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: 10320339
• 负责人: Rexxi Diptya Prasasya
• 金额: $ 6.86万
• 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/10320339
• 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控形式，其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控， 在配子发生过程中获得，在两个亲本等位基因之间的顺式作用元件称为印记 控制区域（ICR）。活性或抑制性组蛋白尾部翻译后修饰的不对称模式 （PTM）进一步加强印迹基因座的单等位基因表达。在人类中， 印记基因与印记疾病有关，如Beckwith-Wiedemann和Prader-Willi 综合征ICR的甲基化在整个发育过程中是稳定的，并且仅在发育期间动态调节。 原始生殖细胞（PGC）发育和配子发生。在哺乳动物中，PGCs来源于体细胞， 表观基因组重编程的目的是：1）防止妊娠期遗传性疾病的传播 获得的表突变到下一代和2）删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。我们以前的工作 实验室和其他人已经暗示了TET1（一种甲基胞嘧啶双加氧酶）在建立 在成熟的雄性和雌性配子中有适当的印记。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需的，它也起到了先前的作用。 保护父系甲基化的ICR在卵母细胞中获得异位甲基化的作用尚未探索。作为 TET1是一种大蛋白，具有与各种染色质重塑物相互作用并影响其活性的能力， 我假设TET1在促进生殖系印记的正确建立中具有一种新的非催化作用， 重塑ICR的更广泛的染色质景观。本提案的目的是调查 PGC发育中ICR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。在目标1中，我将检验TET 1促进 在PGCs中甲基化消除期间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）靶向和释放，以评估Tet1-/-中染色质重编程的效率。 杂交PGCs。在目标2中，我将确定位点，除了父系甲基化的ICR，异位获得 在没有TET1的情况下，卵子发生过程中的甲基化。探讨异位新生的机制， 为了研究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， 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 切和运行。总的来说，这项建议将阐明TET1在细胞内的催化和非催化功能。 在ICR处建立适当的染色质景观和正确获得生殖系印记。 对生殖系印记建立机制的研究将有助于我们更好地理解生殖系印记形成的分子机制。 高质量配子的要求，以及人类印记疾病的病因。