Role of TET1 in germ cell reprogramming and development

哺乳动物的生殖系必须重新编程，以促进正常的发育。这种重新编程，这是 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向后代的传播。虽然许多人 基因组经历复制偶联的被动DNA去甲基化，在Ten-11易位中起关键作用 (Tet)家族酶，特别是TET1，已被证明对基因组的活性去甲基化有作用 序列，如印记控制区(ICR)和生殖系特异基因。拟议的工作将使用 新开发的小鼠品系和测序技术来验证迭代氧化和测序技术 TET1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ICRs和减数分裂特有的基因，在生殖系和体细胞发育期间。Tet酶 可以催化5-甲基胞嘧啶(5mC)的三次连续氧化，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，特别是5hmC，可以 在包括大脑在内的躯体组织中扮演着独立的表观遗传学角色，但被认为是最重要的 起DNA去甲基化中间体的作用。5HmC支持的独特的去甲基化途径 相对于5fC/5caC，混淆了破译TET1确切机制作用的努力。更进一步 TET1的潜在非催化作用带来了挑战，TET1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 Tet1具有去甲基化活性，但其机制、时机和靶序列仍不完整 明白了。因此，我们建议解决(1)是否需要迭代氧化到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将审查TET1在ICRS和 在原始生殖细胞(PGC)中的全基因组。我们设计了一种小鼠，它们要么将5mC的氧化停滞在 5hmC(Tet1v)或缺乏催化功能(Tet1hxd)，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，它可以分辨5mC和5hmC，并在 PGC发展。我们使用新的Infinium鼠标珠芯片的初步数据表明，Tet1突变体 小鼠精子具有非重叠的DNA修饰异常模式。因此，特定目标2将评估 纯合突变配子及其后代中Tet1突变的表观基因组学和表型后果 从这些配子中产生的。最后，特定的目标3将决定表观基因组和表型 Tet1停滞和催化突变在纯合突变成年和老年小鼠中的后果。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 非催化活性和Tet酶的迭代氧化。

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.