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Eukaryotic Nuclear Functions: from Nucleosomes to Chromosomes

真核生物核功能：从核小体到染色体

基本信息

批准号：
10400845
负责人：
PAUL D. KAUFMAN
金额：
$ 33.5万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-01 至 2023-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10400845
关键词：
3-Dimensional Acute Address Affect Architecture Biological Process Cancerous Cell Cycle Cell Cycle Progression Cell Nucleus Cells Characteristics Chromatin Chromatin Structure Chromosome Structures Chromosomes Clinical DNA DNA Damage Development Endogenous Retroviruses Female G1/S Checkpoint Pathway Gene Expression Gene Expression Regulation Genetic Transcription Genome Genotoxic Stress Growth Heterochromatin Histone H3 Histones Human Human Chromosomes Human Genome Impairment Interphase Laboratories Length Link Maintenance Maps Methods Mitotic Chromosome Modification Molecular Mus Mutation Nuclear Nucleosomes Phenotype Play Post-Translational Protein Processing Process Proliferation Marker Protein Isoforms Proteins RNA Regulation Repression Role S phase Saccharomycetales Tail Technology Tertiary Protein Structure Testing Time Tumor Markers X Inactivation Yeasts cancer type centromere protein A design embryonic stem cell epigenome experimental study genetic information histone modification in vivo inhibitor insight neoplastic cell novel protein biomarkers stoichiometry tool

项目摘要

Project Summary/Abstract Eukaryotic genomes must simultaneously be packaged to fit into the cell nucleus, but also provide access at specific loci to allow for fundamental biological processes including gene transcription and genome replication. To accomplish these opposing requirements for packaging and access, eukaryotic genomes are regulated at many levels and length scales, from the nucleosome to the higher-order, three-dimensional interactions among chromosomes. My laboratory is investigating two different levels of regulation along this broad but interconnected spectrum: First, we are testing for the first time the extent of regulation of genome function at the level of nucleosome symmetry. Nucleosomes contain two copies of each core histone, held together by a naturally symmetric, homodimeric histone H3-H3 interface. This symmetry has complicated efforts to determine the regulatory potential of this architecture. In other words, is it important whether one or both tails receives a post- translational modification? Answering this question requires the ability to specifically impair modification on a single tail per nucleosome. Through molecular design and in vivo selection, we have generated obligately heterodimeric H3s, providing a unique tool for discovery of the degree to which histone modification symmetry plays a regulatory role in gene expression and other chromosomal functions in living cells. Having validated an asymmetric H3 pair, we are extending these studies to two additional H3 isoforms. First, we recently generated an asymmetric centromeric H3 (Cse4/CENP-A) pair in budding yeast. Using these, we will address long-standing controversies regarding centromeric nucleosome stoichiometry. Second, we are using an asymmetric replication-independent histone H3.3 pair to probe two histone modifications with key roles in chromatin structure and gene regulation. Histone H3.3 is required for repression of endogenous retrovirus transcription and early differentiation in mouse embryonic stem cells, so we plan to investigate the stoichiometry of regulatory relationships for repressive chromatin mechanisms that are absent in yeast, most notably involving H3K9me3 (characteristic of constitutive heterochromatin) and H3K27me3 (characteristic of facultative heterochromatin that is developmentally regulated). Because dominant H3.3 mutations are implicated in several types of cancer, these studies also provide a novel tool for exploration of how these alterations affect epigenomes in living cells. Second, we are exploring interconnections between the three-dimensional organization of the human genome, cell cycle progression, and protection from genotoxic stress. Our experiments have led us to focus on the clinically important proliferation marker protein Ki-67. Ki-67 is required for normal three- dimensional organization of heterochromatic loci around the nucleoli, protects cells from genotoxic stress, and is essential for forming a proteinaceous layer on mitotic chromosomes. It is not understood how Ki-67 contributes to these processes, or how these functions may be interrelated. We recently discovered that in human cells with intact G1/S cell cycle checkpoints, acute depletion of Ki-67 induces cell cycle inhibitor p21, reduces G1/S-regulated RNA levels, and delays S phase entry. These cell cycle phenotypes are accompanied by reduced maintenance of heterochromatin marks (e.g. H3K27me3) on the inactive X (Xi) chromosome in female checkpoint-proficient cells. Notably, all of these phenotypes are absent in cells lacking G1/S checkpoints. In other words, Ki-67 links cell cycle progression and chromosome maintenance in primary cells, and checkpoint-defective tumor cells evade these mechanisms. To begin molecular exploration of these novel functions, we will therefore test for molecular hallmarks of DNA damage upon Ki-67 depletion in checkpoint-proficient cells. We will also map which Ki-67 protein domains are required for its novel activities, and determine if they are separable from previously described roles in mitotic chromosome structure and interphase heterochromatin localization. In this manner, we will be poised to pursue relevant partner proteins on our path to new insights into the coordination of human chromosome structure and function.

项目摘要/摘要真核基因组必须同时包装成适合细胞核，但也提供了访问允许包括基因转录和基因组复制在内的基本生物学过程的特定基因座。为了实现这些截然相反的包装和获取要求，真核基因组在许多层次和长度的尺度，从核小体到更高层次的，三维的相互作用染色体。我的实验室正在调查两种不同程度的监管互联频谱：首先，我们首次在基因组水平上测试基因组功能的调节程度核小体对称性。核小体包含每个核心组蛋白的两个副本，由一种天然的对称的同源二聚体组蛋白H3-H3界面。这种对称性使确定此架构的监管潜力。换句话说，无论是一条尾巴还是两条尾巴都收到帖子- 翻译修饰？要回答这个问题，需要能够明确地损害对每个核小体有一条尾巴。通过分子设计和体内选择，我们不可避免地产生了异二聚体H3s，为发现组蛋白修饰对称性的程度提供了独特的工具在活细胞中对基因表达和其他染色体功能起着调节作用。在确认了一个不对称的H3对之后，我们将这些研究扩展到另外两个H3亚型。第一, 我们最近在发芽酵母中产生了一对不对称着丝粒H3(Cse4/CENP-A)。使用这些，我们将解决关于着丝粒核小体化学计量的长期争议。第二，我们是使用不对称复制非依赖性组蛋白H3.3对探测两个组蛋白修饰在染色质结构和基因调控中的作用。组蛋白H3.3是内源性阻滞剂所必需的逆转录病毒在小鼠胚胎干细胞中的转录和早期分化，因此我们计划研究酵母中不存在的抑制染色质机制的调节关系的化学计量学特别涉及H3K9me3(构成异染色质的特征)和H3K27me3(特征为异染色质受发育调节的兼性异染色质)。因为显性H3.3突变是这些研究还提供了一种新的工具来探索这些癌症是如何改变会影响活细胞中的表观基因组。第二，我们正在探索人类三维组织之间的相互联系基因组、细胞周期进程和遗传毒性应激的保护。我们的实验使我们关注临床上重要的增殖标记物蛋白Ki-67。正常的三种情况都需要Ki-67- 核仁周围异染色质基因座的空间组织，保护细胞免受遗传毒性应激，以及是在有丝分裂染色体上形成蛋白质层所必需的。尚不清楚Ki-67是如何对这些过程的贡献，或者这些功能如何相互关联。我们最近发现，在G1/S细胞周期检查点完整的人细胞中，Ki-67的急性耗竭诱导细胞周期抑制因子p21，降低G1/S调控的核糖核酸水平，延缓S期进入。这些细胞周期表型伴随着异染色质标记(例如H3K27me3)在女性检查点熟练细胞中不活跃的X(XI)染色体。值得注意的是，所有这些表型都是在缺乏G1/S检查点的细胞中缺失。换句话说，Ki-67将细胞周期进程和染色体联系在一起原代细胞的维持和检查点缺陷的肿瘤细胞逃避这些机制。要开始对这些新功能的分子探索，我们因此将测试DNA损伤的分子特征在检查点熟练的细胞中Ki-67耗尽。我们还将绘制所需的Ki-67蛋白结构域其新的活性，并确定它们是否可与先前描述的有丝分裂中的角色分开染色体结构和间期异染色质定位。通过这种方式，我们将准备继续在我们对人类染色体结构和染色体协调的新见解的道路上的相关伙伴蛋白质功能。