Structural instability and DNA rearrangements in the centromere

描述（由申请人提供）：染色体不稳定性（CIN）描述了染色体数量的破坏，如非整倍体或错聚（nCIN）以及染色体结构的破坏，如染色体易位或重排（sCIN）。两种形式的CIN都与癌症有关。通过CIN逐渐丧失基因组完整性导致杂合性丧失，并可导致或加重疾病。除恶性肿瘤外，染色体结构或数目的破坏也会导致人类发育缺陷。例如，21三体导致唐氏综合症可以通过染色体错分离（nCIN）发生，也可以通过罗伯逊易位（sCIN）发生染色体融合。因此，维持染色体结构和数目的机制在许多层面上对人类健康是不可或缺的。着丝粒对染色体的正常分离至关重要；因此，影响着丝粒功能的突变导致了数字CIN和非整倍体。然而，最近的研究表明，着丝粒也容易受到染色体重排、断裂和DNA损伤的影响，从而产生结构性CIN。这也会导致隔离缺陷。重要的是，阻止sCIN进入着丝粒的机制在很大程度上尚未被探索。本研究研究了与着丝粒相关的结构不稳定性，特别是通常组装成异染色质的高度重复的外重复元件。它采用了一种易于处理的遗传系统：裂变酵母S. pombe，它被公认为高等细胞中着丝粒功能的模型。该项目使用遗传学、分子生物学和新的细胞生物学方法，包括活细胞、单细胞分析和超分辨率显微镜，来研究保护着丝粒并保持其完整性的机制。第一个Aim建立在广泛的初步数据基础上，以检查复制缺陷和异染色质缺失的结合如何增加重排的频率。第二个目的是询问与着丝粒异染色质相关的蛋白质如何通过着丝粒维持或对常染色质作用的影响在DNA损伤反应中起作用。第三个目的是利用一种新的四染色体裂变酵母菌株来研究罗伯逊易位。这是这种常见染色体重排的第一个酵母模型，并将研究着丝粒融合的证据以及减数分裂和有丝分裂中分离和易位的机制。总之，这些方法将建立一个与人类健康直接相关的着丝粒中sCIN的强大机制模型。

DESCRIPTION (provided by applicant): Chromosome instability (CIN) describes both disruptions of chromosome number such as aneuploidy or misegregation (nCIN) as well as disruptions of chromosome structure, such as chromosome translocations or rearrangements (sCIN). Both forms of CIN are associated with cancer. Progressive loss of genome integrity via CIN contributes to loss of heterozygosity, and can cause or exacerbate the disease. In addition to malignancy, disruptions in chromosome structure or number also contribute to defects in human development. For example, trisomy 21 causing Down syndrome can occur via chromosome mis-segregation (nCIN), but also from chromosome fusion via Robertsonian translocation(sCIN). Thus, the mechanisms that maintain chromosome structure and number are integral to human health at many levels. The centromere is essential for the normal segregation of chromosomes; thus, mutations affecting centromere function contribute to numerical CIN and aneuploidy. However, recent work suggests that the centromere is also vulnerable to chromosome rearrangements, fragmentation, and DNA damage, creating structural CIN. This also contributes to segregation defects. Importantly, the mechanisms that prevent sCIN in the centromere are largely unexplored. This proposal investigates structural instability associated with the centromere, specifically at the highly repetitive outer-repeat elements that are usually assembled into heterochromatin. It employs a tractable genetic system: the fission yeast S. pombe, which is well-established as a model for centromere function in higher cells. The project uses genetics, molecular biology, and novel cell biology methods, including live, single cell analysis and super-resolution microscopy, to examine the mechanisms that protect the centromere and preserve its integrity. The first Aim builds on extensive preliminary data to examine how the combination of replication defects and absence of heterochromatin increase the frequency of rearrangements. The second aim asks how proteins that are linked to centromere heterochromatin function in the DNA damage response either through centromere maintenance or effects on the euchromatin effects. The third Aim uses a novel four-chromosome fission yeast strain to study Robertsonian translocation. This is the first yeast model for this common chromosome rearrangement, and will examine evidence for centromere fusion and mechanisms for segregation and translocation in meiosis and mitosis. Together, these approaches will develop a strong mechanistic model for sCIN in the centromere with direct relevance to human health.