Regulation of X-Inactivation by Non-Coding RNA Loci

哺乳动物在雌性早期发育过程中通过一条x染色体的转录沉默实现剂量补偿。x染色体失活（XCI）是由一个被称为“x失活中心”的主位点控制的，这是一个顺式作用的中心，它既必要又充分地指导x染色体的计数、选择和沉默的开始。我们的长期目标是了解每一步的分子基础。目前已知，X-失活中心的非编码产物Xist RNA“描绘”了失活的X，并需要启动该X的全局沉默。上一个资助期的工作导致在X-失活中心发现了两个新的非编码RNA位点。t6是Xist的反义基因，抑制Xist的表达，并指定未来的活性X. Xite是一个与低水平基因间转录相关的位点，正调控t6的表达。因此，所有已知的x -失活中心的调控元件都产生非编码RNA。拟议的研究将剖析Tsix和Xite的作用机制。具体而言，拟制的研究将(1)确定Xite和Tsix如何抑制Xist RNA的传播，(2)确定x失活印迹和选择的分子基础，以及(3)检测Xite是否为Xce（“x控制元件”），Xce是XCI等位基因比例的修饰因子，长期以来一直被认为与x连锁疾病女性携带者的XCI不利偏形有关。Tsix是哺乳动物中为数不多的反义基因座之一，是反义调控的范例。作为哺乳动物中仅有的两个已知的功能性基因间转录元件之一，Xite将推动对这一新兴基因间现象的有限了解。从研究项目中获得的信息也将对人类遗传学、癌症和动物克隆，以及染色体、染色质结构和转录调节的基本生物学产生重大影响。

Mammals achieve dosage compensation by transcriptional silencing of one X-chromosome in early female development. X-chromosome inactivation (XCI) is controlled by a master locus known as the "X-inactivation center," a cis-acting center which is both necessary and sufficient to direct X-chromosome counting, choice, and initiation of silencing. Our long term goal is to understand the molecular underpinnings of each step. It is currently known that Xist RNA, a non-coding product of the X-inactivation center, "paints" the inactive X and is required to initiate global silencing of that X. Work from the last funding period led to the discovery of two novel non-coding RNA loci at the X-inactivation center. Tsix, a gene antisense to Xist, inhibits Xist expression and designates the future active X. Xite, a locus associated with low-level intergenic transcripts, positively regulates Tsix expression. Thus, all known regulatory elements at the X-inactivation center make non-coding RNA. The proposed research will dissect the mechanism of action by Tsix and Xite. Specifically, the proposed research will (1) determine how Xite and Tsix inhibit the spread of Xist RNA, (2) define the molecular basis of X-inactivation imprinting and choice, and (3) test if Xite is the Xce ("X-controlling element"), the long-elusive modifier of XCI allele ratios which has been implicated in unfavorable skewing of XCI in female carriers of X-linked disease. As one of very few antisense loci in mammals, Tsix serves as a paradigm for antisense regulation. As one of only two known functional intergenic transcription elements in mammals, Xite will advance a limited knowledge of this emerging intergenic phenomenon. Information gained from the research program will also have significant impact on human genetics, cancer, and animal cloning, as well as on the basic biology of chromosomes, chromatin structure, and transcriptional regulation.