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

肿瘤抑制因子 BRCA1 在有丝分裂中的基因组稳定功能分析。

• 批准号: 380282559
• 负责人: Professor Dr. Holger Bastians
• 金额: --
• 依托单位: Institute for Molecular Oncology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/380282559?language=en
• 关键词: Analyses; genome; stabilising; function; tumor

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.

人类癌细胞的特征在于分别由全染色体不稳定性（W-CIN）和结构染色体不稳定性（S-CIN）引起的染色体数目和结构畸变。BRCA 1是一种重要的肿瘤抑制因子，已知参与DNA复制和DNA修复的调节。因此，癌细胞中BRCA 1的缺失导致S-CIN。我们最近的工作揭示了BRCA 1和几种BRCA 1调节蛋白在抑制W-CIN所需的有丝分裂中的另一个关键作用。事实上，BRCA 1有丝分裂功能的丧失导致微管加末端组装率异常增加，从而导致整个染色体在分裂后期的错误分离。BRCA 1的有丝分裂和间期功能是否在功能上耦合，以及BRCA 1如何在有丝分裂过程中发挥作用是尚未解决的问题，这些问题将在拟议的研究项目的时间框架内得到解决。在工作计划的第一部分，我们将调查BRCA 1的有丝分裂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BRCA 1-degron系统，该系统允许在间期和有丝分裂期间的特定时间点快速消耗内源性BRCA 1。通过这种方式，我们的目标是检查BRCA 1在确定的细胞周期阶段急性丧失后的不同功能，并探索它们对抑制S-CIN和W-CIN的相对贡献。这些研究的目的是阐明BRCA 1缺失后S-CIN和W-CIN是否以及如何发生联系。由于我们的工作表明BRCA 1的有丝分裂功能涉及其泛素连接酶活性，因此识别与BRCA 1泛素连接酶生理相关的泛素受体蛋白对于理解BRCA 1在有丝分裂过程中如何发挥作用至关重要。基于我们的初步工作，以确定泛素化的蛋白质，我们将使用质谱法来确定蛋白质的泛素化的BRCA 1泛素连接酶在有丝分裂。这些候选蛋白将在体外和体内被验证为BRCA 1靶点，我们将研究泛素修饰对有丝分裂微管组装和有丝分裂染色体分离的调控作用。因此，拟议的项目及其两个互补的工作包旨在揭示BRCA 1如何有助于基因组稳定，特别是在有丝分裂期间。