Eukaryotic Nuclear Functions: from Nucleosomes to Chromosomes

真核生物核功能：从核小体到染色体

• 批准号: 10152614
• 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
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10152614
• 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/antagonist; insight; neoplastic cell; novel; protein biomarkers; stoichiometry; tool

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以适合细胞核，但也提供了在 特定的基因座允许基本生物学过程，包括基因转录和基因组复制。 为了满足包装和访问的这些相反的要求，真核基因组受到调节 从核小体到高阶，三维相互作用的许多级别和长度尺度 染色体。我的实验室正在调查这两个不同级别的监管，但 互连光谱： 首先，我们首次测试基因组功能在水平上的调节程度 核小体对称性。核小体包含每个核心组蛋白的两个拷贝，由自然固定在一起 对称，同型组蛋白H3-H3接口。这种对称性为确定 该体系结构的监管潜力。换句话说，一个或两个尾巴是否接收到后 翻译修改？回答这个问题需要能够在 每个核小体单尾。通过分子设计和体内选择，我们已将 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 在活细胞中的基因表达和其他染色体功能中起调节作用。 在验证了不对称的H3对后，我们将这些研究扩展到另外两个H3同工型。第一的， 我们最近在发芽的酵母中生成了一个不对称的丝粒H3（CSE4/CENP-A）。使用这些，我们 将解决有关丝粒核小体化学计量学的长期争议。第二，我们是 使用非对称复制独立的组蛋白H3.3对探测两个组蛋白的修饰 在染色质结构和基因调节中的作用。组蛋白H3.3是抑制内源性所必需的 逆转录病毒转录和小鼠胚胎干细胞的早期分化，因此我们计划研究 酵母中缺乏抑制性染色质机制的调节关系的化学计量法，大多数 值得注意的是涉及H3K9me3（本构异染色质的特征）和H3K27Me3（特征的特征 受发展调节的兼性异染色质）。因为主要的H3.3突变是 这些研究与几种类型的癌症有关，还为探索这些研究提供了一种新颖的工具 改变会影响活细胞的表观基因组。 其次，我们正在探索人类三维组织之间的互连 基因组，细胞周期进程和免受遗传毒性应激的保护。我们的实验使我们进入 专注于临床上重要的增殖标记蛋白KI-67。正常三 - 需要Ki-67 核核周围异晶性基因座的维度组织，保护细胞免受遗传毒性应激，并 对于在有丝分裂染色体上形成蛋白质层至关重要。尚不了解KI-67 有助于这些过程，或者如何相互关联。 我们最近发现，在具有完整G1/S细胞周期检查点的人类细胞中，KI-67的急性耗竭 诱导细胞周期抑制剂P21，降低G1/S调节的RNA水平，并延迟S相进入。这些细胞 循环表型伴随着杂染色质标记的维持降低（例如H3K27ME3） 女性检查点的细胞中的无活性X（XI）染色体。值得注意的是，所有这些表型都是 缺乏G1/s检查点的细胞中不存在。换句话说，KI-67链接细胞周期的进展和染色体 在原代细胞中维持，检查点缺陷肿瘤细胞逃避了这些机制。开始 因此，我们将对这些新功能的分子探索，因此我们将测试DNA损伤的分子标志 在Ki-67耗尽后，在较高的细胞中耗尽。我们还将绘制需要哪些KI-67蛋白质结构域 为了进行新的活动，并确定它们是否与先前描述的有丝分裂作用可分开 染色体结构和相间异染色质定位。这样，我们将准备追求 相关伴侣蛋白在我们对人类染色体结构协调的新见解的道路上 功能。