In order to understand normal cellular growth and development, and to understand in many instances why growth or development become abnormal, it is necessary to understand the mechanisms that regulate gene expression. this regulation occurs on DNA templates that are complexed with constituents of chromatin, including the core nucleosome histones and histone H1. An early step in the transcription of messenger RNA genes in eukaryotes is the formation of a large transcription complex, containing general transcription factors and RNA polymerase II, at the initiation site of a promoter. It is now clear that nucleosomes inhibit the formation of this complex, and that prevention of that repression is an important function of certain transcriptional regulatory factors. These regulatory factors contain a domain required for sequence specific DNA binding and domain(s) required for transcriptional activation. The transcriptional activation domain of GAL4-VP16 derivatives is responsible for alleviating repression of promoters by nucleosomes, suggesting that an important function of the VP16 activation domain is to help general transcription factors compete with nucleosomes. A biochemical analysis of the mechanism(s) by which activation domains in general, and VP16 in particular, function on nucleosomal templates is proposed in this application. The effects of nucleosomes on binding of general transcription factors to the TATA sequence will be examined by DNase I footprinting and by electrophoretic mobility shift (EMSA) protocols. The ability of the VP16 activation domain to regulate these interactions will be examined. Effects of VP16 on the ability of transcription factors to bind to nucleosomes and on the stability of the underlying nucleosome will be determined. Transcription assays will be used to characterize which transcriptional activation domains can increase the ability of transcription factors to compete with nucleosomes, and to determine the amino acids responsible for this ability. The ability of histone H1 to either alter binding of general transcription factors or to increase the stability of the core nucleosome will be examined on small, fully characterized, nucleosomal DNA segments. Finally, these studies will be extended to a natural promoter region, and the ability of natural regulatory transcription factors to function on nucleosomal templates will be determined. These studies will increase our understanding of how transcription factors interact with nucleosomes and histone H1. These mechanisms are likely to be important to a full understanding of transcriptional regulation in eukaryotes.
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