Iterative oxidation by TET1 is required for reprogramming of imprinting control regions and patterning of mouse sperm hypomethylated regions.

Ten-eleven translocation (TET) enzymes iteratively oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during mammalian germline reprogramming remains unresolved due to the inability to decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 (Tet1-HxD) and TET1 that stalls oxidation at 5hmC (Tet1-V). Tet1 knockout and catalytic mutant primordial germ cells (PGCs) fail to erase methylation at select imprinting control regions and promoters of meiosis-associated genes, validating the requirement for the iterative oxidation of 5mC for complete germline reprogramming. TET1V and TET1HxD rescue most hypermethylation of Tet1-/- sperm, suggesting the role of TET1 beyond its oxidative capability. We additionally identify a broader class of hypermethylated regions in Tet1 mutant mouse sperm that depend on TET oxidation for reprogramming. Our study demonstrates the link between TET1-mediated germline reprogramming and sperm methylome patterning.

Direct enzymatic sequencing of 5-methylcytosine at single-base resolution.

5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes. The ideal method for 5mC localization would be both nondestructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here we present direct methylation sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution using nanogram quantities of DNA. DM-Seq employs two key DNA-modifying enzymes: a neomorphic DNA methyltransferase and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities with deaminase-resistant adapters enables accurate detection of only 5mC via a C-to-T transition in sequencing. By comparison, we uncover a PCR-related underdetection bias with the hybrid enzymatic-chemical TET-assisted pyridine borane sequencing approach. Importantly, we show that DM-Seq, unlike bisulfite sequencing, unmasks prognostically important CpGs in a clinical tumor sample by not confounding 5mC with 5-hydroxymethylcytosine. DM-Seq thus offers an all-enzymatic, nondestructive, faithful and direct method for the reading of 5mC alone.

TET1 Catalytic Activity is Required for Reprogramming of Imprinting Control Regions and Patterning of Sperm-Specific Hypomethylated Regions.

DNA methylation erasure is required for mammalian primordial germ cell reprogramming. TET enzymes iteratively oxidize 5-methylcytosine to generate 5-hyroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxycytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during germline reprogramming remains unresolved due to the lack of genetic models that decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 ( Tet1-HxD ) and TET1 that stalls oxidation at 5hmC ( Tet1-V ). Tet1 -/- , Tet1 V/V , and Tet1 HxD/HxD sperm methylomes show that TET1 V and TET1 HxD rescue most Tet1 -/- hypermethylated regions, demonstrating the importance of TET1’s extra-catalytic functions. Imprinted regions, in contrast, require iterative oxidation. We further reveal a broader class of hypermethylated regions in sperm of Tet1 mutant mice that are excluded from de novo methylation during male germline development and depend on TET oxidation for reprogramming. Our study underscores the link between TET1-mediated demethylation during reprogramming and sperm methylome patterning.