Investigating the roles of active DNA demethylation pathways in epigenetic reprogramming

项目摘要 本提案的目的是确定活性DNA去甲基化途径对 哺乳动物生殖系发育过程中的表观遗传重编程。DNA甲基化的5- 甲基胞嘧啶（5 mC）作为基因表达和细胞特性的基本表观遗传调节剂。 基因组5 mC水平失调导致许多人类发育障碍，包括Rett 综合征、青少年癌症和印记障碍如贝克-威德曼综合征。而DNA 尽管甲基化途径已明确定义，但控制5 mC去除的机制知之甚少。 10 - 11易位（泰特）酶家族的成员通过以下途径调节活性DNA去甲基化 5 mC氧化为5-羟甲基胞嘧啶（5 hmC）、5-甲酰基胞嘧啶（5 fC）或5-羧基胞嘧啶（5caC）。 这些氧化的残基不能被维持甲基转移酶识别，导致它们的损失超过 在称为“被动稀释的主动修饰”（AM-PD）的过程中进行几轮细胞分裂。 或者，5 fC和5caC可以被靶向用于胸腺嘧啶DNA糖基化酶（TDG）切割，触发碱基 切除修复以恢复未修饰的胞嘧啶。该过程被称为“具有活性的活性修饰”。 移除”（AM-AR）。虽然先前的工作表明AM-PD和AM-AR途径进行了不同的 由于这些功能，研究人员缺乏必要的工具来区分体内的两种途径。与 最后，我们的合作者已经开发出了新的泰特突变体，它们精通5 hmC的产生，但不精通5 fC或 5caC是AM-AR去甲基化的必要底物。使用小鼠Tet 1的正向敲入模型， 目的1将测试AM-AR缺陷如何影响生殖系发育。全基因组5 mC和5 hmC水平将 在TET 1突变生殖细胞和配子中测量，并与确定的转录组RNA水平相关 通过RNA-seq。为了检验AM-AR途径的缺失导致女性生育力低下的假设，组织学检查显示， 测定也将用于追踪TET 1突变小鼠中的卵母细胞成熟。目标2将使用一个特征良好的 依赖于泰特酶家族以确定功能差异的iPSC模型系统 在AM-AR和AM-PD通路之间。测试AM-PD通路是否足以驱动iPSC 重新编程后，将用AM-AR缺陷型Tet 1转导泰特三重敲除小鼠胚胎成纤维细胞 并进行OSK重编程。此外，根据我们的假设，AM-AR去甲基化 通过组蛋白修饰剂和染色质重塑剂的募集促进广泛的表观遗传变化， 将进行染色质免疫沉淀实验以监测调节性组蛋白标记的变化 对iPSC重编程很重要的基因总之，这些目标将阐明DNA的独特作用 去甲基化途径在调节细胞的身份和哺乳动物的发展，并可能指出新的 AM-AR通路在促进超过5 mC擦除的表观遗传重编程中的功能。

Project Summary The objective of this proposal is to determine the contribution of active DNA demethylation pathways to epigenetic reprogramming during mammalian germline development. DNA methylation in the form of 5- methylcyosine (5mC) serves as an essential epigenetic regulator of gene expression and cellular identity. Dysregulation of genome 5mC levels contributes to a number of human developmental disorders, including Rett syndrome, juvenile cancers, and imprinting disorders such as Beckwith-Wiedemann syndrome. While DNA methylation pathways are well defined, the mechanisms controlling 5mC removal are poorly understood. Members of the Ten-eleven Translocation (TET) family of enzymes regulate active DNA demethylation through the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). These oxidized residues are not recognized by maintenance methyltransferases, resulting in their loss over several rounds of cellular division in a process known as “active modification with passive dilution” (AM-PD). Alternatively, 5fC and 5caC may be targeted for cleavage by thymine DNA glycosylase (TDG), triggering base excision repair to restore the unmodified cytosine. This process is referred to as “active modification with active removal” (AM-AR). Although previous work suggests the AM-PD and AM-AR pathways carry-out distinct functions, researchers have lacked the necessary tools to distinguish between the two pathways in vivo. To that end, our collaborators have developed novel TET mutants proficient for the generation of 5hmC but not 5fC or 5caC, the necessary substrates for AM-AR demethylation. Using an orthologous knock-in model for mouse Tet1, Aim 1 will test how AM-AR deficiency affects germline development. Genome-wide 5mC and 5hmC levels will be measured in TET1 mutant germ cells an gametes and correlated with transcriptomic RNA levels determined by RNA-seq. To test the hypothesis that loss of the AM-AR pathway leads to female subfertility, histological assays will also be used to track oocyte maturation in TET1 mutant mice. Aim 2 will use a well-characterized iPSC model system that is dependent upon the TET family of enzymes to determine functional differences between the AM-AR and AM-PD pathways. To test whether the AM-PD pathway is sufficient to drive iPSC reprogramming, TET triple-knockout mouse embryonic fibroblasts will be transduced with AM-AR-deficient Tet1 and subjected to OSK reprogramming. Additionally, based on our hypothesis that AM-AR demethylation promotes broad epigenetic changes through the recruitment of histone modifiers and chromatin remodelers, chromatin immunoprecipitation experiments will be performed to monitor for changes in regulatory histone marks at genes important for iPSC reprogramming. Together, these Aims will elucidate the distinct roles of DNA demethylation pathways in regulating cellular identity and mammalian development, and may point to novel functions for the AM-AR pathway in promoting epigenetic reprogramming beyond 5mC erasure.