigenetic inheritance; position effect variegation; locus control as well as silencing at telomeres, repressed mating type loci, and centromeres are all transcriptionally regulated features of the eukaryotic genome for which chromatin structure has been suggested as a possible controlling element. The exact nature of the postulated repressive chromatin domain has not been determined for any of these cases. To explore the role of chromatin in DNA function, the investigator has focused on genes regulated by the mating type locus in a tractable, simple eukaryote, S. cerevisiae. He has shown that a repressive chromatin structure is organized by the alpha2 repressor on alpha-cell specific genes in alpha-cells. There is an absolute concordance of the presence of this organized chromatin structure with complete repression of genes under control of alpha-cell specific promoters in yeast strains bearing mutant histones or mutations in other proteins (Ssn6p and Tuplp) known to be necessary for repression of these genes. A motif of close packed dimeric nucleosomes separated by 43 bp linkers spans the entire repressed STE6 gene. This nucleosome arrangement is considered as a paradigm for organization of repressed chromatin domains. The proposed research will ascertain whether this structure also occurs for another gene controlled by the alpha2 repressor, BARI, other genes which require Ssn6p and Tuplp for repression, and model systems targeting a repressor of Tuplp to a foreign binding site. Ssn6p and Tuplp have domains which suggest their involvement in protein-protein interactions as well as interactions with nuclear matrix structures. Using improvements to the minichromosome system pioneered in the investigator's laboratory for study of chromatin composition and structure, he will isolate minichromosomes containing the STE6 gene and address directly the interaction of Ssn60 and Tuplp with the repressed gene. Biochemical studies of the interactions of these proteins with each other and chromosomal structural proteins will define the composition and structure of the repressed chromatin domain. Finally, the investigator will examine whether silenced domains such as telomeres, centromeres and the silent mating type loci also have the organized chromatin structure of close packed dimers interspersed by long linkers. To address this question, the lab will take advantage of the complete sequence of yeast chromosome III and will map chromatin structure of about 30 kb of this chromosome. While focusing on specific genomic loci in yeast, these studies are relevant to the organization of constitutively repressed genetic loci in other domains, as well as conditionally repressed genes in both yeast and larger eukaryotic cells.