Chromatin Biosynthesis: Tetramer Assembly and the Predeposition Code

该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向后代细胞的转移。为此，本项目重点研究新合成组蛋白（组蛋白是主要的染色体蛋白）的翻译后修饰，以及人类细胞中H3/H4四聚体的组装模式。H3/H4四聚体在体内的组装方式将通过免疫沉淀的单核小体进行检测，该单核小体在同步细胞中含有放射性标记的、表位标记的H4和H3变体。实验还将确定新合成的人类组蛋白H4和H3变体的完全乙酰化、磷酸化和甲基化状态作为细胞周期的功能。这将通过在G1期和S期净化新生的H3/H4预沉积复合物来完成。新合成的组蛋白将通过串联（MS/MS）质谱分析，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）在复制和非复制DNA上组装方式的基本信息。本研究将在三个主要方面产生更广泛的社会影响：(1)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)通过参与科研院所，为高中生提供科研机会；(3)在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，从而有助于刺激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.