Investigating the roles of active DNA demethylation pathways in epigenetic reprogramming

研究活性 DNA 去甲基化途径在表观遗传重编程中的作用

• 批准号: 10212435
• 负责人: Blake Alexander Caldwell
• 金额: $ 0.21万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10212435
• 关键词: Adolescent; Affect; Base Excision Repairs; Beckwith-Wiedemann Syndrome; Biological; Biological Assay; Biological Models; Cell Culture Techniques; Cells; Chromatin; Cytosine; DNA; DNA Methylation; DNA Modification Methylases; DNA biosynthesis; Data; Defect; Derivation procedure; Development; Disease; Embryo; Embryonic Development; Enzymes; Epigenetic Process; Event; Excision; Family; Female; Fertilization; Fibroblasts; Gametogenesis; Gene Expression; Generations; Genes; Genome; Genome Stability; Genomics; Germ Cells; Goals; Histologic; Histones; Human; Human Genome; Knock-in; Knock-in Mouse; Knockout Mice; Laboratories; Link; Maintenance; Malignant Neoplasms; Measures; Meiosis; Methods; Methyltransferase; Modeling; Modification; Monitor; Mus; Mutant Strains Mice; Mutation; Oocytes; Oogenesis; Oxides; Pathway interactions; Pennsylvania; Phenotype; Play; Process; RNA; Regulator Genes; Research Personnel; Rett Syndrome; Role; Shapes; Site; Structure of primordial sex cell; System; Testing; Thymine DNA Glycosylase; Time; Universities; Work; base; chromatin immunoprecipitation; chromatin remodeling; demethylation; developmental disease; epigenome; experimental study; genome-wide; genome-wide analysis; histological stains; imprint; in vivo; induced pluripotent stem cell; mammalian genome; member; mouse model; mutant; novel; oocyte maturation; overexpression; oxidation; pluripotency; recruit; stem cell model; subfertility; success; tool; transcriptome sequencing; transcriptomics; whole genome

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.

项目概要 该提案的目的是确定主动 DNA 去甲基化途径对 哺乳动物种系发育过程中的表观遗传重编程。 DNA甲基化形式为5- 甲基胞嘧啶 (5mC) 是基因表达和细胞身份的重要表观遗传调节因子。 基因组 5mC 水平失调会导致许多人类发育障碍，包括 Rett 综合征、青少年癌症和印记疾病，例如贝克威斯-维德曼综合征。而DNA 甲基化途径已明确，但控制 5mC 去除的机制却知之甚少。 10-11 易位 (TET) 酶家族的成员通过以下方式调节活性 DNA 去甲基化： 5mC 氧化为 5-羟甲基胞嘧啶 (5hmC)、5-甲酰胞嘧啶 (5fC) 或 5-羧基胞嘧啶 (5caC)。 这些氧化残基不被维持性甲基转移酶识别，导致它们丢失 多轮细胞分裂的过程称为“主动修饰与被动稀释”（AM-PD）。 或者，胸腺嘧啶 DNA 糖基化酶 (TDG) 可以靶向 5fC 和 5caC 进行切割，触发碱基 切除修复以恢复未修饰的胞嘧啶。这个过程被称为“主动修改” 去除”（AM-AR）。尽管之前的工作表明 AM-PD 和 AM-AR 途径执行不同的 由于功能不同，研究人员缺乏必要的工具来区分体内的两种途径。对此 最后，我们的合作者开发了新的 TET 突变体，能够熟练生成 5hmC 但不能生成 5fC 或 5caC，AM-AR去甲基化的必要底物。使用小鼠 Tet1 的直系同源敲入模型， 目标 1 将测试 AM-AR 缺陷如何影响种系发育。全基因组 5mC 和 5hmC 水平将 在 TET1 突变生殖细胞和配子中进行测量，并与确定的转录组 RNA 水平相关 通过RNA测序。为了检验 AM-AR 通路缺失会导致女性生育力低下的假设，组织学研究 检测还将用于追踪 TET1 突变小鼠的卵母细胞成熟情况。目标 2 将使用一个特征良好的 iPSC 模型系统依赖于 TET 酶家族来确定功能差异 AM-AR 和 AM-PD 途径之间。测试 AM-PD 通路是否足以驱动 iPSC 重编程，TET 三重敲除小鼠胚胎成纤维细胞将被 AM-AR 缺陷的 Tet1 转导 并进行 OSK 重新编程。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招募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组蛋白标记的变化 对 iPSC 重编程很重要的基因。这些目标将共同阐明 DNA 的独特作用 去甲基化途径在调节细胞身份和哺乳动物发育中的作用，并可能指出新的 AM-AR 通路在促进表观遗传重编程超过 5mC 擦除方面发挥作用。