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 来自性染色体。事实上，雌性哺乳动物使其两条X染色体中的一条保持沉默(X) 在一个称为X失活(XCI)的过程中，这是一种表观遗传基因沉默的范例。在小鼠中，XCI 发生在雌性胚胎发育的早期，分两波进行。在2-4细胞期的胚胎中， XCI的早期印迹形式(IXCI)只会使父亲的X(XP)沉默。在植入前后， 然而，产生体细胞胚胎组织的雌性上皮细胞经历了主要的 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的形式(RXCI)，以相同的概率使父亲或母亲的X沉默。因此，组织中的 成年雌性小鼠一般表现出随机的XCI图案。Xist RNA对X- 在iXCI和rXCI期间都处于静默状态，并将非活动X绘制为 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的功能和 IXCI期间Xist云的维护和Rlim的缺乏中断了预定的胚胎植入 导致滋养层细胞衰竭。然而，Rlim对于RXCI在细胞周期中和细胞周期后的外胚层细胞中是不可缺少的。 植入胚胎。因此，X剂量补偿缺陷如何抑制的原因 在rXCI过程中，滋养细胞的功能以及调节Xist的途径尚不清楚，并且 在上胚层中触发从iXCI到rXCI的发育转换的调控通路仍然存在 神秘莫测。 这项拨款建议的研究是基于我们已发表的初步工作和 将使用小鼠遗传学和新产生的雌性ESC模型相结合 在Rlim缺席的情况下对XCI进行调查。特别是，我们将探索iXCI的影响 对滋养层干细胞命运的失败(目标1)，检查rXCI调节的新机制 女性胚胎干细胞(目标2)与XCR在女性围着床期的体内机制 胚胎(目标3)。 这项拟议的研究将确定滋养层干细胞的新机制。 RXCI和IXCI期间XIST的维护、潜在规则和照明 XCR背后的机制。合并后的结果将允许设计第一个全面的 发育中雌性外胚层细胞中发生iXCI/XCR/rXCI表观遗传转换的模型 老鼠。预期结果将对早期老鼠发展领域产生革命性影响， 干细胞生物学、女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.