Investigating the roles of active DNA demethylation pathways in epigenetic reprogramming

项目摘要 这项建议的目的是确定活跃的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甲基化形式为5- 甲基胞苷(5mC)是基因表达和细胞特性的重要表观遗传调节因子。 基因组5mC水平的失调导致了许多人类发育障碍，包括Rett 综合症、青少年癌症和印记障碍，如Beckwith-Wiedemann综合征。而DNA 甲基化途径被很好地定义，但控制5mC去除的机制却知之甚少。 Ten-11易位(Tet)家族的成员通过 将5mC氧化成5-羟甲基胞嘧啶(5hmC)、5-甲酰胞嘧啶(5fC)或5-羧基胞嘧啶(5caC)。 这些氧化残基不能被维持甲基转移酶识别，导致它们在 在一个被称为“主动修饰被动稀释”(AM-PD)的过程中，细胞分裂了几轮。 或者，5fC和5caC可以被胸腺嘧啶DNA糖基酶(TDG)切割为靶点，触发碱基 切除修复以恢复未修饰的胞嘧啶。这一过程称为“主动修改，主动修改 删除“(AM-AR)。尽管先前的工作表明AM-PD和AM-AR通路执行不同 在功能方面，研究人员缺乏必要的工具来区分体内的这两条途径。到那时候 最终，我们的合作者开发出了新的Tet突变体，它精通于5hmC的产生，但不能产生5fC或 5caC，AM-AR去甲基化所必需的底物。使用小鼠Tet1的同源敲入模型， 目标1将测试AM-AR缺乏如何影响生殖系发育。全基因组的5mC和5hmC水平将 在TET1突变生殖细胞和配子中被测量并与所确定的转录RNA水平相关 作者：RNAseq.为了验证AM-AR途径缺失导致女性不育的假设，组织学 检测还将用于跟踪TET1突变小鼠的卵母细胞成熟情况。《目标2》将使用一部特色化的 依赖于Tet酶家族来确定功能差异的IPSC模型系统 在AM-AR和AM-PD通路之间。为了测试AM-PD通路是否足以驱动IPSC 重新编程，Tet三基因敲除小鼠胚胎成纤维细胞将被AM-AR缺陷的Tet1转导 并接受OSK重新编程。此外，根据我们的假设，AM-AR去甲基化 通过招募组蛋白修饰物和染色质重构体来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 AT基因对IPSC重新编程很重要。总之，这些目标将阐明DNA的不同作用 去甲基化途径在调节细胞特性和哺乳动物发育中的作用，并可能指向新的 AM-AR途径在促进超过5mC缺失的表观遗传重编程中的作用。

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.