Assembly and Function of Human Centromere Chromatin

描述(申请人摘要)：染色体分离是一个关键因素 正常发育和遗传稳定所必需的过程。这里面的瑕疵 过程导致非整倍体，这是导致发育障碍的直接原因 综合症，也是大多数癌症的致病因素。着丝粒是一种独特的 每条染色体上指定染色体附着到有丝分裂的区域 有丝分裂和减数分裂中的纺锤体、染色体运动性和关键信号 监测和维持正常染色体数目的转导途径。 最近的研究结果表明，染色质组装途径用于 复制着丝粒不同于在染色体上工作的着丝粒 手臂。这项提案的工作将检验着丝粒的假设 身份涉及一个关键的自身模板染色质组装途径，通过 鉴定组装着丝粒特异性的染色质组装因子 核心组蛋白H3取代着丝粒特异的染色质结构域 类H3蛋白称为CENP-A。在体外组装的染色质复合体将被 直接测定CENP-A和/或CENP-A和/或 其他着丝粒特异的染色质因子以它们自己的沉积为模板 在体内DNA复制和染色质组装过程中。分子结构 和由CENP-A组成的着丝粒特异性染色质的组成 核小体将通过直接纯化CENP-A染色质进行解剖 复合体之后结合蛋白质的质谱学鉴定， 重组CENP-A核小体的X射线结晶学和电子分析 重组CENP-A染色质纤维的显微镜观察。翻译后 CENP-A的修饰将被表征以确定着丝粒是否 染色质受到与观察到的相似类型的翻译后控制 用于非着丝粒染色质。CENP-A的功能将通过以下方式进行检查 在小鼠和培养的人细胞中建立基因敲除和基因敲除模型 再加上CENP-A的突变分析，来探索这一特定的作用 着丝粒组装和功能中的组蛋白同系物。

DESCRIPTION (APPLICANT'S ABSTRACT): Chromosome segregation is a critical process necessary for normal development and genetic stability. Defects in this process lead to aneuploidy, a direct cause of the developmental disorder Down syndrome and a contributing factor in most cancers. The centromere is a unique region on each chromosome that specifies chromosome attachment to the mitotic spindle, chromosome motility in mitosis and meiosis and critical signal transduction pathways that monitor and maintain normal chromosome number. Recent findings have revealed that the chromatin assembly pathway used to replicate centromeres is distinct from that which operates on the chromosome arms. The work of this proposal will test the hypothesis that centromere identity involves a critical seif-templated chromatin assembly pathway by identifying chromatin assembly factors that assemble a centromere-specific chromatin domain that substitutes the core histone H3 for a centromere specific H3-like protein known as CENP-A. Chromatin complexes assembled in vitro will be introduced into human HT1080 cells to directly determine whether CENP-A and/or other centromere-specific chromatin factors template their own deposition during DNA replication and chromatin assembly in vivo. The molecular structure and composition of centromere-specific chromatin comprised of CENP-A nucleosomes will be dissected by direct purification of CENP-A chromatin complexes followed by mass spectrometric identification of associated proteins, by X-ray crystallography of reconstituted CENP-A nucleosomes and by electron microscopy of reconstituted CENP-A chromatin fibers. Post-translational modifications of CENP-A will be characterized to determine whether centromeric chromatin is subject to similar kinds of post-translational control as observed for non-centromeric chromatin. The function of CENP-A will be examined by developing knock out and knock down models in mouse and cultured human cells coupled with mutational analysis of CENP-A to explore the specific role of this histone homologue in centromere assembly and function.