Epigenetic regulation of X chromosomes during female mouse embryogenesis

摘要 雌性哺乳动物的发育和繁殖依赖于剂量补偿 从性染色体中分离出来事实上，雌性哺乳动物会沉默两条X染色体中的一条（X） 这一过程被称为X染色体失活（XCI），这是一种表观遗传基因沉默的范例。在小鼠中， 在雌性胚胎发育的早期以两个波发生。在2 - 4细胞期的胚胎中， XCI的早期印记形式（iXCI）仅使父体X（Xp）沉默。在植入过程中， 然而，产生体细胞胚胎组织的雌性外胚层细胞经历了一个主要的 表观遗传开关：这些细胞重新激活沉默的Xp（XCR），并经历另一个随机的， XCI（rXCI）的一种形式，以相等的概率沉默父源或母源X。因此， 成年雌性小鼠通常显示随机XCI模式。Xist RNA对X- 在iXCI和rXCI期间都沉默，并将其表达为非活性X的非活性X描绘为 云，触发下游抑制性染色质修饰和X沉默。我们 发现在小鼠中，X-连接的泛素连接酶RLIM对Xist功能至关重要， 在iXCI过程中维持Xist云，缺乏Rlim会破坏胚胎着床， 滋养层衰竭然而，Rlim对于rXCI在体外和体外培养后的上胚层细胞中是无效的。 植入胚胎因此，X射线剂量补偿缺陷抑制的原因 滋养层功能以及在rXCI期间调节Xist的途径是未知的， 在外胚层中，触发从iXCI到rXCI的发育转换的调节途径仍然存在， 神秘莫测 这项资助中提出的研究是基于我们已发表的和初步的工作， 将使用小鼠遗传学与新产生的女性ESC模型相结合， 在不存在Rlim的情况下对XCI的研究。特别是，我们将探讨iXCI的影响 失败的滋养层干细胞的命运（目的1），研究新的机制，rXCI调控， 女性胚胎干细胞（目的2）和解开体内机制XCR在女性围植入期 胚胎（目标3）。 这项研究将确定滋养层干细胞的新机制， 维持，在rXCI和iXCI期间Xist的潜在调节， XCR的潜在机制。综合结果将允许设计第一个全面的 在发育中的女性的上胚层细胞中发生的iXCI/XCR/rXCI表观遗传开关的模型 小鼠预期结果将在小鼠早期发育领域产生变革性影响， 干细胞生物学、雌性生殖和性别特异性表观遗传调控。

Abstract The development and reproduction of female mammals is dependent on dosage compensation from sex chromosomes. Indeed, female mammals silence one of their two X chromosomes (X) in a process called X inactivation (XCI), a paradigm for epigenetic gene silencing. In mice, XCI occurs early during female embryogenesis in two waves. In embryos at the 2-4 cell stage, an early imprinted form of XCI (iXCI) silences exclusively the paternal X (Xp). Around implantation, however, female epiblast cells that give rise to somatic embryonic tissues undergo a major epigenetic switch: These cells reactivate the silenced Xp (XCR) and undergo another, random form of XCI (rXCI), silencing the paternal or maternal X with equal probability. Thus, tissues in the adult female mouse generally display a random XCI pattern. Xist RNA is crucial for X- silencing both during iXCI and rXCI and paints the inactive X from which it is expressed as a cloud, triggering downstream repressive chromatin modifications and X-silencing. We discovered that in mice the X-linked ubiquitin ligase RLIM is crucial for Xist functioning and maintenance of Xist clouds during iXCI and that lack of Rlim disrupts embryo implantation due to trophoblast failure. However, Rlim is dispensable for rXCI in epiblast cells of peri- and post- implantation embryos. Thus, the reasons of how X dosage compensation defects inhibit trophoblast functions as well as the pathways that regulate Xist during rXCI are unknown, and regulatory pathways triggering the developmental switch from iXCI to rXCI in epiblasts remain enigmatic. The research proposed in this grant is based on our published and preliminary work and will use mouse genetics combined with a newly generated female ESC model that permits investigations of XCI in the absence of Rlim. In particular, we will explore the effects of iXCI failure on trophoblast stem cell fates (Aim 1), examine novel mechanisms of rXCI regulation in female ESCs (Aim 2) and unravel in vivo mechanisms of XCR in female peri-implantation embryos (Aim 3). The proposed research will identify novel mechanisms of trophoblast stem cell maintenance, underlying regulation of Xist both during rXCI and iXCI and illuminate mechanisms underlying XCR. The combined results will allow designing the first comprehensive model of a iXCI/XCR/rXCI epigenetic switch that occurs in epiblast cells of developing female mice. Expected results will have transformative impact in the fields of early mouse development, stem cell biology, female reproduction and sex-specific epigenetic regulation.