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Dnmt3b activities in mouse development

Dnmt3b 在小鼠发育中的活性

基本信息

批准号：
10621334
负责人：
Rene Opavsky
金额：
$ 30.5万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10621334
关键词：
Affect Assisted Reproductive Technology Birth Cells Clinical Complex Conceptions DNA DNA Methylation DNA Modification Methylases DNA Transposable Elements DNMT3B gene DNMT3a Data Defect Development Disease Down-Regulation Embryo Embryonic Development Enhancers Enzymes Epiblast Epigenetic Process Ethics Family Fertilization in Vitro Future Gene Expression Gene Expression Regulation Genes Genetic Transcription Genome Genomic Imprinting Genomic Segment Genomics Health Human Human Cell Line Immunoprecipitation In Vitro Individual Knock-out Knockout Mice Knowledge Large Intestine Carcinoma Longitudinal Studies Malignant Epithelial Cell Measures Mediating Medicine Methylation Methyltransferase Modeling Modification Molecular Molecular Analysis Mus Pathogenesis Phenotype Physiological Physiological Processes Play Pre-implantation Embryo Development Preventive care Procedures Process Proteins Reporting Role Techniques Testing Time Tissue-Specific Gene Expression Tissues Totipotency Transcriptional Regulation Western Blotting X Inactivation demethylation embryonic stem cell epigenome genetic approach genome wide methylation genome-wide genomic locus histone modification implantation imprint in vivo methylation pattern mouse development novel therapeutic intervention pluripotency postnatal development prenatal prevent promoter recruit transcriptome sequencing

项目摘要

DNA methylation is an epigenetic modification involved in transcriptional regulation of genes involved in development and differentiation, and its deregulation contributes to human pathogenesis. It is catalyzed by the family of DNA methyltransferases including catalytically active Dnmt1, Dnmt3a, Dnmt3b. DNA methylation plays a major role in preimplantation development in mice. To establish a new epigenome, the mouse zygotic genome undergoes epigenetic reprogramming, including global DNA demethylation at the 8-cell stage. Upon implantation, a wave of de novo methylation in epiblast cells mediated by de novo enzymes Dnmt3a and Dnmt3b results in new methylation patterns maintained by Dnmt1 that form a basis for tissue-specific expression and differentiation. Dnmt3b regulates developmental and imprinted genes, X chromosome inactivation, pericentromeric regions, gene bodies and other genomic regions. Its importance in mouse development was demonstrated by embryonic lethality of Dnmt3b-/- mice. We recently found that Dnmt3bCI/CI mice expressing catalytically inactive Dnmt3bCI protein survived both pre- and postnatal development. Molecular analysis suggested that accessory function - the ability to recruit other Dnmts to proper genomic loci – of Dnmt3b rather than its catalytic activity, is important for methylation and survival. Here we hypothesize that Dnmt3b is a multifaceted protein whose various activities involved in methylation affect pre- and postnatal development and are critical to prevent disease formation in mice. To test this hypothesis, in Aim 1 we analyze global methylation and expression at different stages of development in mice lacking various Dnmt activities to determine the scope of Dnmt3b’s accessory function in Dnmt3a-mediated de novo methylation in vivo as well as regulation of transcription of various genomic features including gene bodies, germline genes and transposons. In Aim 2, will test the ability of Dnmt3b to complex with other Dnmts and contribute to de novo methylation induced by other Dnmts in Dnmt1-/-;Dnmt3a-/-;Dnmt3b-/- triple knockout mouse embryonic stem cells. In addition, we will genetically test the importance of Dnmt3a and Dnmt3l for Dnmt3b’s accessory function and validate our data in a human cell line. In Aim 3, we will perform longitudinal study of Dnmt3b+/+ and Dnmt3bCI/CI mice conceived through the use of in vitro fertilization (IVF) technique to analyze disease development, Dnmt levels, the rate of methylation and gene expression errors, as well as their persistence over time. Collectively, our studies will reveal physiological relevance of Dnmt3b activities in mouse development, uncover basic mechanisms utilizing Dnmt3b functions and their involvement in IVF. Our results could result in changes in Assisted Reproductive Technologies (ART) and affect the focus of preventive care for ART- conceived individuals.

DNA甲基化是一种表观遗传修饰，涉及基因的转录调控发育和分化及其失调有助于人类发病机制。它的催化作用是 DNA 甲基转移酶家族，包括具有催化活性的 Dnmt1、Dnmt3a、Dnmt3b。 DNA甲基化在小鼠植入前发育中起重要作用。为了建立新的表观基因组，小鼠合子基因组经历表观遗传重编程，包括 8 细胞阶段的整体 DNA 去甲基化。之上植入，外胚层细胞中由从头酶 Dnmt3a 介导的从头甲基化波和 Dnmt3b 产生由 Dnmt1 维持的新甲基化模式，形成组织特异性的基础表达和分化。 Dnmt3b 调节发育和印记基因、X 染色体失活、着丝粒周围区域、基因体和其他基因组区域。它在小鼠发育中的重要性通过 Dnmt3b-/- 小鼠的胚胎致死率。我们最近发现 Dnmt3bCI/CI 小鼠表达催化失活 Dnmt3bCI 蛋白在产前和产后发育中均存活。分子分析表明附件功能 - 将其他 Dnmt 招募到适当基因组位点的能力 - Dnmt3b 而不是其催化活性，对于甲基化和生存很重要。在这里，我们假设 Dnmt3b 是一种多面蛋白，其甲基化涉及的各种活动影响产前和产后发育，对于预防至关重要小鼠疾病的形成。为了检验这个假设，在目标 1 中，我们分析了全局甲基化和表达缺乏各种 Dnmt 活性的小鼠的不同发育阶段以确定 Dnmt3b 的范围 Dnmt3a 介导的体内从头甲基化的辅助功能以及转录调节各种基因组特征，包括基因体、种系基因和转座子。在目标2中，将测试能力 Dnmt3b 与其他 Dnmt 复合，并有助于其他 Dnmt 诱导的从头甲基化 Dnmt1-/-;Dnmt3a-/-;Dnmt3b-/- 三重敲除小鼠胚胎干细胞。此外，我们还会进行基因检测 Dnmt3a 和 Dnmt3l 对于 Dnmt3b 辅助功能的重要性并在人体细胞中验证我们的数据线。在目标 3 中，我们将对通过使用孕育的 Dnmt3b+/+ 和 Dnmt3bCI/CI 小鼠进行纵向研究体外受精 (IVF) 技术分析疾病发展、Dnmt 水平、甲基化率和基因表达错误及其随时间的持续存在。总的来说，我们的研究将揭示 Dnmt3b 活性在小鼠发育中的生理相关性，揭示利用 Dnmt3b 功能及其参与 IVF 的基本机制。我们的结果可能会导致辅助生殖技术 (ART) 的变化并影响 ART 预防保健的重点受孕的个体。