Role of TET1 in germ cell reprogramming and development

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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.