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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，参与基因的转录调控，发育和分化，其失调有助于人类发病机制。它是由 DNA甲基转移酶家族，包括催化活性的Dnmt 1、Dnmt 3a、Dnmt 3b。DNA甲基化在小鼠着床前发育中起主要作用。为了建立一个新的表观基因组，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。后植入，由从头酶Dnmt 3a介导的外胚层细胞中的从头甲基化波， Dnmt 3b导致Dnmt 1维持的新甲基化模式，这些模式形成了组织特异性甲基化的基础。表达和分化。 Dnmt 3b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被证明是 Dnmt 3b-/-小鼠的胚胎致死率。我们最近发现，Dnmt 3bCI/CI小鼠表达的催化失活 Dnmt 3bCI蛋白在出生前和出生后的发育中都存活。分子分析表明 Dnmt 3b的功能--将其他Dnmt募集到适当基因组位点的能力--而不是其催化活性，对甲基化和存活很重要。在这里，我们假设Dnmt 3b是一种多面蛋白，参与甲基化的各种活动影响产前和产后发育，在小鼠中形成疾病。为了验证这一假设，在Aim 1中，我们分析了在2000年至2010年期间的总体甲基化和表达。缺乏各种Dnmt活性的小鼠的不同发育阶段，以确定Dnmt 3b的范围在体内Dnmt 3a介导的从头甲基化中的辅助功能以及各种基因组特征，包括基因体、生殖系基因和转座子。在目标2中，将测试 Dnmt 3b与其他Dnmts复合，并有助于由其他Dnmts诱导的从头甲基化， Dnmt 1-/-; Dnmt 3a-/-; Dnmt 3b-/-三重敲除小鼠胚胎干细胞。此外，我们还将进行基因测试， Dnmt 3a和Dnmt 3l对于Dnmt 3b辅助功能的重要性，并验证我们在人类细胞中的数据线在目的3中，我们将对通过使用Dnmt 3b +/+和Dnmt 3bCI/CI小鼠进行纵向研究。体外受精（IVF）技术，以分析疾病发展，Dnmt水平，甲基化率和基因表达错误，以及它们随时间的持续性。总的来说，我们的研究将揭示Dnmt 3b活性在小鼠发育中的生理相关性，揭示利用Dnmt 3b功能的基本机制及其在IVF中的参与。我们的结果可能会导致辅助生殖技术（ART）的变化，并影响ART预防护理的重点- 设想的个人。