Analyses of the genome stabilising function of the tumor suppressor BRCA1 in mitosis.

人类癌细胞的染色体数目异常和结构异常分别是由整体染色体不稳定(W-CIN)和结构染色体不稳定(S-CIN)引起的。BRCA1是一种重要的肿瘤抑制因子，参与DNA复制和DNA修复的调控。因此，癌细胞中BRCA1的缺失导致了S-CIN。我们最近的工作揭示了BRCA1和几个BRCA1调节蛋白在有丝分裂中的另一个关键作用，这是抑制W-CIN所必需的。事实上，BRCA1有丝分裂功能的丧失会导致微管和末端装配率异常增加，导致后期整个染色体的错误分离。BRCA1的有丝分裂功能和间期功能是否在功能上偶联，以及BRCA1在有丝分裂过程中如何发挥作用，这些都是尚未解决的问题，将在拟议的研究项目的时间框架内解决。在工作计划的第一部分，我们将研究BRCA1的有丝分裂功能和间期功能是否在功能上相连。为此，我们将通过CRISPR/Cas9技术在人体细胞中建立BRCA1-degron系统，允许在间期和有丝分裂的特定时间点快速耗尽内源性BRCA1。通过这种方式，我们的目的是检测BRCA1在特定的细胞周期时相急性丢失后的不同功能，并探讨它们在抑制S-CIN和W-CIN中的相对贡献。这些研究的目的是阐明在BRCA1缺失时，S-CIN和W-CIN是否以及如何相连。由于我们的工作表明BRCA1的有丝分裂功能涉及其泛素连接酶的活性，因此确定BRCA1泛素连接酶的生理相关泛素受体蛋白对于了解BRCA1在有丝分裂中如何发挥其作用至关重要。在我们鉴定泛素化蛋白的初步工作的基础上，我们将使用质谱仪来鉴定在有丝分裂过程中被BRCA1泛素连接酶泛素化的蛋白质。然后，这些候选蛋白将在体外和体内被验证为BRCA1靶标，我们将研究泛素修饰在调节有丝分裂微管组装和有丝分裂染色体准确分离方面的作用。因此，拟议的项目及其两个互补的工作包旨在揭示BRCA1如何对基因组稳定做出贡献，特别是在有丝分裂期间。

Human cancer cells are characterized by numerical and structural chromosome aberrations that are caused by whole chromosome instability (W-CIN) and structural chromosome instability (S-CIN), respectively. BRCA1 represents an important tumor suppressor known to be involved in the regulation of DNA replication and DNA repair. Consequently, loss of BRCA1 in cancer cells causes S-CIN. Our most recent work has revealed another key role for BRCA1 and for several BRCA1-regulating proteins in mitosis required for the suppression of W-CIN. In fact, loss of the mitotic function of BRCA1 results in abnormally increased microtubule plus end assembly rates causing whole chromosome missegregation during anaphase. Whether the mitotic and the interphase functions of BRCA1 are functionally coupled and how BRCA1 fulfills its role during mitosis are unresolved questions that will be addressed within the time frame of the proposed research project.In the first part of the working program we will investigate whether the mitotic and interphase functions of BRCA1 are functionally linked. For this, we will establish BRCA1-degron systems in human somatic cells by CRISPR/Cas9 technology that allow the rapid depletion of endogenous BRCA1 at specific time points during interphase and in mitosis. In this way we aim to examine the different functions of BRCA1 after acute loss of BRCA1 in defined cell cycle phases and to explore their relative contributions to the suppression of S-CIN and W-CIN. The goal of these investigations is to elucidate whether and how S-CIN and W-CIN are linked upon loss of BRCA1.Since our work demonstrated that the mitotic function of BRCA1 involves its ubiquitin ligase activity it is pivotal to identify physiologically relevant ubiquitin acceptor proteins for the BRCA1 ubiquitin ligase in order to understand how BRCA1 fulfills its role during mitosis. Based on our preliminary work to identify ubiquitinated proteins we will use mass spectrometry to identify proteins that are ubiquitinated by the BRCA1 ubiquitin ligase during mitosis. Those candidate proteins will then be validated as BRCA1 targets in vitro and in vivo and we will investigate the role of the ubiquitin modification for the regulation of mitotic microtubule assembly and for accurate mitotic chromosome segregation. Thus, the proposed project with its two complementary working packages aims to unravel how BRCA1 contributes to genome stabilization in particular during mitosis.