Within the eukaryotic cell nucleus, DNA is associated with core histones to form nucleosomes; which are further assembled with ancillary proteins into a multi-faceted complex known as chromatin. This complex brings about the orderly packaging of the immense length of DNA within the tiny volume of the nucleus. In addition, elements of the chromatin complex are directly involved in the regulation of multiple processes within the nucleus that involve DNA. Indeed, a large portion of signal transduction within the cell nucleus appears to ultimately direct the post-translational modification of the core histone proteins and, in several cases, mutations in enzymes that carry out these modifications have been linked to various diseases including cancers in humans. In the last 3-5 years, much effort has been devoted to the elucidation and biochemical purification of the regulatory machinery and enzymes that mediate core histone posttranslational modifications. Interestingly, nearly all of these modifications occur within specialized regions known as the core histone tail domains. Biophysical experiments have shown that the tail domains are key components in regulating the structure and function of chromatin at multiple levels. Moreover, posttranslational modifications in these domains are thought to modulate this histone tail-directed regulation. However, the mechanism by which they define chromatin structures - and ultimately the functionality of the underlying DNA - remains unknown. The primary goal of the work described in this proposal is to elucidate the molecular details and the mechanisms by which the core histone tail domains dictate the structural and functional state of the chromatin fiber. The fiber is formed by the folding up of strings of nucleosomes and is a key structure regulated by the tail domains. However little is known regarding molecular interactions of the tail domains. Recently have shown that the tails make precise and localized interactions within nucleosomes. Using a novel site-directed chemical mapping approach, we will examine histone tail interactions in a variety of model chromatin complexes. We will focus on potential inter-nucleosomal interactions likely to be specifically involved in stabilizing the condensed chromatin fiber. Further, we will use a chemical protection approach and NMR of specifically labeled core histones to study the salt-dependent binding stability of individual histone tails within nucleosomes. In addition, we will use these same methods to examine the effects of specific patterns of histone acetylation and a chromatin remodeling activity on histone-DNA interactions.
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