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

项目摘要： 基因组印迹是表观遗传调控的一种形式，它将关键基因子集的表达限制在 一个父母的等位基因。印记基因的表达主要由差异甲基化来调节，这是 在配子发生期间，在顺式作用元件上的两个亲本等位基因之间获得的，称为印迹 控制区域(ICR)。翻译后活性或抑制性组蛋白尾部修饰的不对称模式 (PTMS)进一步增强了印迹基因座的单等位基因表达。在人类中，异常表达 印记基因与印记疾病有关，如Beckwith-Wiedemann和Prader-Willi 综合症。ICRs的甲基化在整个发育过程中是稳定的，只有在 原始生殖细胞(PGC)发育和配子发生。在哺乳动物中，原生殖细胞来源于体细胞。 并进行表观基因组重编程，以：1)防止妊娠传播 获得的表型突变传给下一代，并2)将亲代遗传的体细胞印记抹去 允许获得与胚胎性别一致的新印记。我们之前的工作 实验室和其他人已经证实，TET1，一种甲基胞嘧啶双加氧酶，在建立 成熟的雄配子和雌配子中有适当的印记。我的初步数据显示，虽然TET1 对于PGC中ICR子集的完全甲基化擦除是必需的，它还起到了以前的 保护父系甲基化的ICR在卵母细胞中防止异位甲基化的作用尚未被探索。AS Tet1是一种大蛋白，能够与各种染色质重构体相互作用并影响其活性， 我假设TET1在促进生殖系印迹的正确建立方面发挥了一种新的非催化作用 重塑了ICR更广泛的染色质景观。这项提案的目的是调查 PGC发育中ICRs染色质景观的重塑有助于获得适当的性别- 卵子发生过程中特定的DNA甲基化模式。在目标1中，我将测试TET1有助于 前列腺癌甲基化消除过程中ICRs的染色质重塑。我将使用等位基因特定的切割 使用核酸酶(切割和运行)评估Tet1-/-染色质重编程效率的靶点和释放 混合PGC。在目标2中，我将确定除了父系甲基化的ICR外的基因座，这些基因座异位获得 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 Tet1-/-卵母细胞中的甲基化，我将调查与DNA相关的组蛋白PTM的状态 Tet1-/-卵母细胞父系甲基化ICRs的甲基转移酶抗性染色质图谱 切入并跑动。总而言之，这项建议将阐明TET1在 在ICR建立适当的染色质环境并正确获取生殖系印记。 对生殖系印迹建立的机理研究将加深我们对分子的理解 对高质量配子的要求，以及人类印迹疾病的病因学。

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.