The nucleosome, composed of an octamer of highly conserved histone proteins and associated DNA, is the fundamental unit of eukaryotic chromatin. How arrays of nucleosomes are folded into higher-order structures, and how the dynamics of such compaction is regulated, are questions that remain largely unanswered. This proposal seeks to understand the roles of core histone phosphorylation in regulating higher-order chromatin structure. In particular, histone phosphorylation will be studied in the context of biological events that are associated with dramatic alterations in higher-order chromatin structure, such as DNA damage and apoptosis. A long-range objective of this proposal is to understand core histone phosphorylation mediated chromatin condensation as well as decondensation by two molecular mechanisms: 1) """"""""cis"""""""" effects, whereby internucleosomal contacts and histone/DNA interaction are directly altered, and 2) """"""""trans"""""""" effects, whereby effectors engage specific covalent modifications in a context-dependent fashion. A second long-range goal of this research program is to investigate the role of """"""""basic patches"""""""" and """"""""death motifs"""""""" that reside in the tail domains of H4 and H2B, respectively. We hypothesize that the role of histone phosphorylation at these basic patches and death motifs is to determine compaction states regulated in part by adjacent sequences as well as kinase/phosphase enzyme systems. Understanding the physiological targets and sites phosphorylated by these enzymes is of paramount importance. Just as studies on histone acetylation have led to a wealth of new insights into mechanisms of transcription, we anticipate that insights into histone phosphorylation will pave the way for a better understanding of DNA repair and related phenomena. To achieve these goals, we will employ genetics, biochemistry, and immunocytochemical approaches in a variety of model systems. The highly conserved nature of histone proteins, as well as the phosphorylation events and the relevant enzyme systems involved, underscore the fundamental nature of the chromatin problem for all DNA-templated processes. In addition, the close association of histone kinases, such as aurora kinase, and specialized histone variants, such as H2A.X, with oncogenesis provides strong support for an emerging view that covalent modification of histones plays a vital role in the regulation of chromatin dynamics with far-reaching implications for human biology and disease.
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