Nuclear and Chromatin Packaging of Mammalian X-Chromosome

描述(申请人提供)：哺乳动物的X-失活过程是通过形成兼性异染色质而发生的，这是一种对正常发育至关重要的现象，在一些癌症中被废除。在这个过程中，稳定的XIST RNA的积累在结构上与雌性的一条X染色体相关联，并启动了一系列染色体重塑，从而沉默了不活跃的X(Xi)，形成了一个异染色质Barr小体。我们现在需要知道XIST RNA是如何定位到它的亲本染色体上并“上色”的，以及这是如何导致整个染色体的结构转化和凝聚的。我们解决这些问题的方法是利用分子、生化和结构分析，以及基因组序列组织的生物信息学。我们的目标是处理染色体功能和结构转化的不同但相互关联的方面，重点是XIST RNA与染色体的相互作用以及基因组重复序列的潜在作用。目的1在我们最近成功操纵XIST RNA本地化的基础上，更好地了解XIST结合的调节、所涉及的特定因素，并在特定的 组蛋白修饰，以及异染色质因子和支架附着因子与XI有关。所涉及的因素可能提供对癌症广泛的异色不稳定性的洞察，其中XI/XIST缺陷可能是其中的一个标志。目的2研究了一种新的抑制整个染色体的模型，其中XIST RNA不是在局部或单个基因水平上起作用，而是与整个间期染色体区域有更多的结构关系，特别是与富含重复元件的核心。在这个模型中，XIST首先与X染色体的富含重复区域相互作用，形成一个异染色质核心，然后传播到更外围的蛋白质编码基因。在目标3中，使用系统的转基因方法，结合生物信息学和分子细胞学分析，在一个持续逃避沉默的区域研究了序列上下文与逃避沉默的关系。我们的研究将主要集中在人类X的失活，这方面的研究较少，使用体细胞、转基因和人类ES细胞模型。了解是什么建立和维持人类XI异染色质与 肿瘤中的异染色质不稳定性以及胚胎细胞中兼性异染色质的形成，因此我们的研究具有重要的临床意义。

DESCRIPTION (provided by applicant): The process of X-inactivation in mammals occurs by the formation of facultative heterochromatin, a phenomenon central to normal development and abrogated in some cancers. In this process, an accumulation of stable XIST RNA structurally associates with one X chromosome in females and initiates a cascade of chromosome remodeling that silences the inactive X (Xi), forming a heterochromatic Barr Body. We now need to know how XIST RNA localizes to and "paints" its parent chromosome, and how this leads to the structural transformation and condensation of a whole chromosome. Our approach to these questions utilizes molecular, biochemical and structural analyses, coupled with bioinformatics of genomic sequence organization. Our aims deal with distinct but inter-related aspects of the functional and structural transformation of the chromosome, focusing on the interaction of XIST RNA with the chromosome and the potential role genomic repeat sequences. Aim 1 builds on our recent success in manipulating XIST RNA localization, to better understand the regulation of XIST binding, specific factors involved, and extend strong preliminary results on specific histone modifications, as well as heterochromatin factors and scaffold attachment factors implicated in Xi. The factors involve may provide insight into broad heterochromatic instability in cancer, of which Xi/XIST defects may be one hallmark. Aim 2 investigates a novel model for silencing of an entire chromosome, where XIST RNA is not acting at a local or individual gene level, but has a more architectural relationship with the whole interphase chromosome territory, particularly with an inner core enriched in repeat elements. In this model, XIST first interacts with the repeat-rich regions of the X chromosome, nucleating a heterochromatic core that then propagates to the more peripheral protein coding genes. In Aim 3, the relationship of sequence context to escape from silencing is examined in a region that consistently escapes silencing, using a systematic transgene approach, combined with bioinformatics and molecular cytological analyses. Our studies will focus largely on human X inactivation, which has been less well studied, using somatic cell, transgene and human ES cell models. Understanding what establishes and maintains human Xi heterochromatin has relevance to heterochromatic instability in cancer as well as formation of facultative heterochromatin in embryonic cells, and thus our studies have significant clinical implications.