Nuclear and Chromatin Packaging of Mammalian X-Chromosome

描述（由申请人提供）：哺乳动物的x失活过程发生在兼性异染色质的形成过程中，这是正常发育的核心现象，在某些癌症中被取消。在这一过程中，稳定的XIST RNA的积累在结构上与雌性的一条X染色体结合，并启动染色体重塑的级联，使不活跃的X （Xi）沉默，形成异色的Barr体。我们现在需要知道XIST RNA是如何定位和“描绘”它的亲本染色体的，以及这是如何导致整个染色体的结构转化和凝聚的。我们的方法利用分子，生化和结构分析，再加上基因组序列组织的生物信息学来解决这些问题。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结合的调节，所涉及的特定因素，并扩展特异性的强有力的初步结果

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.