Investigation of the role of TET proteins in epigenetic reprogramming and establishment of germline imprinting

研究TET蛋白在表观遗传重编程和种系印记建立中的作用

• 批准号: 10475225
• 负责人: Rexxi Diptya Prasasya
• 金额: $ 7.41万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10475225
• 关键词: Affect; Affinity; Alleles; Behavior; Binding Proteins; Catalytic Domain; Chromatin; Chromosomes; Common Core; Comprehension; DNA; DNA Methylation; DNA Modification Methylases; Data; Development; Dioxygenases; Disease; Embryo; Embryonic Development; Ensure; Environment; Enzymes; Epiblast; Epigenetic Process; Etiology; Exhibits; Family; Female; Fertilization; Fetal Development; Gametogenesis; Gene Expression; Genes; Genetic Diseases; Genome; Genomic Imprinting; Germ Cells; Gonadal structure; Growth; Histones; Human; Human Genetics; Hybrids; Hypermethylation; Inherited; Intracisternal A-Particle Elements; Investigation; Knockout Mice; Laboratories; Mammals; Meiosis; Methylation; Modification; Molecular; Mus; Oocytes; Oogenesis; Pattern; Play; Post-Translational Protein Processing; Prader-Willi Syndrome; Pregnancy; Protein translocation; Proteins; Regulation; Residual state; Resistance; Rest; Role; Silver-Russell syndrome; Spermatogenesis; Structure of primordial sex cell; Tail; Testing; Tissues; Transcriptional Regulation; Work; blastocyst; chromatin modification; chromatin remodeling; cis acting element; demethylation; epigenetic regulation; epigenome; experimental study; genome-wide; histone modification; imprint; insight; male; methylation pattern; next generation; novel; nuclease; paternal imprint; prevent; promoter; ras-GRF1; sex; transmission process

Project abstract: Genomic imprinting is a form of epigenetic regulation that restricts expression of a critical subset of genes to one parental allele. Imprinted gene expression is primarily regulated by differential methylation, which is acquired during gametogenesis, between the two parental alleles on cis-acting elements known as imprinting control regions (ICRs). Asymmetric patterns of active or repressive histone tail post-translational modifications (PTMs) further reinforce the monoallelic expression of imprinted loci. In humans, abnormal expression of imprinted genes is associated with imprinting disorders such as Beckwith-Wiedemann and Prader-Willi syndromes. Methylation at ICRs is stable throughout development and is only dynamically modulated during primordial germ cell (PGC) development and gametogenesis. In mammals, PGCs are derived from the somatic epiblast and undergo epigenome reprogramming in order to: 1) prevent the transmission of gestationally acquired epimutations to the next generation and 2) erase the parentally-inherited somatic imprinting marks to allow for the acquisition of new imprints that are consistent with the sex of the embryo. Previous work from our laboratory and others have implicated the role of TET1, a methylcytosine dioxygenase, in the establishment of proper imprinting marks in the mature male and female gametes. My preliminary data suggest that while TET1 is necessary for complete methylation erasure of a subset of ICRs in PGCs, it also plays a previously unexplored role of protecting paternally methylated ICRs from gaining ectopic methylation in the oocytes. As TET1 is a large protein with the ability to interact with and influence activities of various chromatin remodelers, I hypothesize a novel non-catalytic role for TET1 in promoting the proper establishment of germline imprints by remodeling the broader chromatin landscape of ICRs. The objective of this proposal is to investigate how the remodeled chromatin landscape of ICRs in PGC development contributes to the proper acquisition of sex- specific DNA methylation patterns during oogenesis. In Aim 1, I will test the hypothesis that TET1 facilitates chromatin remodeling of the ICRs during methylation erasure in PGCs. I will use allele-specific cleavage under targets and release using nuclease (CUT&RUN) to assess the efficiency of chromatin reprogramming in Tet1-/- hybrid PGCs. In Aim 2, I will identify loci, in addition to paternally methylated ICRs, which ectopically gain methylation during oogenesis in absence of TET1. To investigate the mechanism underlying ectopic de novo methylation in Tet1-/- oocytes, I will investigate the status of histone PTMs that are associated with DNA methyltransferase-resistant chromatin landscape at paternally methylated ICRs in Tet1-/- oocytes using CUT&RUN. Collectively, this proposal will elucidate the catalytic and non-catalytic functions of TET1 in the establishment of the proper chromatin landscape at ICRs and the correct acquisition of germline imprints. Mechanistic studies of germline imprinting establishment will enhance our comprehension of the molecular requirements for high quality gametes, as well as etiology of human imprinting disorders.

项目摘要： 基因组印记是一种表观遗传调节的一种形式，它限制了基因的临界子集的表达 一个父母等位基因。印迹基因表达主要由差分甲基化调节，这就是 在配子发生期间获得的，在两个父母的等位基因在顺式作用元素上被称为印迹 控制区（ICR）。活性或抑制性组蛋白尾巴的不对称模式翻译后修饰 （PTM）进一步增强了印迹基因座的单相表达。在人类中，异常表达 印迹基因与诸如贝克维斯·威德曼（Beckwith-Wiedemann）和prader-willi之类的烙印疾病有关 综合征。 ICRS的甲基化在整个开发过程中都是稳定的，只有在过程中动态调制 原始生殖细胞（PGC）发育和配子发生。在哺乳动物中，PGC源自躯体 覆盖层并进行表观基因组重编程以：1）防止妊娠的传播 获得了下一代的表达，2）将父母携带的躯体印记删除 允许获取与胚胎性别一致的新烙印。我们以前的工作 实验室和其他人牵涉到甲基霉素二加氧酶Tet1的作用，在建立中 在成熟的男性和女配子中有适当的印记。我的初步数据表明，当Tet1 对于PGC中ICR的一部分的完全甲基化擦除是必要的，它也播放 未探索的作用是保护含卵母细胞中异位甲基化的亲子甲基化ICR的作用。作为 TET1是一种大蛋白，具有与各种染色质重塑剂相互作用和影响活性的能力， 我假设TET1在促进通过适当建立种系烙印的新型非催化作用 重塑ICR的更广阔的染色质景观。该提议的目的是调查如何 PGC开发中ICRS的重塑染色质景观有助于正确获得性别 卵子发生过程中的特定DNA甲基化模式。在AIM 1中，我将测试TET1促进的假设 PGC中甲基化擦除过程中ICRS的染色质重塑。我将使用特定于等位基因的乳沟 靶标和使用核酸酶（切割和运行）释放，以评估染色质重编程在TET1 - / - 中的效率 混合PGC。在AIM 2中，除了属于亲子甲基化的ICR之外，我还将确定基因座 在没有TET1的情况下，卵子发生过程中的甲基化。研究从头开始的基础机制 TET1 - / - 卵母细胞中的甲基化，我将研究与DNA相关的组蛋白PTM的状态 使用TET1 - / - 卵母细胞中的甲基转移酶抗甲基转移酶的染色质景观 剪切。总的来说，该提案将阐明TET1的催化和非催化功能 在ICRS上建立适当的染色质景观，并正确获得种系烙印。 种系印记的机械研究将增强我们对分子的理解 对人类烙印疾病的高质量配子以及病因的要求。