Dnmt3b activities in mouse development

DNA甲基化是一种表观遗传修饰，涉及基因转录调控，涉及 发育和分化，以及它的去调节作用在人类发病机制中的作用。它是由 DNA甲基转移酶家族，包括具有催化活性的DNMT1、DNMT3A、DNMT3B。DNA甲基化 在小鼠着床前发育中起主要作用。为了建立一个新的表观基因组，小鼠受精卵 基因组经历表观遗传重新编程，包括8-细胞阶段的全球DNA去甲基化。vt.在.的基础上 植入，由从头酶DNMT3A和DNMT3A和DNMT3A介导的外胚细胞从头甲基化浪潮 Dnmt3b导致由Dnmt1维持的新的甲基化模式，形成组织特异性的基础 表达和分化。 Dnmt3b调控发育和印记基因、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以下几个方面得到证明 Dnmt3b-/-小鼠的胚胎致死性。我们最近发现Dnmt3bCI/CI小鼠表达的是催化失活 DNMT3bCI蛋白在出生前和出生后的发育中都存活了下来。分子分析表明，辅助物 Dnmt3b的功能--将其他DNMT招募到适当的基因组位置的能力--而不是它的催化活性， 对甲基化和生存很重要。这里我们假设Dnmt3b是一种多方面的蛋白质，它 甲基化所涉及的各种活动会影响出生前和出生后的发育，对预防 小鼠的疾病形成。为了验证这一假设，在目标1中，我们分析了全球甲基化和在 缺乏不同DNMT活性的小鼠不同发育阶段确定DNMT3b的范围 DNMT3A介导的体内从头甲基化中的辅助功能及其转录调控 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，将测试能力 DNMT3b与其他DNMT形成复合体，并参与其他DNMT诱导的从头甲基化。 DNMT1-/-；DNMT3A-/-；DNMT3b-/-三基因敲除小鼠胚胎干细胞。此外，我们还将进行基因检测 DNMT3A和Dnmt3l对Dnmt3b辅助功能的重要性以及在人类细胞中验证我们的数据 排队。在目标3，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展、DNMT水平、甲基化率和 基因表达错误，以及它们随时间的持久性。 总而言之，我们的研究将揭示Dnmt3b活动与小鼠发育的生理相关性， 揭示利用Dnmt3b功能的基本机制及其在体外受精中的参与。我们的结果可能会导致 辅助生殖技术(ART)的变化和影响ART预防保健的重点-- 受孕的个体。

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.