Epigenetic Regulation of a non-coding RNA, nc886

项目摘要 异常的表观遗传调控是人类疾病的主要机制。可变DNA甲基化 编码转录的聚合酶III的基因座的101个碱基对的非编码RNA，nc 886，与 AML患者的存活率、体重指数、帕金森病和各种癌症。多 独立的研究表明，nc 886基因座是母系印记的， 这种印记的变化。75%的个体具有单等位基因DNA甲基化，或印记， nc 886和25%在两个等位基因上都缺乏DNA甲基化。以前的工作集中在DNA甲基化在 nc 886启动子。然而，多态性印迹区域延伸到nc 886侧翼的1.7kb，包括一个 潜在的增强子元件和在着丝粒侧上的一个甲基化CTCF结合位点（cent-CTCF）。 这个较大的多态性印迹区域被定义为nc 886差异甲基化区域（DMR）。 有趣的是，C/A SNP位于中心-CTCF结合基序中的CpG位点，并影响局部DNA 甲基化和CTCF结合。核心假设是C/A SNP影响了基因突变的可能性。 nc 886 DMR将被印迹，从而提醒局部染色质结构和基因表达模式。 具体目的1是表征nc 886 DMR的DNA甲基化如何改变全基因组基因 表情虽然以前的研究已经调节了nc 886的表达，并检查了基因组范围内的变化， 基因表达，结果是矛盾的。此外，nc 886相关增强子和cent-CTCF位点 还没有被调查。为了确定nc 886 DMR处DNA甲基化的后果， 将在具有已知DNA的细胞系中系统地去除（使用CRISPR/Cas9）以下元件 甲基化状态：（1）cent-CTCF结合位点（2）nc 886编码序列（3）nc 886相关增强子，和 (4)整个DMR nc 886基因座的破坏元件的后果将由RNA- 测序具体目标2是定义可变DNA甲基化如何影响CTCF结合和染色质 架构使用具有可变DNA甲基化和表达nc 886、染色质 免疫沉淀和核小体占据和甲基化组测序（NOMe-Seq）将用于 确定DNA甲基化和SNP状态如何改变CTCF占用。杂交捕获测序（Hi-C2） 将被用来确定染色质循环到我们感兴趣的区域的细胞系中， 在nc 886 DMR处甲基化、单等位基因甲基化和双等位基因未甲基化。这种系统性 阐明常见SNP、DNA甲基化、染色质结构 和基因表达将提供对nc 886 DMR的潜在功能的理解。考虑到 nc 886的可变DNA甲基化与几种人类疾病有关，我们的发现可以提供 对于肥胖症、癌症、帕金森氏病和其他潜在疾病的发展有价值的信息。 确定的疾病。

PROJECT SUMMARY Aberrant epigenetic regulation is a major contributing mechanism to human disease. Variable DNA methylation of a locus encoding a polymerase III transcribed, 101 base pair non-coding RNA, nc886, is associated with survival of AML patients, body-mass index, Parkinson's disease, and a variety of cancers. Multiple independent studies have demonstrated that the nc886 locus is maternally imprinted, and there is natural variation of this imprinting. Seventy-five percent of individuals have monoallelic DNA methylation, or imprinting, of nc886 and 25% lacked DNA methylation on both alleles. Previous work focused on DNA methylation at the nc886 promoter. However, the region of polymorphic imprinting extends to 1.7 kb flanking nc886 to include a potential enhancer element and a variably methylated CTCF binding site on the centromeric side (cent-CTCF). This larger region of polymorphic imprinting was defined as the nc886 differentially methylated region (DMR). Intriguingly, a C/A SNP lies in a CpG site in the binding motif of the cent-CTCF and influences local DNA methylation and CTCF binding. The central hypothesis is that a C/A SNP influences the likelihood that the nc886 DMR will be imprinted, therefore alerting local chromatin structure and gene expression patterns. Specific Aim 1 is to characterize how DNA methylation of the nc886 DMR alters genome-wide gene expression. While previous studies have modulated nc886 expression and examined changes in genome-wide gene expression, results are contradictory. Additionally, the nc886-associated enhancer and cent-CTCF site have not been investigated. To determine the consequences of DNA methylation at the nc886 DMR, the following elements will be systematically removed (using CRISPR/Cas9) in cell lines with known DNA methylation status: (1) cent-CTCF binding site (2) nc886 coding sequence (3) nc886-associated enhancer, and (4) entire DMR. Consequences of disrupting elements of the nc886 locus will be determined by RNA- sequencing. Specific Aim 2 is to define how variable DNA methylation affects CTCF binding and chromatin architecture. Using cell lines with variable DNA methylation and expression of nc886, Chromatin Immunoprecipitation and Nucleosome Occupancy and Methylome Sequencing (NOMe-Seq) will be used to determine how DNA methylation and SNP status alters CTCF occupancy. HYbrid Capture Sequencing (Hi-C2) will be utilized to determine chromatin looping to our region of interest in cells lines that are biallelically methylated, monoallelically methylated, and biallelically unmethylated at the nc886 DMR. This systematic approach to elucidating the relationship between a common SNP, DNA methylation, chromatin architecture, and gene expression will provide understanding for the potential function of the nc886 DMR. Considering that variable DNA methylation of nc886 is associated with several human diseases, our findings could provide valuable information for the development of obesity, cancer, Parkinson's disease and potentially other yet identified maladies.