Nuclear and Chromatin Packaging of Mammalian X-Chromosome

描述（由申请人提供）：哺乳动物中的X-失活过程通过兼性异染色质的形成而发生，这是正常发育的核心现象，在某些癌症中被废除。在此过程中，稳定XIST RNA的积累在结构上与女性中的一条X染色体相关联，并启动染色体重塑的级联反应，使失活的X（Xi）沉默，形成异染色质巴尔体。我们现在需要知道XIST RNA是如何定位并“描绘”其亲本染色体的，以及这是如何导致整个染色体的结构转变和浓缩的。我们的方法来解决这些问题，利用分子，生物化学和结构分析，再加上基因组序列组织的生物信息学。我们的目标是处理染色体的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和基因组重复序列的潜在作用。目的1建立在我们最近成功操纵XIST RNA定位的基础上，以更好地理解XIST结合的调控，涉及的特定因素，并在特异性转录因子上扩展强有力的初步结果。 组蛋白修饰以及与Xi有关的异染色质因子和支架附着因子。所涉及的因素可能提供了对癌症中广泛的异染色质不稳定性的深入了解，其中Xi/XIST缺陷可能是一个标志。目的2研究了一种新的沉默整个染色体的模型，其中XIST RNA不是在局部或个体基因水平上起作用，而是与整个间期染色体领域，特别是与富含重复元件的内核具有更多的结构关系。在这个模型中，XIST首先与X染色体的重复序列丰富的区域相互作用，使异染色质核心成核，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，在持续逃避沉默的区域中检查序列背景与逃避沉默的关系。 我们的研究将主要集中在人类X染色体失活，这已经很少研究，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异染色质具有相关性， 因此，我们的研究对癌症中异染色质不稳定性以及胚胎细胞中兼性异染色质的形成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.