The long-term objective of the applicant's research program is to elucidate the mechanisms of somatic chromatin biosynthesis, and to understand the transfer of epigenetic information to progeny cells. To this end, the present proposal focuses on posttranslational modifications of newly synthesized histones (the major chromosomal proteins), and the propagation of histone modifications at the replication fork, during chromatin replication in human cells. The ability to faithfully replicate genetic and epigenetic information is essential for normal cellular growth and function. Loss of this capability can result in increased developmental abnormalities, and pathological cell proliferation. ? ? Experiments will be performed to determine the complete acetylation, phosphorylation, and methylation states of newly synthesized human histones. This will be accomplished by immunoprecipitating nascent histones from cytosolic extracts (which contain new histones prior to deposition onto DNA), using anti-histone antibodies that are specific for site-specific histone modifications. Newly synthesized histones will also be microsequenced, using methods that reveal site-specific posttranslational modifications. The ability of pre-existing histone modifications to persist during DNA replication will also be examined, by performing immunoprecipitation assays on chromatin replicated in the absence of concurrent de novo nucleosome assembly. These experiments will provide fundamental information on the manner in which epigenetic information is established, and maintained from generation to generation. ? ? As part of the above studies, the properties of the HAT-B histone acetyltransferase, which very likely is involved in acetylating newly synthesized histone H4, will be examined. The activity and specificity of HAT-B will be analyzed, using modified histones and H4 N-terminal peptides as substrates. Experiments will also be performed using yeast mutants, deleted of the Had gene, to determine whether these mutants are capable of acetylating newly synthesized H4 in vivo. Finally, the function of the HAT-B enzyme will be studied through whole-genome mRNA analysis, in a hatidelete mutant of the yeast Schizosaccharomyces pombe.
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