有丝分裂期姐妹染色体粘连疲劳的分子机制研究

Numerical, whole-chromosome aneuploidy is a common characteristics of tumors and has been proposed to drive tumor progression. Aneuploidy is often caused by errors in chromosome segregation during mitosis. Sister chromatid cohesion is an essential prerequisite for faithful chromosome segregation, defects in which lead to chromosome missegregation and the subsequent production of aneuploid daughter cells. Recent studies showed that cells arrested or delayed at metaphase undergo asynchronous and unscheduled separation of sister chromatids. This phenomenon is termed cohesion fatigue which constitutes a previously overlooked type of cohesion defects. Given its conceivable contribution to chromosome instability, a lot of important questions associated with cohesion fatigue remain to be addressed. Here, we aim to investigate how frequently cohesion fatigue occurs at least in certain types of cancer cells, as well as the causes and potential consequences of cohesion fatigue. Importantly, we found severe chromosome cohesion fatigue in our preliminary studies of targeted genome engineering by CRISPR-Cas9 in the gene encoding the serine/threonine kinase Haspin, which has been implicated in chromosome cohesion regulation with unknown mechanism. We hypothesize that Haspin plays a key role in preventing chromosome cohesion fatigue in a way independent of its kinase activity. Combining co-immunoaffinity purification and identification of Haspin-interacting proteins involved in cohesion regulation with functional studies of separation-of-function mutants, we aim to dissect the molecular mechanism by which Haspin functions directly in promoting chromosome cohesion. These studies will be carried out by emerging techniques including small molecule inhibitors, RNAi complementation, genome engineering with CRISPR-Cas9, super-resolution imaging and time-lapse microscopy of live cells. Upon successful conclusion of these studies, we will have revealed the mechanisms associated with chromosome cohesion fatigue, and have provided novel insights into the sophisticated regulation of mitosis which is also important for the development of new anti-mitotic cancer therapeutics.

染色体数目的非整倍性是癌细胞的重要特征和癌变的潜在诱因，主要由有丝分裂期染色体的异常分离所致。姐妹染色体之间的粘连是染色体精确分离的根本前提。染色体粘连疲劳，即有丝分裂中期滞留导致染色体非同步化错误分离的现象，是近几年报道的染色体粘连缺陷的新形式，受到同行关注。基于染色体粘连疲劳对基因组稳定性的威胁，一系列相关重要问题亟待解决。本课题拟利用小分子抑制剂、RNA干扰、CRISPR-Cas9基因组编辑、超高分辨率显微镜和活细胞实时成像等关键技术，调查染色体粘连疲劳的普遍性和严重程度，描述粘连疲劳造成染色体异常分离的特点，探讨粘连疲劳的成因与后果。基于Haspin基因突变严重加剧染色体粘连疲劳的预实验结果，我们推测并将验证Haspin以不依赖于其蛋白激酶活性的方式促进染色体粘连的分子机制。课题的顺利完成将揭示染色体粘连调控的新机制，丰富有丝分裂研究的基础理论，并为相关抗癌药物的研发提供新的依据。

有丝分裂是一个严密的过程，通过复杂而精细的调控来实现配对姐妹染色单体均等地分配至子细胞，任何细微的错误都可能导致染色体的错误分离以及非整倍体细胞的产生。黏连蛋白复合体（cohesin）维持姐妹染色单体配对，并促进染色体与纺锤体的正确连接。在有丝分裂的初期，染色体臂上的cohesin在其调节亚基Wapl的作用下被大量地去除，而着丝粒区的cohesin必须得以保留才能防止染色体的错误分离。研究发现，蛋白激酶Haspin通过位于氨基端非激酶域的一个保守基序PIM与cohesin的调节亚基Pds5B直接结合。而且，Haspin与Pds5B的结合能竞争性地阻碍Wapl以类似的方式与Pds5B相结合。因此，Haspin通过结合Pds5B拮抗了Wapl对着丝粒区cohesin的去除，保证了姐妹染色单体的正常粘连和正确分离，从而保护了染色体的稳定性。此外，Haspin的激酶域对于姐妹染色体粘连具有重要保护作用：一方面，Haspin的激酶域以较强的亲和力结合Wapl的PIM基序，从而竞争性地阻碍Pds5B结合Wapl的PIM；另一方面，Haspin磷酸化Wapl的PIM并直接削弱Wapl结合Pds5B的能力。进而发现，HP1α和HP1γ对于着丝粒区染色体的粘连发挥着相互冗余的保护作用，HP1的CSD与蛋白激酶Haspin氨基端的PxVxL基序相结合，促进Haspin在着丝粒区的定位，从而抑制Wapl介导的粘连去除。研究还提出了染色体乘客复合体保护着丝粒区姐妹染色体粘连的工作模型：一方面，通过不依赖于Aurora B激酶活性的方式，INCENP结合并招募HP1至着丝粒，促进Haspin在着丝粒区的富集并拮抗Wapl对cohesin的破坏作用；另一方面，Aurora B以激酶依赖的机制促进Mps1激酶在动粒区的定位，从而使得Sgo1富集于着丝粒区并拮抗Wapl。研究还发现，当细胞进入有丝分裂时，Bub1对组蛋白H2A的磷酸化修饰招募TOP2A至着丝粒，促进着丝粒区DNA连环的去除，进而保证了染色体的精确分离和基因组的稳定性。这一系列论文解析了姐妹染色体粘连的重要调控网络，阐明了多个关键环节的分子机制，揭示了染色体不稳定性的重要发生原因，拓展了相关研究领域的基础理论。研究成果多次被国外同行专文评述，产生了一定的国际影响。