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

• 批准号: 10467364
• 负责人: MARISA S. BARTOLOMEI
• 金额: $ 30.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 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/10467364
• 关键词: Address; Adult; Aging; Alleles; Base Excision Repairs; Behavioral Assay; Biological Assay; Brain; 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; Lead; Link; Maintenance; Mediating; Meiosis; Methylation; Mouse Strains; Mus; Mutant Strains Mice; Mutation; Oocytes; Oxides; 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; mouse genome; mutant; neurobehavior; new technology; next generation; offspring; oxidation; prevent; pup; response; sexual dimorphism; sperm cell; sperm viability; tool; transcriptome sequencing; transmission process

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.

必须重编程哺乳动物种系以促进适当的发育。此重新编程，其中 包括擦除DNA甲基化和组蛋白修饰，确保建立配子 适当的表观遗传模式，并最大程度地降低了对后代的传播。虽然大部分 基因组经历了复制耦合的无源DNA脱甲基化，这是十个易位的关键作用 （TET）家族酶，特别是TET1，已被证明用于基因组的主动脱甲基化 诸如印迹控制区（ICR）和种系特异性基因之类的序列。拟议的工作将使用 新开发的小鼠菌株和测序技术，以测试迭代氧化和 TET1的非催化功能是DNA甲基化擦除和小鼠重编程所必需的 在种系和躯体发育期间，基因组，包括ICR和减数分裂特异性基因。 TET酶 可以催化5-甲基环肽（5MC）连续的三连续氧化，产生5-羟基甲基胞嘧啶 （5HMC），5-甲基环胞嘧啶（5FC）或5-羧基氨酸（5CAC）。氧化的5MC碱基，尤其是5HMC，可以 在包括大脑在内的体细胞组织中扮演独立的表观遗传角色，但最明显地认为 功能充当DNA脱甲基化中间体。 5HMC支持的独特去甲基化途径 相对于5FC/5CAC与破译TET1的精确机理作用的努力混淆了。进一步 挑战是由TET1潜在的非催化作用提出的，该作用已知与染色质相互作用 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 脱甲基化的TET1活性，但机制，时机和目标序列仍然不完全 理解。因此，我们建议解决（1）是否需要迭代氧化为5FC/5CAC 重新编程，（2）TET1是否具有非催化重编程角色，（3）哪些序列需要 各种TET活动。具体目标1将检查TET1在重新编程中的确切作用，并且 原始生殖细胞（PGC）中全基因组。我们已经设计了小鼠，要么停滞5mc氧化 5HMC（TET1V）或缺乏催化功能（TET1HXD），并将测试其对DNA甲基化重编程的影响 使用我们解决5MC和5HMC的新技术，以及配置文件在期间相关的染色质动力学 PGC开发。我们使用新的Infinium Mouse Beadchip的初步数据表明TET1突变体 小鼠精子具有非重叠的DNA修饰模式。因此，具体目标2将评估 纯合突变配子和后代中TET1突变的表观基因组和表型后果 这些配子引起的。最后，特定的目标3将确定表观基因组和表型 纯合成年和衰老小鼠中TET1失速和催化突变的后果。这项工作 将能够评估TET酶在基因组重编程中的作用，并剖析要求 TET酶的非催化活性和迭代氧化。