Regulation of X-Chromosome Inactivation and Imprinting by Non-coding Elements

描述（由申请人提供）：在雌性哺乳动物中，两条x染色体中的一条被灭活，以确保雄性和雌性具有相同的x染色体剂量。X染色体失活（XCI）可以随机发生，也可以在父本X染色体（XP）上以印记方式发生。在小鼠胚胎体（体细胞）中观察到随机XCI，在胎盘中观察到印迹XCI。体细胞和胎盘的XCI都需要“x失活中心”（Xic），这是一个已知的x连锁区域，用于制造非编码RNA （ncRNA）的基因。Xic产生Xist RNA，这是一种大的未翻译RNA，它“描绘”X并顺式地向X招募沉默因子。我们的实验室先前发现反义t6基因和第二个非编码元件Xite是Xist的必要抑制因子。在之前的融资周期中，我们关注的是Tsix和Xite调控XCI不同步骤的分子手段。这项研究有三个主要发现：(i)在雌性中，x ‘对’在XCI开始时短暂地相互结合。我们认为配对介导了x染色体之间的“串扰”，从而确保了互斥的染色体命运。t6和Xite介导配对相互作用。（ii） Tsix通过反义转录驱动染色质结构的抢先性改变来控制Xist的表达。Tsix RNA也结合Dnmt3a并将DNA从头甲基化引导到Xist启动子。剂量补偿在发育过程中发生的时间比以前认为的要早得多。我们认为，印迹XCI在受孕时已经发生，并在此基础上提出，XP是作为父系种系的“预失活”染色体遗传的。这些发现提出了许多新的问题，我们现在打算在三个不同的目标中进行研究。在目标1中，我们将定义x染色体配对所需的分子因子。在Aim 2中，我们打算研究Tsix转录如何指导染色质修饰以及Tsix RNA招募了哪些染色质修饰剂。在Aim 3中，我们将进行遗传和生化测试，以区分印迹XCI的预失活和新生模型。从研究项目中获得的信息将对人类遗传学、癌症和表观遗传重编程产生广泛的影响。它也将增强我们对基本生物学过程的理解，特别是那些涉及染色体动力学、ncRNA调控和跨代遗传的过程。

DESCRIPTION (provided by applicant): In the female mammal, one of two X-chromosomes is inactivated to ensure that males and females have equal X-chromosome dosage. X-chromosome inactivation (XCI) can take place randomly or in an imprinted manner on the paternal X (XP). In the mouse, random XCI is observed in the embryo proper (soma) and imprinted XCI in the placenta. Both somatic and placental XCI require the 'X-inactivation center' (Xic), an X-linked region known for genes making noncoding RNA (ncRNA). The Xic produces Xist RNA, a large untranslated RNA that 'paints' the X and recruits silencing factors to the X in cis. Our laboratory previously identified the antisense Tsix gene and a second noncoding element, Xite, as essential repressors of Xist. In the previous funding cycle, we focused on the molecular means by which Tsix and Xite regulate different steps of XCI. This investigation has resulted in three major discoveries: (i) In females, the Xs 'pair' transiently with each other at the onset of XCI. We believe that pairing mediates 'cross-talk' between the Xs to ensure mutually exclusive chromosome fates. Tsix and Xite mediate the pairing interaction. (ii) Tsix controls Xist expression through preemptive changes in chromatin structure driven by its antisense transcription. Tsix RNA also binds Dnmt3a and directs de novo DNA methylation to the Xist promoter. (iii) Dosage compensation occurs much earlier in development than previously thought. We believe that imprinted XCI has already taken place by the time of conception and, on this basis, propose that the XP is inherited as a 'pre-inactivated' chromosome from the paternal germline. These discoveries have raised many new questions, which we now intend to pursue in three distinct Aims. In Aim 1, we will define molecular factors required for X-chromosome pairing. In Aim 2, we intend to investigate how chromatin modifications are directed by Tsix transcription and what chromatin modifiers are recruited by Tsix RNA. In Aim 3, we will carry out genetic and biochemical tests to distinguish between the pre-inactivation and de novo models for imprinted XCI. Information gained from the research program will have broad implications for human genetics, cancer, and epigenetic reprogramming. It will also enhance our understanding of basic biological processes, especially those involving chromosome dynamics, ncRNA regulation, and transgenerational inheritance.