X-chromosome Inactivation Catalyzed by Genes That Escape X-inactivation

母药R 01摘要（R 01 GM 124571） 表观遗传转录调控是无数发育过程所必需的， 人类疾病的重要贡献者。X染色体失活提供了一种实验上易于处理的 表观遗传的解剖系统。X染色体失活导致有丝分裂遗传的 雌性哺乳动物中一条X染色体的转录失活，从而使X连锁基因 雄性和雌性之间的剂量。X失活需要表达的Xist长非编码RNA 只有不活跃的X染色体目前的模型证实，Xist RNA的诱导和X- 顺式染色体触发一系列表观遗传事件，最终导致X失活。值得注意的是，Xist如何 在雌性中选择性地诱导而在雄性中完全不诱导，以及它如何触发沉默仍然没有解决 问题.该提案的目的是解决Xist表达和X连锁基因沉默是如何在 在X射线灭活过程中触发基于我们已发表的工作和初步数据，我们的中心假设是： 逃避X失活的基因作为诱导Xist的剂量敏感因子发挥作用， 在女性中选择性增强X连锁基因沉默。X-失活逃逸分子表达于X- XX女性中的染色体，包括其他失活的X染色体;因此，它们的表达 女性比XY男性更高。我们开发并使用了一种定制的等位基因特异性RNA 测序管道，以编制小鼠外胚层干细胞中逃逸基因的列表，这些细胞含有一个 非活性X。然后，我们优先考虑那些被预测为进化上保守的， 作为转录/染色质调节因子在诱导X失活中发挥作用。在本提案中，我们 系统地测试这些逃逸基因中的两个在触发中的剂量依赖性和生物化学活性， Xist表达和X连锁基因沉默使用公正和整合的高通量方法。的 这些结果有望告诉我们，X染色体上沿着长度的基因是如何沉默的，以及为什么女性 经历X-失活，而雄性则不会。X染色体失活的表观遗传因素和机制 已知与调节胚胎发育和疾病进展的基因重叠。因此，在本发明中， 了解表征X失活的表观遗传事件的级联提供了一个窗口， 确定广泛建立表观遗传表达模式的共同因素和机制。

Abstract of Parent R01 (R01GM124571) Epigenetic transcriptional regulation is required for a myriad of developmental processes as well as being a significant contributor to human diseases. X-chromosome inactivation provides an experimentally tractable system for the dissection of epigenetic inheritance. X-inactivation results in the mitotically-heritable transcriptional inactivation of one X-chromosome in female mammals, thereby equalizing X-linked gene dosage between males and females. X-inactivation requires the Xist long non-coding RNA that is expressed only from the inactive X-chromosome. Current models posit that Xist RNA induction and coating of the X- chromosome in cis triggers a series of epigenetic events that culminates in X-inactivation. Notably, how Xist is selectively induced in females and not at all in males and how it triggers silencing are still unresolved questions. The objective of this proposal is to address how Xist expression and X-linked gene silencing are triggered during X-inactivation. Our central hypothesis, based on our published work and preliminary data, is that genes that escape X-inactivation function as dose-sensitive factors that induce Xist and, separately, potentiate X-linked gene silencing selectively in females. X-inactivation escapees are expressed from both X- chromosomes in XX females, including from the otherwise inactivated X-chromosome; hence, their expression is higher in females compared to XY males. We developed and employed a custom allele-specific RNA- sequencing pipeline to compile a list of the escape genes in mouse epiblast stem cells, which harbor an inactive-X. We then prioritized validated escapees that are predicted to be evolutionarily conserved and which function as transcriptional/chromatin regulators for a role in inducing X-inactivation. In this proposal, we systematically test the dose-dependent and biochemical activities of two of these escape genes in triggering Xist expression and X-linked gene silencing using unbiased and integrated high-throughput approaches. The results are expected to inform how genes along the length of the X-chromosome are silenced and why females undergo X-inactivation and males do not. The epigenetic factors and mechanisms that execute X-inactivation are known to overlap with those that regulate embryonic development and disease progression. Thus, understanding the cascade of epigenetic events that characterizes X-inactivation offers a window into identifying the common factors and mechanisms that establish epigenetic expression patterns broadly.