Molecular Analysis of Pericentric Sister Chromatid Cohesion

描述（由申请人提供）：准确的染色体分离对于遗传信息成功传递给子细胞至关重要，该过程中的缺陷与流产、先天性疾病和肿瘤发生有关。染色体正确分离的关键是复制的姐妹染色单体的内聚或物理结合，这是由进化上保守的内聚复合物介导的。内聚蛋白不仅参与染色体分离，还在DNA修复和基因调控中发挥重要作用。内聚蛋白在出芽酵母染色体上的空间分布具有高度的可重复性，表明其在染色体结构和功能中起着重要作用。内聚蛋白经常出现在聚合转录基因之间的基因间区域，这表明内聚蛋白分布与转录之间的相互作用目前尚未被表征。值得注意的是，广泛的内聚蛋白结构域聚集在着丝粒周围（着丝粒侧翼）区域，在那里它们促进染色体双向定向并抵抗姐妹染色单体的过早分离。我们之前已经证明出芽酵母着丝点直接指导着粒内聚结构域的组装。我们的初步数据表明，周中心点内聚蛋白结构域是通过成核和扩散机制进行表观遗传组装的。我们在Specific Aim 1中通过确定着丝粒附近的环形成是否会阻碍远端序列的内聚蛋白募集来测试该模型的准确性。此外，还将分离周中心点染色质，并对其蛋白质组成进行无偏筛选，以确定着丝酶相关因子或表观遗传染色质修饰直接内聚结构域组装。最后，限制内聚蛋白募集的绝缘子将用于描绘高保真染色体传递所必需的最低有效的中心点周围结构域。初步数据还表明，黏附蛋白的分布受Scc2/Scc4黏附蛋白装载物与基因间序列的先验关联的影响。在第二个目的中，我们试图确定染色体臂上装载物和内聚物定位的机制和意义。RSC atp依赖性染色质重塑子、Scc2/Scc4和黏结蛋白的关联顺序和相互依赖性将在染色体臂黏结蛋白相关区域进行测定，以剖析黏结蛋白沉积的途径。在聚合酶失活后，我们将研究RNA聚合酶II依赖性转录在建立内聚蛋白分布中的贡献，并研究RNA聚合酶II转录终止突变体中内聚蛋白重新分布的机制和意义，以描述转录与内聚蛋白定位之间关系的本质。出芽酵母内聚蛋白在促进RNA聚合酶II转录终止中的作用将通过条件内聚蛋白突变体来确定。

DESCRIPTION (provided by applicant): Accurate chromosome segregation is essential for the successful transmission of genetic information to daughter cells, and deficiencies in this process are associated with miscarriages, congenital disorders, and tumorigenesis. Integral to proper chromosome segregation is the cohesion or physical association of replicated sister chromatids, which is mediated by the evolutionarily conserved cohesin complex. Cohesins are involved not only in chromosome segregation, but also play important roles in DNA repair and gene regulation. Cohesin's spatial distribution on budding yeast chromosomes is highly reproducible, suggesting that it plays important roles in chromosome structure and function. Cohesins are frequently found in intergenic regions between convergently transcribed genes, indicating interplay between cohesin distributions and transcription that is currently not characterized. Notably, extensive cohesin-enriched domains assemble in pericentromeric (kinetochore-flanking) regions, where they promote chromosome biorientation and resist precocious sister chromatid separation. We showed previously that budding yeast kinetochores direct pericentromeric cohesin domain assembly. Our preliminary data indicate that pericentromeric cohesin domains are assembled epigenetically by a nucleation and spreading mechanism. We test the veracity of this model in Specific Aim 1 by determining whether loop formation adjacent to the centromere impedes cohesin recruitment in distal sequences. Pericentromeric chromatin will also be isolated and its protein composition characterized in an unbiased screen for kinetochore-associated factors or epigenetic chromatin modifications that direct cohesin domain assembly. Lastly, insulators that limit cohesin recruitment will be used to delineate the minimally effective pericentromeric domain necessary for high fidelity chromosome transmission. Preliminary data also indicate that cohesin distributions are directed by the prior association of the Scc2/Scc4 cohesin loader with intergenic sequences. In the second aim, we endeavor to determine the mechanisms and significance of loader and cohesin localization on chromosome arms. The order and interdependence of association of the RSC ATP-dependent chromatin remodeler, Scc2/Scc4, and cohesin will be determined at chromosome arm cohesin-associated regions to dissect the pathway for cohesin deposition. The contribution of RNA polymerase II-dependent transcription in the establishment of cohesin loader distributions will be examined following polymerase inactivation and the mechanism and significance of cohesin redistribution in an RNA polymerase II transcription termination mutant are examined to delineate the nature of the relationship between transcription and cohesin localization. The role of budding yeast cohesins in the promotion of RNA polymerase II transcription termination will be determined using conditional cohesin mutants. PUBLIC HEALTH RELEVANCE: Chromosomes must be accurately duplicated and segregated to daughter cells during each cell division, and errors in these events can lead to cancer or genetic disease, such as Down syndrome. The accurate segregation of replicated chromosomes, or sister chromatids, to daughter cells requires that sisters physically associate with one another throughout much of the cell cycle. In this application, we endeavor to understand how the proteins that mediate the physical association of sister chromatids, called cohesins, are recruited to proper locations on the chromosome to mediate chromosome segregation, DNA repair, and gene regulation, each of which is important for the maintenance of genomic integrity.