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甲基化是参与参与基因的转录调节的表观遗传修饰发育与分化及其放松管制有助于人类的发病机理。它被 DNA甲基转移酶的家族，包括催化活性DNMT1，DNMT3A，DNMT3B。 DNA甲基化在小鼠的植入前发育中起主要作用。为了建立新的表观基因组，小鼠合子基因组经历了表观遗传重编程，包括在8细胞阶段的全局DNA脱甲基化。之上植入，在新酶DNMT3A和 DNMT3B导致DNMT1维持的新甲基化模式，构成了组织特异性的基础表达和分化。 DNMT3B调节发育和印迹基因，X染色体灭活，周围质粒区域，基因体和其他基因组区域。它在鼠标开发中的重要性已通过 DNMT3B - / - 小鼠的胚胎致死性。我们最近发现DNMT3BCI/CI小鼠表达催化性无活性 DNMT3BCI蛋白在产后和产后发育中幸存下来。分子分析表明附件功能 - 将其他DNMT募集到适当基因组的能力 - 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是一种多面蛋白甲基化涉及的各种活动会影响产后和产后发展，并且对于防止小鼠疾病形成。为了检验这一假设，在AIM 1中，我们分析了全局甲基化和表达在缺乏各种DNMT活动的小鼠中，不同的发育阶段可以确定DNMT3B的范围 DNMT3A介导的从体内从头甲基化中的辅助功能以及调节的调节各种基因组特征，包括基因体，种系基因和转座子。在AIM 2中，将测试能力 DNMT3B与其他DNMT的复合物，并有助于其他DNMT诱导的从头甲基化 dnmt1 - / - ; dnmt3a - / - ; dnmt3b - / - 三重基因敲除小鼠胚胎干细胞。此外，我们将基因测试 DNMT3A和DNMT3L对DNMT3B附属功能的重要性，并在人类细胞中验证我们的数据线。在AIM 3中，我们将对DNMT3B+/+和DNMT3BCI/CI小鼠进行纵向研究体外受精（IVF）技术，用于分析疾病发育，DNMT水平，甲基化速率和基因表达误差及其持久性随着时间的流逝。总的来说，我们的研究将揭示DNMT3B活动在小鼠发育中的身体相关性，使用DNMT3B功能及其参与IVF的基本机制。我们的结果可能导致辅助生殖技术（ART）的变化，并影响艺术预防护理的重点构思的个人。