Identify and characterize genes involved in X-chromosome inactivation

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、全染色体基因沉默的最引人注目的例子。XCI在早期胚胎发生时启动x -失活特异性转录物（Xist）非编码RNA的表达，该RNA以顺式方式包裹在失活的x染色体（Xi）上，并促进沉默因子向整个Xi的扩散。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，Xi上富集了多种表观遗传标记，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，有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、基因转录和染色质等方面发挥作用。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个基因中哪些参与Xi沉默，并确定每个已验证候选基因的耗尽如何影响Xi沉默的已知机制。其次，我们将确定在屏幕中验证的Orc2和Lrwd1如何影响Xi沉默的维持。Orc2是起源识别复合物（Origin-Recognition- Complex， ORC）的一个亚基，Lrwd1是ORC结合蛋白。除了DNA复制外，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。我们期望这些研究能够深入了解Xi的表观遗传沉默是如何维持的，有助于揭示Orc2和Lrwd1在Xi沉默中的作用机制，并为进一步研究Xi沉默的维持奠定坚实的基础。

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.