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: 8846616
• 负责人: Zhiguo Zhang
• 金额: $ 31.27万
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8846616
• 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; 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; histone methylation; 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.

描述（由申请人提供）：X染色体失活（XCI）对于雌性小鼠发育至关重要，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、全染色体基因沉默的最引人注目的例子。 XCI 在早期胚胎发生中起始于 X 失活特异性转录物 (Xist) 非编码 RNA 的表达，该 RNA 以顺式方式包裹失活的 X 染色体 (Xi)，并促进沉默因子向整个 Xi 的扩散。一旦建立，Xi 在随后的所有体细胞分裂中都保持沉默。细胞遗传学研究表明 Xi 上富集了多种表观遗传标记，包括 Xist 非编码 RNA 和 DNA 高甲基化。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尚不清楚。通过进行全基因组 shRNA 筛选，我们鉴定了 94 个可能参与 Xi 沉默维持的基因。对 94 个候选基因中的 46 个进行验证后，有 32 个基因被证实参与沉默位于 Xi 上的内源基因。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。为了检验这一假设，我们将首先验证其余 48 个基因中的哪些基因参与 Xi 沉默，并确定每个经过验证的候选基因的耗尽如何影响已知的 Xi 沉默机制。其次，我们将确定 Orc2 和 Lrwd1（两者均在筛选中得到验证）如何影响 Xi 沉默的维持。 Orc2 是起源识别复合体 (ORC) 的一个亚基，Lrwd1 是 ORC 结合蛋白。除了 DNA 复制之外，ORC 在表观遗传沉默中也发挥着作用。然而，此前并不知道 ORC 和 Lrwd1 在 Xi 沉默中发挥任何作用。我们希望这些研究能够深入了解 Xi 的表观遗传沉默是如何维持的，有助于阐明 Orc2 和 Lrwd1 在 Xi 沉默中发挥作用的机制，并为未来维持 Xi 沉默的研究奠定坚实的基础。