Role of TET1 in germ cell reprogramming and development

哺乳动物的生殖细胞必须重新编程以促进正常发育。这种重新编程， 包括DNA甲基化和组蛋白修饰的消除，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对后代的传播。在很大 基因组经历复制偶联的被动DNA去甲基化，这是10 - 11易位的关键作用 (TET)家族酶，特别是TET 1，已被证明可用于基因组DNA的主动去甲基化， 序列，如印记控制区（ICR）和生殖系特异性基因。拟议的工作将使用 新开发的小鼠品系和测序技术，以测试迭代氧化和 TET 1的非催化功能是小鼠DNA甲基化消除和重编程所必需的 基因组，包括ICR和减数分裂特异性基因，在生殖系和体细胞发育。泰特酶 可催化5-甲基胞嘧啶（5 mC）的三次连续氧化，生成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）。氧化的5 mC碱，特别是5 hmC，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去甲基化的中间体。5 hmC支持的独特去甲基化途径 相对于5 fC/5caC，已经混淆了破译TET 1的精确机制作用的努力。又进一步 TET 1的潜在非催化作用带来了挑战，已知TET 1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 TET 1具有去甲基化活性，但机制、时间和靶序列仍不完全 明白因此，我们建议解决（1）对于以下问题是否需要迭代氧化至5 fC/5caC： 重编程，（2）TET 1是否具有非催化重编程作用，以及（3）需要什么序列 各种泰特活动。具体目标1将检查TET 1在ICR重编程中的确切作用， 原始生殖细胞（PGCs）。我们已经设计了小鼠， 5 hmC（Tet 1v）或缺乏催化功能（Tet 1hxd），并将测试它们对DNA甲基化重编程的影响 使用我们的新技术，解决了5 mC和5 hmC，并分析了相关的染色质动力学， PGC开发。我们使用新的Infinium小鼠微珠芯片的初步数据表明，Tet 1突变体 小鼠精子具有非重叠的异常DNA修饰模式。因此，具体目标2将评估 Tet 1突变在纯合突变配子和后代中的表观基因组和表型后果 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 Tet 1停滞和催化突变在纯合突变成年和衰老小鼠中的后果。这项工作 将能够评估泰特酶在基因组重编程中的作用， 非催化活性和泰特酶的反复氧化。

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.