Mechanisms of Kinetochore Assembly

项目摘要 细胞必须在每次分裂时准确地将染色体分离成子细胞， 让细胞得以繁殖和增殖染色体分离的错误导致不平衡 染色体拷贝数称为非整倍体，是癌症的标志， 发育性疾病，如唐氏综合症，是流产的主要原因。 这项工作的重点是了解细胞如何维持正常的染色体拷贝数 在染色体分离的过程中。染色体分离的关键调节因子 是染色体动粒。动粒是每个染色体上的位点， 到有丝分裂纺锤体的微管上，这样染色体就可以在分裂过程中移动到子细胞中。 细胞分裂动粒还通过有丝分裂过程监测染色体的正确排列。 检查点，以确保每个子细胞得到每个染色体的一个副本。的 动粒形成的基础是染色体上称为着丝粒的区域。 着丝粒是一个染色质结构域，它是由变异体的存在唯一决定的 组蛋白称为着丝粒蛋白A（CENP-A）。着丝粒的形成是表观遗传学上的 这取决于染色质中CENP-A的存在，一旦着丝粒形成， 无论其构建的DNA序列如何，都能稳定地维持。CENP-A染色质是 对于着丝粒和动粒的形成至关重要，CENP-A的突变或缺失导致 着丝粒丢失和染色体错误分离。我们正在研究 组装和维持CENP-A染色质以及CENP-A染色质如何被识别以构建 着丝粒和动粒。在我们的第一个具体目标，我们剖析了生化 在染色体中的正确时间和位置构建CENP-A核小体的机制。在 特别是，我们专注于了解新CENP-A核小体的关键调节因子如何 Mis18复合体的形成，正确地针对着丝粒，以及它如何在 CENP-A组件。在我们的第二个目的中，我们探讨了DNA序列在着丝粒中的功能 阵虽然CENP-A是着丝粒功能的主要表观遗传决定因子， 着丝粒处的DNA序列促进有效的着丝粒形成， 上维护我们测试特定的DNA序列是否刺激CENP-A组装， 无论是DNA还是RNA分子主动调节新CENP-A核小体的形成。 总之，我们的方法定义了着丝粒形成的基础，以及着丝粒的蛋白质是如何形成的。 着丝粒产生有丝分裂中染色体分离所需的性质。

Project Summary Cells must accurately segregate their chromosomes into daughter cells at each division in order for cells to reproduce and proliferate. Mistakes in chromosome segregation lead to imbalances in chromosome copy number termed aneuploidies that are hallmarks of cancer, that cause developmental diseases such as Down syndrome and that are a primary cause of miscarriage. This work is focused on understanding how cells maintain a normal chromosome copy number during the process of chromosome segregation. The key regulator of chromosome segregation is the chromosomal kinetochore. The kinetochore is the site on each chromosome that attaches to the microtubules of the mitotic spindle so that chromosome can move to daughter cells during cell division. Kinetochores also monitor proper chromosome alignment through the mitotic checkpoint to ensure that each daughter cell gets one copy of each chromosome. The foundation for kinetochore formation is a region of the chromosome termed the centromere. Centromeres are a chromatin domain that is uniquely determined by the presence of a variant histone termed centromere protein A (CENP-A). The formation of centromeres is epigenetically determined by the presence of CENP-A in chromatin and once a centromere is formed it can be stably maintained regardless of the DNA sequence on which it is built. CENP-A chromatin is essential for centromere and kinetochore formation and mutation or loss of CENP-A results in centromere loss and chromosome missegregation. We are studying the mechanisms that assemble and maintain CENP-A chromatin and how CENP-A chromatin is recognized to build the centromere and kinetochore. In our first specific Aim we dissect the biochemical mechanisms that build CENP-A nucleosomes at the right time and place in the chromosome. In particular, we focus on understanding how a key regulator of new CENP-A nucleosome formation, the Mis18 complex, is properly targeted to centromeres and how it functions in CENP-A assembly. In our second Aim we explore the function of DNA sequences in centromere formation. Although CENP-A is the primary epigenetic determinant of centromere function the sequence of the DNA at centromeres promotes efficient centromere formation and maintenance. We test whether specific DNA sequences stimulate CENP-A assembly and whether DNA or RNA molecules actively regulate new CENP-A nucleosome formation. Together our approach defines the basis for centromere formation and how the proteins of the centromere give rise to the properties required for chromosome segregation in mitosis.