Chromatin Biosynthesis: Tetramer Assembly and the Predeposition Code

Boston College, Chestnut Hill MA

Investigators

Abstract

The long-term objective of the Principal Investigator'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 project focuses on posttranslational modifications of newly synthesized histones (histones are the major chromosomal proteins), and the mode(s) of H3/H4 tetramer assembly in human cells. The manner in which H3/H4 tetramers are assembled in vivo will be examined by immunoprecipitating mononucleosomes containing radiolabeled, epitope-tagged H4 and H3 variants in synchronized cells. Experiments will also be performed to determine the complete acetylation, phosphorylation, and methylation states of newly synthesized human histones H4 and H3 variants as a function of the cell cycle. This will be accomplished by purifying nascent H3/H4 predeposition complexes during G1 and S phases. Newly synthesized histones will analyzed by tandem (MS/MS) mass spectrometry, to uncover all of the modifications of human H3 and H4 prior to deposition onto DNA. The project will provide essential information on the manner in which nucleosomes (the fundamental subunits of eukaryotic chromosomes) are assembled, onto both replicating and non-replicating DNA. The research will have a broader societal impact in three major areas: (1) It will provide excellent research experience for undergraduates, thereby fostering their scientific development; (2) it will offer research opportunities for high school students, through participation in the Research Science Institute; and (3) cell lines developed in the course of the project will be made available to other researchers in the field, thus helping to stimulate further research at other national institutions.