Regulation and function of developmentally programmed 3 CpG island methylation

项目总结 在分化过程中控制和稳定细胞命运的表观遗传状态的建立和维持 对多细胞生物体的发展至关重要。DNA甲基化，这是有丝分裂遗传，是一种 哺乳动物表观遗传基因调控的重要组成部分。在过去的十年里，人类的表观基因组 项目已经全面分析了组织和细胞类型特定的DNA甲基化，并确定了动态 甲基化差异。发育程序性DNA甲基化的时间及其相关 然而，早期细胞谱系特化过程中的转录调控机制仍然很差。 明白了。这一建议建立在我们最近的发现之上，除了规范的转录 启动子CpG岛(CGI)上DNA甲基化的抑制，一组 人胚胎干细胞发育基因与基因体CGI甲基化的关系 差异化。一个特别感兴趣的基因是癌症1(HIC1)基因的高甲基化，这是一种肿瘤 抑制子和发育障碍Miller-Dieker综合征的候选基因。我们发现了CGI HIC1基因3‘端的甲基化(3’CGI甲基化)在人类和小鼠中高度保守，可能 明确胎儿发育过程中间充质Hic1的表达。更重要的是，我们的初步研究 提示3‘CGI甲基化通过依赖CCCTC结合因子(CTCF)调控Hic1转录 机制。基于这些发现，这项拟议的研究使用小鼠模型来调查是否和 3‘CGI甲基化如何调控Hic1在胚胎发育过程中的时空表达 具体地说，我们将：1研究3‘cGI甲基化调控hic1基因的机制 激活。使用Hic1-Citine(一种黄色荧光蛋白)报告小鼠，我们将进行染色质 免疫沉淀和定量聚合酶链式反应(ChIP-qPCR)系统地定位CTCF结合在 已知在小鼠胚胎中表达Hic1的组织中的Hic1基因座。2研究Hic1 3‘cGI的功能 小鼠发育过程中的甲基化。利用一种新的小鼠模型实现基于CRISPR的靶向DNA 去甲基化，我们将确定Hic13‘CGI去甲基化是否影响Hic1的转录调控 从胚胎发育到出生，以及表观遗传调控缺陷是否会导致发育 缺陷。总之，这一探索性项目将为活体研究建立新技术的组合。 来剖析表观遗传的转录调控。这些研究的成功完成将产生重要的 洞察DNA甲基化对哺乳动物发育的功能作用。

PROJECT SUMMARY Establishment and maintenance of epigenetic states that govern and stabilize cell fate upon differentiation are crucial for the development of multicellular organisms. DNA methylation, which is mitotically heritable, is an important component of mammalian epigenetic gene regulation. Over the past decade, the Human Epigenome Projects have comprehensively profiled tissue- and cell-type-specific DNA methylation and identified dynamic methylation differences. The timing of developmentally programmed DNA methylation and associated mechanisms of transcriptional regulation during early cell-lineage specification, however, remain poorly understood. This proposal builds upon our recent discovery that, in addition to canonical transcriptional repression by DNA methylation at promoter CpG islands (CGIs), transcriptional activation of a group of developmental genes is related to gene body CGI methylation during human embryonic stem cell (hESC) differentiation. One particular gene of interest is Hypermethylated in cancer 1 (HIC1) gene, which is a tumor suppressor and a candidate gene for a developmental disorder Miller-Dieker syndrome. We found that CGI methylation at the 3' end of HIC1 (3' CGI methylation) is highly conserved in human and mouse, and may specify mesenchymal Hic1-expressing during fetal development. More importantly, our preliminary studies suggest that 3' CGI methylation regulates Hic1 transcription via a CCCTC-binding factor (CTCF)-dependent mechanism. Based on these findings, the proposed research uses mouse models to investigate whether and how the 3' CGI methylation controls the temporal and spatial expression of Hic1 in developing embryos. Specifically, we will: 1  Investigate the mechanism by which 3' CGI methylation regulates Hic1 gene activation. Using Hic1-citrine (a yellow fluorescent protein) reporter mice, we will perform chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) to systemically map CTCF-bindings at the Hic1 locus in tissues known to express Hic1 in mouse embryos. 2  Investigate the function of Hic1 3' CGI methylation during mouse development. Using a novel mouse model to enable CRISPR-based targeted DNA demethylation, we will determine whether Hic1 3' CGI demethylation affects transcriptional regulation of Hic1 from embryonic development into birth, and whether defective epigenetic regulation leads to developmental defects. Altogether, this exploratory project will establish a combination of novel technologies for in vivo studies to dissect epigenetic transcriptional regulation. The successful completion of these studies will yield important insights into the function role of DNA methylation for mammalian development.