Eukaryotic Nuclear Functions: from Nucleosomes to Chromosomes

项目概要/摘要 真核生物的基因组必须同时被包装，以适应细胞核，但也提供访问， 特定的基因座，以允许基本的生物过程，包括基因转录和基因组复制。 为了实现包装和获取的这些相反的要求，真核基因组被调节为 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沿着这个广泛的， 互联频谱： 首先，我们第一次测试了基因组功能在基因水平上的调节程度。 核小体对称性核小体包含两个拷贝的每个核心组蛋白，由一个天然的 对称的同二聚体组蛋白H3-H3界面。这种对称性使得确定 这种架构的监管潜力。换句话说，重要的是一个或两个尾巴收到后， 翻译修饰要解决这个问题，需要有能力具体损害对 每个核小体单尾。通过分子设计和体内选择，我们已经产生了专性的 异二聚体H3 s，提供了一个独特的工具，发现的程度，组蛋白修饰对称性 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H3对之后，我们将这些研究扩展到另外两种H3亚型。第一、 我们最近在芽殖酵母中产生了不对称着丝粒H3（Cse 4/CENP-A）对。利用这些，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 使用不对称的不依赖复制的组蛋白H3.3对来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是抑制内源性 逆转录病毒转录和小鼠胚胎干细胞的早期分化，所以我们计划研究 抑制性染色质机制的调节关系的化学计量学在酵母中是不存在的， 特别是涉及H3 K9 me 3（组成型异染色质的特征）和H3 K27 me 3（组成型异染色质的特征）。 发育调节的兼性异染色质）。因为显性H3.3突变是 与几种类型的癌症有关，这些研究也为探索这些癌症如何发生提供了一种新的工具。 改变影响活细胞中的表观基因组。 其次，我们正在探索人类的三维组织之间的相互联系， 基因组、细胞周期进程和基因毒性应激保护。我们的实验让我们 关注临床上重要的增殖标志物蛋白Ki-67。Ki-67是正常的三- 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， 是有丝分裂染色体上形成蛋白质层所必需的。目前尚不清楚Ki-67 有助于这些过程，或者这些功能如何相互关联。 我们最近发现，在具有完整的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期进入。这些细胞 周期表型伴随着异染色质标记（如H3 K27 me 3）的维持减少， 女性检查点熟练细胞中的非活性X（Xi）染色体。值得注意的是，所有这些表型都是 在缺乏G1/S检查点的细胞中不存在。换句话说，Ki-67将细胞周期进程和染色体 在原代细胞中的维持，和检查点缺陷的肿瘤细胞逃避这些机制。开始 为了探索这些新功能，我们将测试DNA损伤的分子标志 在检查点熟练细胞中Ki-67耗竭后。我们还将绘制需要哪些Ki-67蛋白结构域 其新的活动，并确定他们是否是从先前描述的有丝分裂中的作用分离 染色体结构和间期异染色质定位。我们将以这种方式 相关的伙伴蛋白质在我们的道路上新的见解协调人类染色体结构和 功能

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.