Elements of chromatin are now known to be directly involved in the control of gene expression. Indeed, a large portion of signal transduction within the cell nucleus appears to ultimately direct the posttranslational modification of the core histone proteins and, in several cases, mutations in enzymes that carry out these modifications have been linked to various cancers in humans. In the last 2-3 years much effort has been devoted to the elucidation and biochemical purification of the regulatory machinery and enzymes that mediate core histone modifications, especially acetylation. Interestingly, practically all of these posttranslational modifications occur within specialized regions known as the core histone 'tail' domains. Biophysical experiments have shown that the tail domains are key components in the assembly of strings of nuclesomes into the highly compacted and functionally inert chromatin fiber. For example, acetylation of the tails greatly destabilizes the fiber and thus affords greater accessibility of the DNA to trans-acting factors and enzymes that carry out transcription, replication, or DNA repair. The primary goal of this proposal is to elucidate the molecular details of the mechanisms by which the core histone tail domains dictate the structure and functional state of the chromatin fiber. Recent evidence suggests that these tails make precise and localized intra- and inter-nucleosomal interactions within chromatin. A novel site-directed chemical mapping approach will be used to map histone tail interactions in a variety of model chromatin complexes. Tail interactions will be mapped in extended and condensed nucleosomal arrays to determine those interactions specifically involved in stabilizing the condensed chromatin fiber. A chemical protection approach and NMR of specifically labeled core histones will be used to study the salt-dependent binding stability of individual histone tails within the nucleosome. In addition, these same methods will be used to examine the effects of specific patterns of histone acetylation related to transcriptional activation and DNA replication. These results will help elucidate the mechanisms of tail function in chromatin.
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