Regulation and function of developmentally programmed 3 CpG island methylation

发育程序 3 CpG 岛甲基化的调节和功能

基本信息

批准号：
10064151
负责人：
Miaohsueh Chen
金额：
$ 17.98万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10064151
关键词：
17p13.3 3&apos Untranslated Regions Affect Alleles Antibodies Applications Grants BTB/POZ Domain Binding Birth CCCTC-binding factor Candidate Disease Gene Catalogs Cell Lineage Cells Chromosome Deletion Clustered Regularly Interspaced Short Palindromic Repeats CpG Islands DNA DNA Methylation DNA Methylation Regulation Defect Development Developmental Gene Developmental Gene Expression Regulation Embryo Embryonic Development Epigenetic Process Epitopes Exhibits Exons Fetal Development Gene Activation Gene Expression Gene Expression Profile Gene Expression Regulation Genes Genetic Transcription Goals Growth Heritability Human Impairment Knock-in Knockout Mice Lead Maintenance Malignant Neoplasms Maps Mesenchymal Mesenchyme Methylation Miller-Dieker Syndrome Mitotic Modeling Mus Organism Organogenesis Patients Peptides Perinatal Process Proteins Regulation Reporter Research Robin bird Role Specific qualifier value Testing Tissues Transcription Repressor Transcriptional Activation Transcriptional Regulation Tumor Suppressor Genes Tumor Suppressor Proteins Zinc Fingers base cell type chromatin immunoprecipitation demethylation density design developmental disease disorder prevention effective intervention embryo tissue epigenetic regulation epigenome gene function gene repression human embryonic stem cell in vivo insight interest methylation pattern mouse development mouse model new technology novel prevent promoter recruit stem cell differentiation

项目摘要

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.

项目概要控制和稳定细胞分化命运的表观遗传状态的建立和维持是 DNA甲基化对于多细胞生物的发育至关重要，具有有丝分裂遗传性。在过去的十年中，人类表观基因组是哺乳动物表观遗传基因调控的重要组成部分。项目全面分析了组织和细胞类型特异性 DNA 甲基化，并确定了动态发育程序性 DNA 甲基化的时间差异以及相关的甲基化差异。然而，早期细胞谱系规范期间的转录调控机制仍然很差这个提议建立在我们最近的发现之上，除了规范转录之外。启动子 CpG 岛 (CGI) 上 DNA 甲基化的抑制，一组基因的转录激活发育基因与人胚胎干细胞（hESC）过程中基因体CGI甲基化有关一个特别感兴趣的基因是癌症 1 (HIC1) 基因，它是一种肿瘤。我们发现了 CGI 的抑制基因和发育障碍米勒-迪克综合征的候选基因。 HIC1 3' 末端的甲基化（3' CGI 甲基化）在人类和小鼠中高度保守，可能更重要的是，我们的初步研究明确了胎儿发育过程中间充质 Hic1 的表达。表明 3' CGI 甲基化通过 CCCTC 结合因子 (CTCF) 依赖性调节 Hic1 转录基于这些发现，拟议的研究使用小鼠模型来研究是否和。 3' CGI 甲基化如何控制发育中胚胎中 Hic1 的时间和空间表达。具体来说，我们将： 1 – 研究3'CGI甲基化调节Hic1基因的机制使用 Hic1-citrine（一种黄色荧光蛋白）报告小鼠，我们将进行染色质激活。免疫沉淀，然后进行定量 PCR (ChIP-qPCR)，以系统地绘制 CTCF 结合图谱已知在小鼠胚胎中表达 Hic1 的组织中的 Hic1 位点 2 - 研究 Hic1 3' CGI 的功能。使用新型小鼠模型实现基于 CRISPR 的靶向 DNA 甲基化。去甲基化，我们将确定 Hic1 3' CGI 去甲基化是否影响 Hic1 的转录调控从胚胎发育到出生，表观遗传调控缺陷是否会导致发育障碍总而言之，这个探索性项目将建立用于体内研究的新技术组合。剖析表观遗传转录调控的成功完成这些研究将产生重要的成果。深入了解 DNA 甲基化对哺乳动物发育的功能作用。