Regulation and function of developmentally programmed 3 CpG island methylation

项目摘要 在分化时控制和稳定细胞命运的表观遗传状态的建立和维持是 对多细胞生物的发育至关重要。DNA甲基化是有丝分裂遗传的， 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，人类表观基因组 项目已经全面分析了组织和细胞类型特异性DNA甲基化，并确定了动态的 甲基化差异发育程序性DNA甲基化的时间和相关的 然而，在早期细胞谱系特化过程中的转录调控机制仍然很差， 明白这一建议建立在我们最近的发现，除了典型的转录， 启动子CpG岛（CGIs）的DNA甲基化抑制，一组 人胚胎干细胞（hESC）发育过程中基因体CGI甲基化与发育基因相关 分化一个特定的感兴趣的基因是癌症中的高甲基化1（Hypermethylated in cancer 1，HM 1）基因，其是一种肿瘤， 抑制基因和发育障碍Miller-Dieker综合征的候选基因。我们发现CGI 在人和小鼠中，在HAL 1的3'端的甲基化（3' CGI甲基化）是高度保守的，并且可能 说明胎儿发育期间间充质Hic 1表达。更重要的是，我们的初步研究 提示3' CGI甲基化通过CCCTC结合因子（CTCF）依赖性 机制基于这些发现，拟议中的研究使用小鼠模型来调查是否和 3' CGI甲基化如何控制Hic 1在发育胚胎中的时空表达。 具体而言，我们将：1.研究3' CGI甲基化调控Hic 1基因的机制 activation.使用Hic 1-citrine（黄色荧光蛋白）报告小鼠，我们将进行染色质 免疫沉淀，然后进行定量PCR（ChIP-qPCR），以系统性地绘制CTCF结合， Hic 1基因座在已知表达Hic 1的小鼠胚胎组织中。2.探讨Hic 1 3' CGI的功能 在小鼠发育过程中的甲基化。使用新型小鼠模型实现基于CRISPR的靶向DNA 去甲基化，我们将确定Hic 1 3' CGI去甲基化是否影响Hic 1的转录调节 从胚胎发育到出生，以及有缺陷的表观遗传调控是否会导致发育障碍， 缺陷总之，这个探索性的项目将建立一个新的技术组合，用于体内研究 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 深入了解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.