The precise topological organization of DNA in chromatin has important functional consequences such as the regulation of gene expression. Thus, advances in our understanding of histones, components of chromatin, and their modifications are likely to impinge on a wide variety of basic issues in molecular and cell biology and genetics. The long range goal of this proposal is to provide an in-depth understanding of the biological function(s) of linker histone (LH) phosphorylation. We propose that LH phosphorylation allows a transient decondensation of chromatin allowing specific factors to gain access to specific DNA sequences. To test this hypothesis, we will exploit the nuclear dimorphism (micro- vs. macronuclei) exhibited by the ciliated protozoan, Tetrahymena. Aspects of chromatin structure dealing with transcription take place in macronuclei, but not in micronuclei. Conversely, aspects of chromatin structure dealing with mitosis are found in micronuclei, but not in macronuclei. Methods have been developed to prepare homogeneous fractions of phosphorylated and dephosphorylated macronuclear H1. Reconstitution studies will determine if phosphorylated H1 preferentially promotes the unfolding of chromatin in vitro. Novel antibodies have been obtained that recognize highly phosphorylated forms of H1 in all organisms tested. """"""""Contol"""""""" antibodies selective for dephosphorylated H1 are being generated. These antibodies will be used to fractionate chromatin by immunoaffinity methods. By determining the DNA sequences associated with phosphorylated and dephosphorylated H1, we will directly test the hypothesis that phosphorylated H1 is preferentially associated with active genes. Antibodies will be used to examine condensed vs. decondensed chromatin in a variety of systems including Tetrahymena, Drosophila and yeast from which an H1 homologue has yet to be identified. Micronuclei contain a unique set of LH polypeptides that are phosphorylated by protein kinase A. Efforts will be made to perturb the PKA-driven phosphorylation of micronuclear LH's in vivo. Antibodies will also be generated to phosphorylated and dephosphorylated forms of micronuclear LHs to analyze micronuclear chromatin during synchronized mitotic and meiotic divisions. Transformation of Tetrahymena with modified H1 genes will be used to determine the in vivo phenotype of cells lacking specific phosphorylation sites. Understanding how higher-order chromatin structure modulated to accommodate gene expression, DNA replication, recombination and segregation in normal cells is essential if we are to understand abnormal growth and development.
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