Analysis of Imprinting Control of H19/Igf2 and Kcnq1

描述(申请人提供)：表观遗传机制的研究将有助于理解基因组如何作为发育蓝图发挥作用，以及基因表达模式的扰动如何导致癌症。这项建议的目的是阐明导致两组印记基因的等位基因特异性基因沉默的表观遗传学机制。印迹基因具有亲本特异性的单等位基因表达。特定序列上的差异DNA甲基化是与印记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。关于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，人们知之甚少。以小鼠为模型，该方案将首先通过染色体构象捕获技术阐明H19/lgf2基因座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基因差异甲基化结构域(DMD)发生定向突变的小鼠将与野生型小鼠进行比较。其次，靶向实验将测试Kcnq1ot1基因是否需要转录，通过在Kcnq1ot1基因中插入多聚腺苷酸化位点来维持该基因的印迹。Kcnq1ot1是从KCNQ1外显子10产生的印记反义非编码RNA。第三个实验将利用转基因RNA干扰来靶向Kcnq1ot1转录本，并测试Kcnq1ot1 RNA本身是否在建立和维持等位基因特异性基因沉默方面发挥作用。H19/lgf2和Kcnq1结构域的基因都与Beckwith-Wiedemann综合征有关，后者会导致产前过度生长和易患癌症。这两个领域的印记丢失也与几种人类肿瘤有关。因此，这些研究将提供对基因沉默中部署的调控机制的更彻底的理解，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.