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

基本信息

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
项目类别：
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

项目摘要

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：基因组印记是表观遗传调控的一种形式，它限制基因的关键子集的表达一个亲本等位基因。印记基因表达主要受差异甲基化调节，即在配子发生过程中获得的，在称为印记的顺式作用元件上的两个亲本等位基因之间控制区域（ICR）。活性或抑制性组蛋白尾翻译后修饰的不对称模式 (PTM) 进一步强化了印记位点的单等位基因表达。在人类中，异常表达印记基因与 Beckwith-Wiedemann 和 Prader-Willi 等印记疾病有关综合症。 ICR 的甲基化在整个发育过程中保持稳定，并且仅在发育过程中动态调节。原始生殖细胞（PGC）发育和配子发生。在哺乳动物中，PGC 来源于体细胞外胚层并进行表观基因组重编程，以便：1）防止妊娠传播获得下一代的表突变，2）消除父母遗传的体细胞印记标记允许获得与胚胎性别一致的新印记。我们之前的工作实验室和其他人暗示了 TET1（一种甲基胞嘧啶双加氧酶）在建立成熟雄性和雌性配子中的适当印记。我的初步数据表明，虽然 TET1 对于 PGC 中 ICR 子集的完全甲基化擦除是必要的，它也发挥了先前的作用保护父系甲基化 ICR 免于在卵母细胞中获得异位甲基化的尚未探索的作用。作为 TET1 是一种大蛋白质，能够与各种染色质重塑因子相互作用并影响其活性，我假设 TET1 在促进种系印记的正确建立方面具有新颖的非催化作用重塑更广泛的 ICR 染色质景观。该提案的目的是研究如何 PGC 发育过程中 ICR 染色质景观的重塑有助于正确获得性别卵子发生过程中特定的DNA甲基化模式。在目标 1 中，我将检验 TET1 促进的假设 PGC 甲基化擦除过程中 ICR 的染色质重塑。我将使用等位基因特异性切割使用核酸酶 (CUT&RUN) 定位和释放，以评估 Tet1-/- 中染色质重编程的效率混合 PGC。在目标 2 中，除了父系甲基化 ICR 之外，我还将识别位点，这些位点异位获得在没有 TET1 的情况下，卵子发生过程中发生甲基化。研究异位从头发生的机制 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-/- 卵母细胞中父系甲基化 ICR 的甲基转移酶抗性染色质景观剪切并运行。总的来说，该提案将阐明 TET1 在在 ICR 处建立适当的染色质景观并正确获取种系印记。种系印记建立的机制研究将增强我们对分子印记的理解对高质量配子的要求，以及人类印记障碍的病因学。