Molecular Basis of Centromere Specification and Inheritance

项目摘要/摘要 真核细胞中一个基本但鲜为人知的过程是细胞如何将它们的基因组结构成 不同的功能域。这个项目通过研究着丝粒来解决这一知识差距，着丝粒是一种 存在于所有真核生物中的特定染色质结构域。这个稳定传播的轨迹引导着 在有丝分裂和减数分裂过程中确保染色体适当分离的动粒。监管不善 着丝粒对染色体分离产生不利影响，导致非整倍体，这种情况在更多的 占所有癌症的90%以上。非整倍体导致许多疾病的发展，如癌症和 唐氏综合症。这一项目的目标是了解潜在的分子机制 着丝粒的规范和遗传。在大多数真核生物中，着丝粒是由表观遗传学控制的 着丝粒特异的组蛋白H3变异体CENP-A。CENP-A部分取代正则组蛋白H3 着丝粒，为动粒的组装提供了基础。着丝粒通常是 嵌入在表观遗传不同的异染色质中，转录沉默的染色质结构域。 CENP-A在着丝粒的组装受细胞周期调控。亲本CENP-A平均分配给 DNA复制后的子体着丝粒，而将新合成的CENP-A装载到 着丝粒与DNA复制解偶联。着丝粒的CENP-A染色质是如何组装的 在整个细胞周期中，人们对此知之甚少。CENP-A错误定位于非着丝粒区域 对染色体分离有毁灭性的影响，并与多种癌症有关。泛素- 介导的CENP-A蛋白降解是防止CENP-A错误定位的一种保守机制。但怎么不- 着丝粒区域受到CENP-A的保护，在正常细胞中的误掺入在很大程度上是未知的。在……里面 此外，着丝粒中的CENP-A与典型的组蛋白H3点缀在一起。体内的组蛋白H3 着丝粒实际上对CENP-A染色质的正确组装至关重要。CENP-A和H3水平如何 着丝粒的适当平衡是未知的。我们建议使用裂解酵母(裂殖酵母) 来解决这些悬而未决的问题。裂解酵母是一种简单的真核模式生物，具有许多 着丝粒调控的某些方面在人类中是保守的。它特别适合于跨学科的 方法包括遗传学、基因组学、细胞学、生物化学和结构生物学。我们建议：1) 定义细胞周期依赖的CENP-A在着丝粒组装的潜在机制，2)确定如何 防止异位CENP-A染色质的形成，3)确定CENP-A和CENP-A的调控机制 组蛋白H3水平在着丝粒上是平衡的。我们的研究也提供了重要的新见解， 着丝粒功能中的异染色质。鉴于分裂酵母的表观遗传调控是保守的，我们的 研究将阐明人类细胞中控制染色体分离的过程，并有助于 更好地理解着丝粒错误调节引起的人类疾病。

PROJECT SUMMARY/ABSTRACT A fundamental but poorly understood process in eukaryotic cells is how cells structure their genomes into distinct functional domains. This project addresses this gap in knowledge by studying the centromere, a specific chromatin domain found in all eukaryotes. This stably propagated locus guides the assembly of kinetochores to ensure proper segregation of chromosomes during mitosis and meiosis. Mis-regulation of centromeres adversely affects chromosome segregation resulting in aneuploidy, a condition found in more than 90% of all cancers. Aneuploidy contributes to the development of many diseases, such as cancer and Down syndrome. The goal of this project is to understand the molecular mechanisms underlying the specification and inheritance of centromeres. In most eukaryotes, centromeres are epigenetically governed by the centromere-specific histone H3 variant, CENP-A. CENP-A partially replaces canonical histone H3 at centromeres, and provides the foundation for the assembly of kinetochores. Centromeres are usually embedded in epigenetically distinct heterochromatin, the transcriptionally silenced chromatin domain. Assembly of CENP-A at centromeres is cell cycle-regulated. Parental CENP-A is partitioned equally among daughter centromeres following DNA replication, whereas loading of newly synthesized CENP-A to centromeres is uncoupled from DNA replication. How CENP-A chromatin at centromeres is assembled throughout the cell cycle remains poorly understood. Mislocalization of CENP-A to non-centromeric regions has a devastating impact on chromosome segregation, and has been linked to a variety of cancers. Ubiquitin- mediated proteolysis of CENP-A is a conserved mechanism to prevent CENP-A mislocalization. But how non- centromeric regions are protected from CENP-A mis-incorporation in normal cells is largely unexplored. In addition, CENP-A in centromeres is interspersed with the canonical histone H3. The histone H3 within centromeres is actually vital for proper assembly of CENP-A chromatin. How CENP-A and H3 levels are properly balanced in centromeres is unknown. We propose to use fission yeast (Schizosaccharomyces pombe) to address these outstanding questions. Fission yeast is a simple eukaryotic model organism with many aspects of centromere regulation conserved with humans. It is particularly suited to an interdisciplinary approach that includes genetics, genomics, cytology, biochemistry, and structural biology. We propose to: 1) define the mechanisms underlying cell cycle-dependent CENP-A assembly at centromeres, 2) determine how formation of ectopic CENP-A chromatin is prevented, 3) identify regulatory mechanism for how CENP-A and histone H3 levels are balanced at centromeres. Our study also provides important new insights into the role of heterochromatin in centromere function. Given that epigenetic regulation in fission yeast is conserved, our studies will shed light on the processes governing chromosome segregation in human cells, and contribute to a better understanding of human diseases resulting from centromere misregulation.