Germline DNA demethylation dynamics and imprint erasure through 5-hydroxymethylcytosine

Abstract

Mouse primordial germ cells (PGCs) undergo sequential epigenetic changes and genome-wide DNA demethylation to reset the epigenome for totipotency. Here, we demonstrate that erasure of CpG methylation (5mC) in PGCs occurs via conversion to 5-hydroxymethylcytosine (5hmC), driven by high levels of TET1 and TET2. Global conversion to 5hmC initiates asynchronously among PGCs at embryonic day (E) 9.5 to E10.5 and accounts for the unique process of imprint erasure. Mechanistically, 5hmC enrichment is followed by its protracted decline thereafter at a rate consistent with replication-coupled dilution. The conversion to 5hmC is an important component of parallel redundant systems that drive comprehensive reprogramming in PGCs. Nonetheless, we identify rare regulatory elements that escape systematic DNA demethylation in PGCs, providing a potential mechanistic basis for transgenerational epigenetic inheritance.

Figure 1. Global dynamics of 5mC, 5hmC & TETs in PGCs.

(A) Single cell RNA-seq analysis of Tet1, Tet2 and Tet3 expression. Shown is Log2 reads per million (RPM). (B) Expression of TET1 and TET2 in E10.5 PGCs (arrowheads) and soma. (C) Dynamics of DNA methylation (5mC) in PGCs shows 5mC erasure between E9.5-E11.5. (D) 5-hydroxymethylcytosine (5hmC) kinetics in PGCs. TG1/STELLA mark PGCs. Scale bar = 10μm.

Figure 2. 5mC erasure is coupled to 5hmC conversion.

(A) Enrichment of 5mC and 5hmC in E10.5-E13.5 PGCs and Epiblast stem cells (EpiSC) over internal exons. (B) 5mC and 5hmC distribution relative to a metagene. (C) Profiles of 5mC (blue) and 5hmC (red) at the Dazl promoter. (D) Glucosyltransferase-qPCR showing quantitative levels of 5mC and 5hmC at a CCGG site in the Dazl promoter. Error bars represent S.E.M. (E & F) DNA methylation (%) by bisulfite sequencing of uninduced (−DOX) or induced (+DOX) Tet1/Tet2 miR or non-targeting (NT) miR PGCLCs at (E) gene promoters and (F) repeat elements. Open and closed circles represent unmethylated and methylated CpGs, respectively. (G) DNA methylation in PGCLCs stably expressing catalytically active (WT) or mutant (Mut) TET1 and TET2. Error bars represent S.E.M of allelic methylation.

Figure 3. Imprint erasure in PGCs.

(A) Average (h)meDIP-seq enrichment of 5mC and 5hmC across all imprinted gDMRs in PGCs. EpiSC represent monoallelic methylation. (B & C) 5mC and 5hmC profiles of DMRs that undergo (B) early or (C) late 5mC erasure and corresponding delay in 5hmC enrichment. (D) Glu-qPCR analysis of 5mC and 5hmC levels at early (Kcnq1ot1 and Igf2r) and late (Peg10 and Peg3) reprogrammed DMRs. Error bars represent S.E.M. (E) Rate of demethylation to unmodified cytosine. Shown is the predicted rate of passive demethylation and observed rates for Peg10 and Peg3.

Figure 4. Inheritance of 5mC through reprogramming.

(A) The Vmn2r29, Sfi1 and Srrm2 CGIs escape reprogramming in PGCs. Open and closed circles represent unmethylated and methylated CpGs, respectively. Dazl is representative of demethylation at most loci. (B) Model for the mechanisms and dynamics of DNA demethylation in PGCs.