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

• 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
• 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

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染色体失活（XCI）是雌性小鼠发育所必需的，是确保XX雌性和XY雄性之间X连锁基因的基因剂量相等的分子机制。XCI是哺乳动物细胞中长期染色体范围基因沉默的最引人注目的例子。XCI在早期胚胎发生中起始，表达X失活特异性转录物（Xist）非编码RNA，其顺式包覆失活的X染色体（Xi），并促进沉默因子扩散到整个Xi。一旦建立，Xi在所有随后的体细胞分裂中保持沉默。细胞遗传学研究表明，一些表观遗传标记富集在Xi上，包括Xist非编码RNA和DNA超甲基化。此外，Xi还富含抑制性组蛋白标记，包括H3K27me3，H3K9me3和H4K20me。然而，参与维持Xi沉默的蛋白质因子在很大程度上是未知的。通过进行全基因组范围的shRNA筛选，我们确定了94个可能参与维持Xi沉默的基因。在对94个候选基因中的46个进行验证后，32个基因被证实参与了位于Xi上的内源基因的沉默。基因本体分析表明，大多数基因在RNA加工、细胞周期调控、基因转录和染色质中发挥作用。这些结果表明，Xi沉默是通过不同的机制维持的。为了验证这一假设，我们将首先验证其余48个基因中的哪些参与Xi沉默，并确定每个验证的候选基因的缺失如何影响Xi沉默的已知机制。其次，我们将确定Orc2和Lrwd 1（两者都在筛选中得到验证）如何影响Xi沉默的维持。Orc2是起源识别复合物（ORC）的亚基，Lrwd 1是ORC结合蛋白。除了DNA复制，ORC在表观遗传沉默中也有作用。然而，以前并不知道ORC和Lrwd1在Xi沉默中有任何作用。我们期望这些研究将有助于深入了解Xi的表观遗传沉默是如何维持的，有助于阐明Orc 2和Lrwd 1在Xi沉默中的作用机制，并为未来Xi沉默维持的研究奠定坚实的基础。 公共卫生相关性：X染色体失活（XCI）是表观遗传沉默最引人注目的例子，导致XX雌性哺乳动物两条X染色体之一（Xi）上大多数基因的表达受到抑制。XCI在早期胚胎发生期间建立，然后通过随后的细胞分裂维持。已知多种沉默机制，包括非编码RNA、组蛋白甲基化（组蛋白H3赖氨酸9和27、组蛋白H4赖氨酸20的甲基化）和DNA甲基化协同作用以维持沉默。这些沉默机制也被癌细胞用来沉默肿瘤抑制基因（TSG）。参与维持Xi沉默的蛋白质因子在很大程度上是未知的。在这项提案中，我们计划识别和表征参与维持Xi沉默的基因。这些研究不仅将增加我们对Xi沉默是如何维持的理解，这是一个知之甚少的关键细胞过程，而且还将阐明肿瘤抑制基因的表观遗传沉默是如何维持的。