Understanding gene regulation in eukaryotes is one of the key problems in biology, affecting virtually all aspects of cell biology and our understanding of many disease states. While changes in chromatin structure are the result of gene activity, it was not known whether these structures also help regulate the transcription of eukaryotic genes. Recently, it has been shown that the N-termini of different histone proteins, in particular H4 and H3, have unique positive and negative regulatory roles in yeast (Saccharomyces cerevisiae) transcription. These N-termini help regulate not only transcriptional activation but also the repression of uninduced, basal transcription. The goal of this project is to determine how different core histones interact with promoters and regulatory factors in order to help regulate gene activity. The effects of deleting the N-termini of the different core histones on nucleosome positioning will be compared using high resolution nucleosome mapping procedures. The effects of histone N-terminal deletions on promoter topology as measured by linking number measurements will be examined. In addition, promoter mutations will be used to determine which promoter sequences and regulatory factors respond to the effects of H4 and H3 deletions on transcription. The histone H4 and H3 N- terminal regions required for the repression of basal transcription will be defined using histone mutations and sensitive genetic assays for basal transcription. Genetic selections and screens will be employed to identify the regulatory factors which cause nucleosome displacement and which interact with individual core histones. Finally, two proteins which interact biochemically with H4 and one protein which interacts with H3 will be purified. The sites at which these histones interact will be determined using selected histone deletions and mutations. The non- histone proteins will be sequenced partially. This information will be used to clone them and genetically alter their function in order to determine their roles in H4 and H3 function. These studies will be extended to the other core histones. The experiments described above will allow us to map at high resolution the exact histone, promoter and regulatory factor elements involved in gene regulation. This information promises to provide a new understanding of histones as components of the transcriptional machinery.
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