Identify and characterize genes involved in X-chromosome inactivation

鉴定和表征参与 X 染色体失活的基因

基本信息

批准号：
8216410
负责人：
Zhiguo Zhang
金额：
$ 31.48万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-06-11 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8216410
关键词：
Adopted Affect Binding Binding Proteins Candidate Disease Gene Cell Cycle Regulation Cell Line Cell division Cell physiology Chromatin Chromatin Structure Chromosomes Cytogenetics DNA DNA Methylation DNA Methyltransferase Inhibitor DNA biosynthesis DNA replication origin Development Embryo Embryonic Development Ensure Epigenetic Process Female Fibroblasts Foundations Functional RNA Future Gene Dosage Gene Silencing Genes Genetic Transcription Goals Green Fluorescent Proteins Heterochromatin Histone Deacetylase Histone H3 Histone H4 Histones Hypermethylation Inherited Knowledge Light Link Lysine Maintenance Mammalian Cell Mammals Mediating Methylation Molecular Mus Normal Cell Ontology Process Proteins RNA Processing Recruitment Activity Repression Role S Phase Solid Somatic Cell Structure Testing Transcript Translating Tumor Suppressor Genes Untranslated RNA Validation Work X Chromosome X Inactivation cancer cell gene function genome-wide heterochromatin-specific nonhistone chromosomal protein HP-1 insight male mouse development origin recognition complex protein function small hairpin RNA

项目摘要

DESCRIPTION (provided by applicant): X-chromosome inactivation (XCI), essential for female mouse development, is the molecular mechanism that ensures equivalent gene dosage of the X-linked genes between XX females and XY males. XCI is the most dramatic example of long-term, chromosome-wide gene silencing in mammalian cells. XCI initiates in early embryogenesis with the expression of the X-inactive specific transcript (Xist) noncoding RNA, which coats the inactivated X-chromosome (Xi) in cis and facilitates the spreading of silencing factors to the entire Xi. Once established, Xi remains silenced through all subsequent somatic cell divisions. Cytogenetic studies indicate that several epigenetic marks are enriched on Xi, including the Xist non-coding RNA and DNA hypermethylation. Moreover, Xi is also enriched with repressive histone marks including H3K27me3, H3K9me3 and H4K20me. However, the protein factors involved in the maintenance of Xi silencing are largely unknown. By performing a genome-wide shRNA screen, we identified 94 genes that are potentially involved in the maintenance of Xi silencing. Following validation of 46 of the 94 candidate genes, 32 genes were verified to be involved in silencing endogenous genes located on Xi. Gene ontology analysis reveals that most of the genes function in RNA processing, cell cycle regulation, gene transcription and chromatin. These results indicate that Xi silencing is maintained via distinct mechanisms. To test this hypothesis, we will first validate which of the remaining 48 genes are involved in Xi silencing and determine how depletion of each verified candidate affects known mechanisms of Xi silencing. Second, we will determine how Orc2 and Lrwd1, both of which were validated in the screen, impact the maintenance of Xi silencing. Orc2 is a subunit of the Origin-Recognition- Complex (ORC), and Lrwd1 is an ORC binding protein. In addition to DNA replication, ORC has a role in epigenetic silencing. However, it was not previously known that ORC and Lrwd1 had any role in Xi silencing. We expect that these studies will provide insight into how epigenetic silencing of Xi is maintained, help delineate the mechanism by which Orc2 and Lrwd1 function in Xi silencing, as well as lay a solid foundation for future studies on the maintenance of Xi silencing. PUBLIC HEALTH RELEVANCE: X-chromosome inactivation (XCI), the most dramatic example of epigenetic silencing, results in repression of expression of most genes on one of the two X chromosomes (Xi) in XX female mammals. XCI is established during early embryogenesis and is then maintained through subsequent cell divisions. It is known that multiple silencing mechanisms, including non-coding RNA, histone methylation (methylation of histone H3 lysine 9 and 27, histone H4 lysine 20) and DNA methylation, work in synergy to maintain silencing. These silencing mechanisms are also adopted by cancer cells to silence tumor suppressor genes (TSG). The protein factors involved in the maintenance of Xi silencing are largely unknown. In this proposal, we plan to identify and characterize genes involved in maintaining Xi silencing. These studies will not only increase our understanding of how Xi silencing is maintained, a poorly understood and critical cellular process, but also shed light on how epigenetic silencing of tumor suppressor genes is maintained.

描述（由申请人提供）：X染色体灭活（XCI），对于雌性小鼠发育所必需的，是确保XX雌性和XY雄性之间X连锁基因的等效基因的分子机制。 XCI是哺乳动物细胞中长期，全染色体基因沉默的最戏剧性的例子。 XCI通过X-不活性转录本（XIST）非编码RNA的表达在早期的胚胎发生中启动，该RNA在顺式中co骨cosected cos cos的X染色体（XI）并促进了沉默因子对整个XI的传播。建立后，XI在随后的所有体细胞分裂中保持沉默。细胞遗传学研究表明，在XI上富含几种表观遗传标记，包括XIST非编码RNA和DNA高甲基化。此外，XI还具有抑制性组蛋白标记，包括H3K27me3，H3K9me3和H4K20Me。但是，维持XI沉默的蛋白质因子在很大程度上未知。通过执行全基因组shRNA筛选，我们确定了94个基因，这些基因可能参与XI沉默的维持。在验证了94个候选基因中的46个后，证实了32个基因与位于XI上的沉默内源基因有关。基因本体分析表明，大多数基因在RNA加工，细胞周期调节，基因转录和染色质中起作用。这些结果表明，XI沉默是通过不同的机制维持的。为了检验这一假设，我们将首先验证其余48个基因参与XI沉默，并确定每个经过验证的候选者的耗尽如何影响XI沉默的已知机制。其次，我们将确定ORC2和LRWD1如何在屏幕上验证，影响XI沉默的维护。 ORC2是原点识别复合物（ORC）的亚基，LRWD1是兽人结合蛋白。除了DNA复制外，ORC还在表观遗传沉默中发挥作用。但是，以前不知道兽人和LRWD1在XI沉默中起作用。我们预计这些研究将提供有关如何维持XI表观遗传沉默的洞察力，有助于描述ORC2和LRWD1在XI沉默中功能的机制，并为维持XI沉默的未来研究奠定了坚实的基础。公共卫生相关性：X染色体灭活（XCI），这是表观遗传沉默的最戏剧性的例子，导致在XX雌性哺乳动物中两个X染色体之一（XI）中的大多数基因表达抑制。 XCI是在早期胚胎发生期间建立的，然后通过随后的细胞分裂来维持。众所周知，包括非编码RNA，组蛋白甲基化（组蛋白H3赖氨酸9和27的甲基化，组蛋白H4赖氨酸20）和DNA甲基化的多种沉默机制和协同作用以保持沉默。这些沉默机制也被癌细胞采用以使肿瘤抑制基因（TSG）沉默。维持XI沉默的蛋白质因子在很大程度上未知。在此提案中，我们计划识别和表征与维持XI沉默有关的基因。这些研究不仅会增加我们对如何保持XI沉默的理解，这是一个鲜为人知的细胞过程，而且还阐明了如何保持肿瘤抑制基因的表观遗传沉默。