Eukaryotic Nuclear Functions: from Nucleosomes to Chromosomes

真核细胞核功能：从核小体到染色体

• 批准号: 9923723
• 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/9923723
• 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.

项目概要/摘要 真核基因组必须同时被包装以适合细胞核，但也提供访问 允许基本生物过程（包括基因转录和基因组复制）的特定位点。 为了实现这些对包装和获取的相反要求，真核基因组受到监管 许多层次和长度尺度，从核小体到高阶、三维相互作用 染色体。我的实验室正在研究两个不同级别的监管，沿着这个广泛但 互连频谱： 首先，我们首次在基因组水平上测试基因组功能的调控程度。 核小体对称性。核小体包含每个核心组蛋白的两个副本，通过天然结构结合在一起 对称的同源二聚体组蛋白 H3-H3 界面。这种对称性使得确定 该架构的监管潜力。换句话说，一个或两个尾巴是否接受后处理重要吗？ 翻译修饰？回答这个问题需要有能力专门削弱对 每个核小体单尾。通过分子设计和体内选择，我们已经产生了 异二聚体 H3，为发现组蛋白修饰对称性程度提供了独特的工具 在活细胞的基因表达和其他染色体功能中发挥调节作用。 验证了不对称 H3 对后，我们将这些研究扩展到另外两个 H3 同工型。第一的， 我们最近在芽殖酵母中生成了一对不对称着丝粒 H3 (Cse4/CENP-A)。利用这些，我们 将解决有关着丝粒核小体化学计量学的长期争议。其次，我们是 使用不对称复制独立组蛋白 H3.3 对来探测两个具有关键组蛋白修饰 在染色质结构和基因调控中的作用。组蛋白 H3.3 是抑制内源性 小鼠胚胎干细胞中的逆转录病毒转录和早期分化，因此我们计划研究 酵母中不存在的抑制染色质机制的化学计量调节关系 特别是涉及 H3K9me3（组成型异染色质的特征）和 H3K27me3（组成型异染色质的特征） 受发育调节的兼性异染色质）。因为显性 H3.3 突变是 这些研究还与多种类型的癌症有关，还提供了一种新的工具来探索这些癌症是如何产生的。 改变影响活细胞中的表观基因组。 其次，我们正在探索人类三维组织之间的相互联系。 基因组、细胞周期进程和免受遗传毒性应激的保护。我们的实验使我们发现 重点关注临床上重要的增殖标记蛋白 Ki-67。正常三线需要 Ki-67 核仁周围异染色质位点的空间组织，保护细胞免受基因毒性应激，以及 对于在有丝分裂染色体上形成蛋白质层至关重要。目前尚不清楚 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，并延迟进入 S 期。这些细胞 周期表型伴随着异染色质标记（例如 H3K27me3）维持的减少 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 缺乏 G1/S 检查点的细胞中不存在。换句话说，Ki-67 将细胞周期进程和染色体联系起来 原代细胞中的维持，而检查点缺陷的肿瘤细胞则逃避这些机制。开始 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 检查点熟练细胞中 Ki-67 耗尽后。我们还将绘制所需的 Ki-67 蛋白结构域图 其新颖的活性，并确定它们是否与先前描述的有丝分裂中的作用可分离 染色体结构和间期异染色质定位。以此方式，我们将做好准备去追求 相关的伙伴蛋白在我们对人类染色体结构和协调的新见解的道路上 功能。