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）：在有丝分裂过程中，染色体通过称为有丝分裂纺锤体的基于微管的结构将染色体准确地隔离到子细胞上。长期以来，人们已经认识到，未检测到的微管损伤会导致有丝分裂缺陷和遗传不稳定性，从而使细胞容易受到肿瘤发生的影响。一条公认的途径，即主轴组件检查点途径，可确保姐妹染色单体相等的分离，并在所有真核生物中保守。最近，我们发现了脊椎动物独有的新途径的证据，该途径也可以感觉到微管应激。结合的遗传，细胞和生化分析导致发现CUL7和CUL9，两个细胞质局部局部的E3泛素连接酶可以通过感测微管应激与p53结合，对照有丝分裂和细胞因子。小鼠中CUL9的缺失导致广泛的多倍体，自发的肿瘤发育，并使小鼠易受致癌作用，表明CUL9是肿瘤抑制器。相反，CUL9的功能增益促进了p53依赖性凋亡。我们进一步发现，CUL7或其结合伴侣obsl1的耗竭会导致严重的微管损伤，异常的染色体比对，以及细胞因子和广泛的有丝分裂细胞死亡的缺陷，所有这些死亡都可以通过同时耗尽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一种obsl1-Cul9-Cul9-p53途径，该途径在有丝分裂过程中感受到微管应激，并且在功能上与已建立的纺锤体组装检查点具有分离。我们在这里提出了一系列严格的遗传和生化实验，以检查这种新型的Castl1-Cul7-Cul9-P53途径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双基因敲除小鼠，以遗传测试CUL7和CUL9的功能关系以及Vivo中的obsl1和Cul9之间。在第二个目标中，我们将通过产生两只独立的敲入小鼠来从基因测试p53对途径的贡献，每只小鼠在CUL7和CUL9的p53结合结构域中都有一个点突变。最后，我们将在生化上测试泛素化如何有助于obsl1-cul7-cul9-p53途径的机理。