CHROMATIN STRUCTURE IN LIVING CELLS

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DESCRIPTION (verbatim from the application): Histone-DNA interactions modulate the activity of the nucleic acid in many functions. Both activation (by histone acetyltransferases or chromatin remodeling machines) and repression (by histone deacetylases or corepressors) of gene activity have been attributed to changes in chromatin structure. The physical alterations in chromatin that result from these genetically defined engines are largely unknown. The long term objective of this project is understanding the structural basis for chromatin-related gene regulation. Chromatin will be modified either by endogenous cloned methyltransferases or DNase I in whole yeast cells or by exogenous nucleases in isolated nulcei. The modification of chromatin DNA will be analyzed by methods that quantify the kinetics at specific sites in the genome. One goal of the project is a quantitative analysis of the micrococcal nuclease and DNase I digestion kinetics of about 85 sites, each 25 nucleotides long, that are located in the 5' flanking regions of nine regulated genes or four non-transcribed loci. Each of these analyses will be performed for the two S. cerevisiae mating types, a- and alpha-cells, as well as for yeast that are mutant for genes of histone acetyltransferases, deacetylases, members of the SWI/SNF remodeling complex, and the SIR or Ssn6p/Tup1p corepressor proteins. A second goal is to extend this experimental approach to a genome wide set of 12,000 oligonucleotides located at the 5' and 3' borders of every yeast open reading frame. Hybridization of DNase I digested DNA labeled at the cutting sites to this oligonucleotide set in a high density array will locate all the intergenic DNase hypersensitive sites in the yeast genome. Since DNase I hypersensitive sites mark regions of the genome which are critical in function, such as promoters, enhancers, replication origins, recombination hotspots, etc., this analysis will provide a map indicating regions of the genome for investigation of DNA function in various physiological and genetic situations. All the experimental data from genome wide studies will be available for others in a curated database. Additionally, the present project is a proof of concept study for a massively parallel approach to the micrococcal nuclease based structural analysis of the entire yeast chromosome using high density oligonucleotide arrays.