Deciphering the role of XACT lncRNA in human development

解读 XACT lncRNA 在人类发育中的作用

• 批准号: 10460910
• 负责人: Anna Afasizheva
• 金额: $ 5.18万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10460910
• 关键词: 3-Dimensional; Address; Affect; Cells; Chromatin; Chromosome Territory; DNA sequencing; Development; Dosage Compensation (Genetics); Epigenetic Process; Female; Gene Deletion; Gene Dosage; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic Transcription; Genomic Segment; Goals; Human; Human Development; Image; Interference Microscopy; Link; Mediating; Microscopy; Modeling; Molecular Mechanisms of Action; Mus; Nuclear; Pattern; Phenotype; Pluripotent Stem Cells; Primates; Process; RNA; Regulation; Resolution; Role; System; Testing; Transcript; Untranslated RNA; X Chromosome; X Inactivation; base; blastocyst; density; experimental study; genome-wide; genomic locus; human embryonic stem cell; human female; implantation; knockout gene; male; natural Blastocyst Implantation; novel; pluripotency; preimplantation; premature; prevent; programs; transcriptome; transcriptome sequencing

Project Summary / Abstract The goal of this proposal is to define the role of the long non-coding RNA (lncRNA) XACT (X-active coating transcript). XACT is a primate-specific and X-chromosome-linked lncRNA expressed exclusively in pluripotent stem cells. In naïve human embryonic stem cells (hESCs), which model the pluripotent state of the pre-implantation embryo, the XACT RNA forms two clouds in female and one cloud in male cells over the respective X-chromosome territories. At this stage of development, female and male cells express X-linked genes at comparable levels despite both female X-chromosomes being transcriptionally active. This gene dosage compensation mechanism, known as X-chromosome dampening (XCD), is unique to human development. Upon implantation, female pluripotent cells switch to complete silencing of one X-chromosome via X-chromosome inactivation (XCI), a process mediated by the well-studied chromatin-associated lncRNA XIST. In primed hESCs, which capture the pluripotent state of the post-implantation embryo, XACT expression is silenced on the inactive X-chromosome (Xi) of female cells, but is retained on the active X-chromosome in female and male cells. Considering that many lncRNAs function as transcriptional regulators, we hypothesize that XACT modulates the naïve and primed pluripotent states in early human development, potentially by regulating X- chromosome dosage compensation. Intriguingly, preliminary findings in our lab have shown that XIST also controls X-chromosome dampening in naïve pluripotent cells, but does not induce complete silencing in this developmental state. One possible function of XACT may be to counteract the silencing capacity of XIST in naïve pluripotent cells, in which XCD is characterized by concurrent expression of XACT and XIST from the X- chromosome. We posit that XACT antagonizes the activity of XIST during XCD through competitive chromatin association, thus preventing premature XCI prior to implantation. To unveil the role of XACT in pluripotent states and in the regulation of X-chromosome dosage compensation, we will: 1) delete XACT in naïve and primed hESCs and analyze the effect on cell states and transcriptomes; and 2) define its molecular mechanism of action as a chromatin-associated lncRNA. Taken together, these approaches will elucidate the contribution of XACT to the control of pluripotent states and its role in X-chromosome gene dosage regulation. By exploring the link between XACT function and the modulation of XIST in early human development, we may uncover a novel, primate-specific strategy for gene dosage compensation and define fundamental features of human pluripotency.

项目概要/摘要 该提案的目标是定义长非编码 RNA (lncRNA) XACT（X-active 涂层转录本）。 XACT 是一种灵长类动物特异性、X 染色体连接的 lncRNA，仅在 多能干细胞。在幼稚的人类胚胎干细胞 (hESC) 中，它模拟了多能状态 在植入前胚胎中，XACT RNA 在雌性细胞中形成两朵云，在雄性细胞中形成一朵云。 各自的X染色体区域。在这个发育阶段，雌性和雄性细胞表达 X 连锁 尽管两条女性 X 染色体都具有转录活性，但基因的水平相当。这个基因 剂量补偿机制，称为 X 染色体抑制 (XCD)，是人类独有的 发展。植入后，雌性多能细胞通过以下方式切换到一条 X 染色体的完全沉默： X 染色体失活 (XCI)，这是一个由经过充分研究的染色质相关 lncRNA XIST 介导的过程。 在捕获植入后胚胎多能状态的引发 hESC 中，XACT 表达为 在雌性细胞的非活性 X 染色体 (Xi) 上沉默，但在雌性细胞的活性 X 染色体上保留 和雄性细胞。考虑到许多lncRNA作为转录调节因子，我们假设XACT 可能通过调节 X- 来调节早期人类发育中的幼稚和启动多能状态 染色体剂量补偿。有趣的是，我们实验室的初步研究结果表明 XIST 还 控制幼稚多能细胞中的 X 染色体抑制，但不会诱导该细胞的完全沉默 发育状态。 XACT 的一种可能功能可能是抵消 XIST 的沉默能力 多能细胞，其中 XCD 的特征是来自 X-的 XACT 和 XIST 同时表达 染色体。我们假设 XACT 在 XCD 过程中通过竞争性染色质拮抗 XIST 的活性 关联，从而防止植入前过早的 XCI。揭示 XACT 在多能状态中的作用 在X染色体剂量补偿的调节中，我们将：1）删除naïve和primed中的XACT hESC 并分析对细胞状态和转录组的影响； 2）定义其分子作用机制 作为染色质相关的 lncRNA。总而言之，这些方法将阐明 XACT 对 多能状态的控制及其在 X 染色体基因剂量调节中的作用。通过探索链接 在 XACT 功能和早期人类发育中 XIST 的调节之间，我们可能会发现一个新的， 灵长类动物特异性基因剂量补偿策略并定义人类多能性的基本特征。