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。网站的缺陷 过程中导致非整倍体，直接导致发育障碍 综合征和大多数癌症的促成因素。着丝粒是一种独特的 每个染色体上指定染色体附着于有丝分裂细胞的区域 纺锤体、有丝分裂和减数分裂中染色体运动和临界信号 转导途径，监测和维持正常的染色体数目。 最近的研究结果表明，用于 复制的着丝粒与作用于染色体的着丝粒不同 怀里这项提议的工作将检验着丝粒 同一性涉及一个关键的自模板染色质组装途径， 鉴定组装着丝粒特异性染色体的染色质组装因子， 一种染色质结构域，它将核心组蛋白H3替换为一个着丝粒特异性的 H3类蛋白质称为CENP-A。在体外组装的染色质复合物将 引入人HT 1080细胞中以直接确定CENP-A和/或 其他着丝粒特异性染色质因子模板自身沉积 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 将通过直接纯化CENP-A染色质来解剖核小体 复合物随后质谱鉴定相关蛋白质， 通过重组CENP-A核小体的X射线晶体学和电子衍射， 重组CENP-A染色质纤维的显微镜检查。翻译后 CENP-A的修饰将被表征以确定是否存在着丝粒修饰。 染色质受到类似的翻译后控制， 非着丝粒染色质。CENP-A的职能将由 在小鼠和培养的人类细胞中开发敲除和敲低模型 再加上CENP-A的突变分析，以探索这种基因的具体作用。 组蛋白同源物在着丝粒组装和功能中作用。

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.