Identify and characterize genes involved in X-chromosome inactivation

描述(申请人提供)：X染色体失活(XCI)，对雌性小鼠发育至关重要，是确保XX雌性和XY雄性之间X连锁基因的等量基因剂量的分子机制。XCI是哺乳动物细胞中长期的、染色体范围的基因沉默的最戏剧性的例子。XCI在早期胚胎发育中以X失活特异性转录本(Xist)非编码RNA的表达启动，它包裹着顺式染色体中失活的X染色体(Xi)，促进沉默因子向整个xi的传播。一旦建立，Xi在随后的所有体细胞分裂过程中保持沉默。细胞遗传学研究表明，一些表观遗传标记在XI上富含，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，维持xi沉默的蛋白质因素在很大程度上是未知的。通过全基因组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，32个基因被证实与沉默位于xi上的内源基因有关。基因本体论分析表明，这些基因大多在RNA加工、细胞周期调控、基因转录和染色质等方面发挥功能。这些结果表明，Xi沉默是通过不同的机制维持的。为了验证这一假说，我们将首先验证剩余的48个基因中哪些参与了xi沉默，并确定每个已验证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Lrwd1，两者都在屏幕上得到验证，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，ORC还在表观遗传沉默中发挥作用。然而，之前还不知道ORC和Lrwd1在xi沉默中起到了什么作用。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Orc2和Lrwd1在Xi沉默中的作用机制，并为未来维持xi沉默的研究奠定坚实的基础。

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.