Analysis of Imprinting Control of H19/Igf2 and Kcnq1

描述（由申请人提供）：表观遗传机制的研究将导致了解基因组如何作为发育蓝图以及基因表达模式的扰动如何导致癌症。本提案的目的是阐明导致等位基因特异性基因沉默的两簇印迹基因的表观遗传机制。印迹基因具有亲本特异性的单等位基因表达。在特定序列上的差异DNA甲基化是与印迹基因最一致的表观遗传修饰。许多印迹结构域也表现出非编码rna的表达。我们对等位基因特异性沉默的分子机制以及非编码rna在调节这一过程中的作用知之甚少。本提案将以小鼠为模型，首先通过染色体构象捕获技术阐明H19/lgf2位点调控元件之间的物理相互作用，验证存在等位基因特异性相互作用的假设。H19上差异甲基化结构域（DMD）靶向突变的小鼠将与野生型小鼠进行比较。其次，通过在kcnq10t1基因中插入聚腺苷化位点来检测kcnq10ot是否需要转录来维持该位点的印迹。kcnq10t1是Kcnq1外显子10产生的一种反义非编码RNA。第三个实验将利用转基因RNA干扰kcnq10t1转录本，并测试kcnq10t1 RNA本身是否在建立和维持等位基因特异性基因沉默中发挥作用。H19/lgf2和Kcnql结构域的基因都与导致产前过度生长和癌症易感性的beckwithwithwiedemann综合征有关。这两个区域的印迹缺失也与几种人类肿瘤有关。因此，这些研究将为基因沉默的调控机制提供更全面的理解，并将深入了解这些机制如何在癌细胞中出错。

DESCRIPTION (provided by applicant): The study of epigenetic mechanisms will lead to understanding of how the genome functions as a developmental blueprint and how perturbations of gene expression patterns can lead to cancer. The goal of this proposal is to elucidate epigenetic mechanisms leading to allele-specific gene silencing at two clusters of imprinted genes. Imprinted genes have parental-specific monoallelic expression. Differential DNA methylation at specific sequences is the epigenetic modification most consistently associated with imprinted genes. Many imprinted domains also exhibit expression of non-coding RNAs. Very little is known of the molecular mechanisms involved in allele-specific silencing and the role of the non-coding RNAs in modulating this process. Using the mouse as a model, this proposal will first elucidate the physical interactions between regulatory elements at the H19/lgf2 locus through chromosome conformation capture technology, testing the hypothesis that there are allele-specific interactions. Mice with targeted mutations in the differentially methylated domain (DMD) at H19 will be compared to the wild-type mice. Second, a targeting experiment will test whether transcription of Kcnq1ot, an imprinted antisense non-coding RNA produced from exon 10 of Kcnq1, is required to maintain the imprinting at this locus by inserting a polyadenylation site in the Kcnq1ot1 gene. A third experiment will exploit transgenic RNA interference to target the Kcnq1ot1 transcript and test whether the Kcnq1ot1 RNA itself plays a role in establishing and maintaining allele-specific gene silencing. Genes in both the H19/lgf2 and the Kcnql domains are involved in Beckwith-Wiedemann syndrome, which causes prenatal overgrowth and predisposition to cancer. Loss of imprinting at both domains has also been implicated in several human neoplasias. Thus, these studies will provide a more thorough understanding of the regulatory mechanisms deployed in gene silencing and will give insight into how these mechanisms can go awry in cancerous cells.