Epigenetic control of the human X chromosome

项目总结 这一建议的总体目标是研究长染色体对X染色体剂量补偿的调节。 人类胚胎干细胞(HESCs)中的非编码(LNC)RNA从根本上促进了对 人类早期发育，多能状态的调节，以及通过lncRNAs进行基因调节的机制。 X染色体剂量补偿是平衡X连锁基因表达的重要过程 雌性和雄性，在发育早期就开始了。在哺乳动物中，它是由X染色体介导的 失活(XCI)，女性两条X染色体中的一条上的基因转录沉默。 已经在小鼠身上进行了XCI的机制研究，并证实编码在XCI上的IncRNA Xist X染色体，沿X染色体传播，介导XCI。尽管XCI同时发生在小鼠和 人类着床后发育，着床前胚胎中X染色体剂量的调节 在这两个物种之间的进化是不同的。在植入前的发育中，小鼠胚胎经历了 印记形式的XCI，而人类胚胎缺乏印记XCI，而是通过以下方式调节基因表达 抑制两条X染色体上的转录。X染色体抑制(XCD)不同于XCI，因为它 发生在两条X染色体上，与一条X染色体的转录完全沉默形成对比。 人和老鼠的显著区别包括前所未有的表达和积累 X染色体活性(抑制)与灵长类和多能性特异性基因的表达 人类植入前胚胎中来自X的IncRNA XACT。因此，人类中X连锁基因的剂量是 首先受到XCD的调节，然后受到XCI的调节，值得注意的是，XIST的表达是从 当XCD发生时静音。对XCD的机械理解，XIST在前几年缺乏沉默 植入胚胎，XIST向介导XCI的转变，而lncRNA XACT的作用是缺乏的，它是 尚不清楚XIST在人类植入前细胞或XCD中是否有任何作用。我们发现幼稚的人类胚胎干细胞 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的表达，以及分化时XCI的启动。这里, 我们将利用幼稚的hESCs来研究XIST表达如何不会在早期诱导沉默 发育和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函数。我们有以下具体目标：1)定义XIST的功能 在幼稚的HESCs中，它对XCD的要求，以及XCD的机制。2)我们将定义染色质靶标和 XIST的蛋白质相互作用，以了解其在幼稚和启动的hESCs中的差异沉默能力。3)我们会 鉴定lncRNA XACT的功能和定位。理解人类X-射线损伤的机制 染色体剂量补偿将允许我们推进对表观遗传特征的理解 并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.