Role of TET1 in germ cell reprogramming and development

TET1 在生殖细胞重编程和发育中的作用

基本信息

批准号：
10689734
负责人：
MARISA S. BARTOLOMEI
金额：
$ 30.76万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10689734
关键词：
Address Adult Aging Alleles Base Excision Repairs Behavioral Assay Biological Assay Brain Cell Reprogramming Chromatin Coupled DNA DNA Methylation DNA Modification Methylases DNA Modification Process DNA biosynthesis Data Development Disease Embryo Engineering Ensure Enzymes Epigenetic Process Excision Failure Family Fertilization Fibroblasts Generations Genes Genetic Genetic Transcription Genome Genomics Germ Cells Gonadal structure Heterogeneity Infertility Link Maintenance Mediating Meiosis Methylation Mouse Strains Mus Mutant Strains Mice Mutation Oocytes Partner in relationship Pathway interactions Pattern Phenotype Play Production Publishing Role Shapes Somatic Cell Structure of primordial sex cell Syndrome Technology Testing Tissues Wild Type Mouse Work base bead chip chromatin protein demethylation developmental disease egg epigenome epigenomics fetal genome-wide genomic locus histone modification imprint in vivo induced pluripotent stem cell mammalian genome methylation pattern migration mouse genome mutant neurobehavior new technology next generation offspring oxidation prevent pup response sexual dimorphism sperm cell tool transcriptome sequencing transmission process

项目摘要

The mammalian germline must be reprogrammed to facilitate proper development. This reprogramming, which includes the erasure of DNA methylation and histone modifications, ensures the establishment of gamete- appropriate epigenetic patterns and minimizes the transmission of epimutations to offspring. While much of the genome undergoes replication-coupled passive DNA demethylation, a critical role for Ten-eleven Translocation (TET) family enzymes, specifically TET1, has been demonstrated for active demethylation of genomic sequences such as imprinting control regions (ICRs) and germline-specific genes. The proposed work will use newly developed mouse strains and sequencing technologies to test the hypothesis that iterative oxidation and noncatalytic functions of TET1 are required for DNA methylation erasure and reprogramming of the mouse genome, including ICRs and meiosis-specific genes, during germline and somatic development. TET enzymes can catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Oxidized 5mC bases, particularly 5hmC, can play independent epigenetic roles in somatic tissues including the brain, but are most significantly thought to function as DNA demethylation intermediates. The distinctive demethylation pathways supported by 5hmC versus 5fC/5caC have confounded efforts to decipher the precise mechanistic role for TET1. Yet further challenges are posed by potential non-catalytic roles for TET1, which is known to interact with chromatin modifying enzymes. Published work and our preliminary data suggest that a role for catalytic and non-catalytic TET1 activities for demethylation, but the mechanism, timing and target sequences remain incompletely understood. Thus, we propose to address (1) whether iterative oxidation to 5fC/5caC is required for reprogramming, (2) whether TET1 has a noncatalytic reprogramming role, and (3) what sequences require various TET activities. Specific Aim 1 will examine the precise role of TET1 in reprogramming at ICRs and genome-wide in primordial germ cells (PGCs). We have engineered mice that either stall 5mC oxidation at 5hmC (Tet1v) or lack catalytic function (Tet1hxd) and will test their effects on DNA methylation reprogramming using our new technology which resolves 5mC and 5hmC, and profile associated chromatin dynamics during PGC development. Our preliminary data using the new Infinium Mouse BeadChip suggest that the Tet1 mutant mice sperm have non-overlapping aberrant patterns of DNA modification. Thus, Specific Aim 2 will assess the epigenomic and phenotypic consequences of Tet1 mutations in homozygous mutant gametes and the offspring that arise from these gametes. Finally, Specific Aim 3 will determine the epigenomic and phenotypic consequences of Tet1 stalling and catalytic mutations in homozygous mutant adult and aging mice. This work will enable an assessment of the role of TET enzymes in genome reprogramming, dissecting the requirement of noncatalytic activity and iterative oxidation by TET enzymes.

哺乳动物种系必须重新编程以促进正常发育。这次重新编程，包括消除 DNA 甲基化和组蛋白修饰，确保配子的建立适当的表观遗传模式并最大限度地减少表观突变向后代的传播。虽然大部分基因组经历复制耦合被动 DNA 去甲基化，这对 10-11 易位至关重要 (TET) 家族酶，特别是 TET1，已被证明可以主动进行基因组去甲基化序列，例如印记控制区 (ICR) 和种系特异性基因。拟议的工作将使用新开发的小鼠品系和测序技术来检验迭代氧化和 TET1 的非催化功能是小鼠 DNA 甲基化擦除和重编程所必需的种系和体细胞发育过程中的基因组，包括 ICR 和减数分裂特异性基因。四环素酶可以催化 5-甲基胞嘧啶 (5mC) 最多三次连续氧化，生成 5-羟甲基胞嘧啶 (5hmC)、5-甲酰胞嘧啶 (5fC) 或 5-羧基胞嘧啶 (5caC)。氧化的 5mC 碱基，特别是 5hmC，可以在包括大脑在内的体细胞组织中发挥独立的表观遗传作用，但最重要的是被认为作为 DNA 去甲基化中间体。 5hmC 支持的独特去甲基化途径与 5fC/5caC 相比，破译 TET1 精确机制作用的努力变得混乱。更进一步 TET1 的潜在非催化作用带来了挑战，已知 TET1 会与染色质相互作用修饰酶。已发表的工作和我们的初步数据表明催化和非催化的作用 TET1具有去甲基化活性，但机制、时间和靶序列仍不完整明白了。因此，我们建议解决（1）是否需要迭代氧化至 5fC/5caC 重编程，(2) TET1 是否具有非催化重编程作用，以及 (3) 需要什么序列各种 TET 活动。具体目标 1 将检查 TET1 在 ICR 重编程中的精确作用，原始生殖细胞（PGC）中的全基因组。我们设计了小鼠，可以在以下条件下阻止 5mC 氧化： 5hmC (Tet1v) 或缺乏催化功能 (Tet1hxd)，将测试它们对 DNA 甲基化重编程的影响使用我们的新技术解析 5mC 和 5hmC，并分析相关染色质动态 PGC 开发。我们使用新的 Infinium Mouse BeadChip 的初步数据表明 Tet1 突变体小鼠精子具有不重叠的 DNA 修饰异常模式。因此，具体目标 2 将评估 Tet1 突变对纯合突变配子及其后代的表观基因组和表型影响由这些配子产生。最后，具体目标 3 将确定表观基因组和表型 Tet1 停滞和催化突变对纯合突变成年小鼠和衰老小鼠的影响。这部作品将能够评估 TET 酶在基因组重编程中的作用，剖析需求 TET 酶的非催化活性和迭代氧化。