Control of Microtubule and Genome Integrity by the OBSL1-CUL7-CUL9-p53 Pathway

通过 OBSL1-CUL7-CUL9-p53 途径控制微管和基因组完整性

基本信息

批准号：
9421591
负责人：
YUE XIONG
金额：
$ 7.03万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-07-17 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9421591
关键词：
Alpha Cell Antimitotic Agents Apoptosis Area Back Binding Biochemical CCL18 gene CDKN2A gene CUL9 gene Cell Cycle Regulation Cell Death Cell Proliferation Cells Chromatids Chromosomes Collection Cyclin-Dependent Kinase Inhibitor Gene Cytokinesis Cytoplasm Cytoskeletal Proteins Defect Development Disease Ensure Eukaryota Failure Family Funding Generations Genes Genetic Genetic Transcription Goals Growth Human Investigation Knock-in Knock-in Mouse Knockout Mice Lead Link Microtubules Mitosis Mitotic Mitotic spindle Molecular Mus Mutant Strains Mice Mutate Nuclear Paper Partner in relationship Pathway interactions Point Mutation Polyploidy Positioning Attribute Process Proteins Publishing Regulation Research Research Peer Review Role Series Sister Chromatid Stress Structure Syndrome TP53 gene Testing Tetraploidy Therapeutic Agents Transcriptional Regulation Tumor Suppression Tumor Suppressor Proteins Vertebrates base cancer therapy carcinogenesis daughter cell embryonic stem cell experimental study gain of function genetic analysis genome integrity in vivo mouse model mutant novel overexpression postnatal prevent tool tumor tumorigenesis ubiquitin-protein ligase vector

项目摘要

DESCRIPTION (provided by applicant): During mitosis, chromosomes are accurately segregated to daughter cells by a microtubule-based structure called the mitotic spindle. It has long been recognized that undetected microtubule damage causes mitotic defects and genetic instability which renders cells susceptible to tumorigenesis. A well-established pathway, the spindle assembly checkpoint pathway, ensures equal separation of sister chromatids and is conserved in all eukaryotes. We recently uncovered evidence for a new pathway, unique to vertebrates, which can also sense microtubule stress. Combined genetic, cellular and biochemical analyses have led to the finding that CUL7 and CUL9, two cytoplasmically localized E3 ubiquitin ligases which can bind to p53, control mitosis and cytokinesis by sensing microtubule stress. Deletion of Cul9 in mice resulted in wide spread polyploidy, spontaneous tumor development, and rendered mice susceptible to carcinogenesis indicating that Cul9 is a tumor suppressor. Conversely, gain of function in CUL9 promoted a p53-dependent apoptosis. We further found that depletion of CUL7 or its binding partner OBSL1 caused severe microtubule damage, abnormal chromatid alignment, and defects in cytokinesis and widespread mitotic cell death, all of which can be rescued by simultaneous depletion of CUL9. These extensive preliminary results led us to propose that there exists in vertebrates an OBSL1-CUL7-CUL9-p53 pathway that senses microtubule stress during mitosis and is functionally separate from the well-established spindle-assembly checkpoint. We propose here a series of rigorous genetic and biochemical experiments to examine the role of this novel OBSL1-CUL7-CUL9-p53 pathway in maintaining genome integrity. In the first Aim, we will generate Cul7-Cul9 and Obsl1-Cul9 double knockout mice to genetically test the functional relationship of CUL7 and CUL9 and between OBSL1 and CUL9 in vivo. In the second Aim, we will genetically test the contribution of p53 to pathway by generating two separate knock-in mice, each with a point mutation in the p53 binding domain of CUL7 and CUL9. Finally, we will biochemically test how ubiquitylation contributes to the mechanism of the OBSL1-CUL7-CUL9-p53 pathway.

描述（由申请人提供）：在有丝分裂期间，染色体通过称为有丝分裂纺锤体的微管结构准确地分离到子细胞。人们早就认识到，未被检测到的微管损伤会导致有丝分裂缺陷和遗传不稳定，从而使细胞容易发生肿瘤。纺锤体组装检查点途径是一条完善的途径，可确保姐妹染色单体的平等分离，并且在所有真核生物中都是保守的。我们最近发现了脊椎动物特有的新途径的证据，该途径也可以感知微管压力。结合遗传、细胞和生化分析，我们发现 CUL7 和 CUL9 这两种细胞质定位的 E3 泛素连接酶可以与 p53 结合，通过感知微管应激来控制有丝分裂和胞质分裂。小鼠中 Cul9 的缺失导致广泛传播的多倍体、自发性肿瘤发展，并使小鼠易患癌，表明 Cul9 是一种肿瘤抑制因子。相反，CUL9 的功能获得促进了 p53 依赖性细胞凋亡。我们进一步发现，CUL7 或其结合伴侣 OBSL1 的缺失会导致严重的微管损伤、染色单体排列异常、胞质分裂缺陷和广泛的有丝分裂细胞死亡，所有这些都可以通过同时缺失 CUL9 来挽救。这些广泛的初步结果使我们提出，脊椎动物中存在 OBSL1-CUL7-CUL9-p53 通路，该通路在有丝分裂期间感知微管应力，并且在功能上与成熟的纺锤体组装检查点分开。我们在这里提出了一系列严格的遗传和生化实验来检验这种新的 OBSL1-CUL7-CUL9-p53 通路在维持基因组完整性中的作用。在第一个目标中，我们将生成Cul7-Cul9和Obsl1-Cul9双敲除小鼠，以在体内对CUL7和CUL9以及OBSL1和CUL9之间的功能关系进行基因测试。在第二个目标中，我们将通过生成两只单独的敲入小鼠来对 p53 对通路的贡献进行基因测试，每只小鼠的 CUL7 和 CUL9 的 p53 结合域都有一个点突变。最后，我们将通过生化测试泛素化如何促进 OBSL1-CUL7-CUL9-p53 通路的机制。