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

摘要 表观遗传转录调控是无数发育过程所必需的，也是一种 人类疾病的重要贡献者。X染色体失活提供了一种实验上容易处理的 表观遗传解剖系统。X-失活导致有丝分裂可遗传 雌性哺乳动物中一条X染色体的转录失活，从而使X连锁基因相等 男性和女性之间的剂量。X失活需要表达的Xist长的非编码RNA 仅来自不活跃的X染色体。目前的模型假设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是如何 选择性地诱导雌性和完全不诱导雄性，以及它是如何触发沉默的，仍然没有解决 问题。这项建议的目的是解决Xist表达和X连锁基因沉默是如何 在X停用期间触发。基于我们已发表的工作和初步数据，我们的中心假设是 逃脱X失活的基因作为剂量敏感因素分别诱导Xist和Xist， 选择性地增强女性X连锁基因沉默。X-失活逃逸表示来自X- XX雌性的染色体，包括来自其他未激活的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- 测序流水线，以汇编小鼠上皮细胞干细胞中逃逸基因的清单，该干细胞含有 非活动-X。然后，我们对经过验证的逃逸对象进行了优先排序，这些逃逸对象被预测为进化保守的，并且 作为转录/染色质调节器，在诱导X失活的过程中发挥作用。在这项提案中，我们 系统地检测其中两个逃逸基因在触发过程中的剂量依赖和生化活性 使用无偏见和集成的高通量方法进行Xist表达和X连锁基因沉默。这个 这一结果有望揭示X染色体长度上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。执行X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， 了解表征X失活的一系列表观遗传事件为我们提供了一扇了解 确定广泛建立表观遗传表达模式的共同因素和机制。

Abstract 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.