Epigenetic control of the human X chromosome

项目摘要 这项提案的总体目标是研究X染色体剂量补偿的调节， 人类胚胎干细胞（hESC）中的非编码（lnc）RNA，从根本上推进对 人类早期发育、多能性状态的调节以及lncRNA的基因调节机制。 X染色体剂量补偿是平衡X连锁基因表达的一个重要过程 女性和男性，并在发育早期启动。在哺乳动物中，它由X染色体介导 失活（XCI），女性两条X染色体之一上的基因转录沉默。 已经在小鼠中进行了XCI的机制研究，并确定了在小鼠中编码的lncRNA Xist。 X染色体，沿着X染色体传播介导XCI。虽然XCI发生在小鼠和 人类植入后发育，植入前胚胎X染色体剂量的调控 在这两个物种中进化的方式不同在植入前发育中，小鼠胚胎经历了一个 XCI的印记形式，而人类胚胎缺乏XCI印记，而是通过 抑制两条X染色体的转录X染色体阻尼（XCD）与XCI不同，因为它 发生在两个X染色体上，与一个X染色体的完全转录沉默相反。额外 人类和小鼠之间的显著差异包括： XIST在活性（抑制）X染色体上的表达以及灵长类和多能性特异性 人类植入前胚胎中来自X的lncRNA XACT。因此，人类的X连锁基因剂量是 首先由XCD调节，植入后由XCI调节，值得注意的是，XIST表达与XCD和XCI解偶联。 当XCD发生时进行静默。对XCD的机械性理解，XIST在预处理中缺乏沉默， 植入胚胎，XIST向介导XCI的转变，以及lncRNA XACT的作用缺乏， 尚不清楚XIST是否在人类植入前细胞或XCD中有任何作用。我们发现幼稚的hESC 在体外概括了人类X染色体剂量补偿的许多独特特征，包括XCD， XIST在活性X染色体上的表达、XACT表达和分化时XCI的起始。在这里， 我们将利用幼稚的hESC研究XIST表达如何不诱导早期沉默， 发展，后来获得沉默的能力，揭示人类XCD和XCI的调节机制， 如果XACT控制XIST功能。具体目标如下：1）明确XIST的功能 在幼稚hESC中，其对XCD的需求以及XCD的机制。2)我们将定义染色质靶点， XIST的蛋白质相互作用物，以了解其在幼稚和引发的hESC中的差异沉默能力。3)我们将 表征lncRNA XACT的功能和定位。了解人类X- 染色体剂量补偿将使我们能够推进我们对表观遗传特征的理解， 人类发展和揭示关键的表观遗传过程是如何在进化中发生变化的。

PROJECT SUMMARY The overall goal of this proposal is to study the regulation of X-chromosome dosage compensation by long- noncoding (lnc) RNAs in human embryonic stem cells (hESCs) to fundamentally advance the understanding of early human development, the regulation of pluripotency states, and mechanisms of gene regulation by lncRNAs. X-chromosome dosage compensation is an essential process that equalizes X-linked gene expression between females and males and is initiated early in development. In mammals, it is mediated by X-chromosome inactivation (XCI), the transcriptional silencing of genes on one of the two X-chromosomes in females. Mechanistic studies of XCI have been carried out in mice and established that the lncRNA Xist, encoded on the X chromosome, spreads along the X chromosome to mediate XCI. Although XCI occurs in both mouse and human post-implantation development, the regulation of X-chromosome dosage in pre-implantation embryos has evolved differently between the two species. In pre-implantation development, mouse embryos undergo an imprinted form of XCI, while human embryos lack imprinted XCI and instead regulate gene expression by dampening transcription on both X-chromosomes. X-chromosome dampening (XCD) is different from XCI as it occurs on both X chromosomes in contrast to the complete transcriptional silencing of one X. Additional remarkable differences between human and mouse include the unprecedented expression and accumulation of XIST on the active (dampened) X-chromosomes and the expression of the primate- and pluripotency-specific lncRNA XACT from the X in human pre-implantation embryos. Thus, X-linked gene dosage in humans is regulated first by XCD and upon implantation by XCI, and remarkably, XIST expression is uncoupled from silencing when XCD takes place. A mechanistic understanding of XCD, lack of silencing by XIST in pre- implantation embryos, XIST's transition to mediating XCI, and the role of the lncRNA XACT is lacking, and it is not known if XIST has any role in human pre-implantation cells or XCD. We discovered that naïve hESCs recapitulate in vitro many of the unique features of human X-chromosome dosage compensation, including XCD, expression of XIST on active X-chromosomes, XACT expression, and initiation of XCI upon differentiation. Here, we will take advantage of naïve hESCs to investigate how XIST expression does not induce silencing early in development and later acquires the ability to silence, to reveal regulatory mechanisms of human XCD and XCI, and if XACT controls XIST functions. We have the following Specific Aims: 1) We will define the function of XIST in naïve hESCs, its requirement for XCD, and mechanisms of XCD. 2) We will define chromatin targets and protein interactors of XIST to understand its differential silencing ability in naïve and primed hESCs. 3) We will characterize the function and localization of the lncRNA XACT. Understanding the mechanisms of human X- chromosome dosage compensation will allow us to advance our understanding of epigenetic features unique to human development and reveal how key epigenetic processes are changing in evolution.