Chromatin Biosynthesis: Tetramer Assembly and the Predeposition Code

主要研究者的研究计划的长期目标是阐明体细胞染色质生物合成的机制，并了解表观遗传信息转移到后代细胞。为此，该项目的重点是新合成的组蛋白（组蛋白是主要的染色体蛋白质）的翻译后修饰，以及人类细胞中H3/H4四聚体组装的模式。H3/H4四聚体在体内组装的方式将通过在同步化细胞中免疫沉淀含有放射性标记的、表位标记的H4和H3变体的单核体来检查。还将进行实验以确定新合成的人组蛋白H4和H3变体的完整乙酰化、磷酸化和甲基化状态作为细胞周期的函数。这将通过在G1和S阶段期间纯化新生H3/H4预沉积复合物来实现。新合成的组蛋白将通过串联（MS/MS）质谱法进行分析，以揭示人类H3和H4在沉积到DNA上之前的所有修饰。 该项目将提供核小体（真核染色体的基本亚基）组装到复制和非复制DNA上的方式的基本信息。这项研究将在三个主要领域产生更广泛的社会影响：（1）它将为本科生提供良好的研究经验，从而促进他们的科学发展;（2）它将通过参与研究科学研究所为高中生提供研究机会;以及（3）在该项目过程中开发的细胞系将提供给该领域的其他研究人员，从而有助于促进其他国家机构的进一步研究。

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.