Germline DNA Demethylation Dynamics and Imprint Erasure Through 5-Hydroxymethylcytosine

Abstract

Mouse primordial germ cells (PGC) 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-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 a significant 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.Specification of primordial germ cells (PGCs) from epiblast cells at ~E6.25 is linked with extensive epigenetic reprogramming, including global DNA demethylation, chromatin reorganisation and imprint erasure, that is vital for generating totipotency (1, 2). The erasure of CpG methylation (5mC) is a key component of this program, but the dynamics and underlying mechanisms of the process remain unclear (3). Here we report a comprehensive analysis of PGCs by combining immunofluorescence, genome-wide (h)meDIP-seq, single cell RNA-seq, bisulfite-seq and functional analyses to address the mechanistic basis of epigenetic reprogramming in PGCs.We investigated Tet expression using single cell RNA-seq, which revealed that Tet1 and Tet2 are expressed in PGCs and peak between E10.5-E11.5, but that Tet3 is undetectable (Fig. 1A). Immunofluorescence (IF) showed that TET1 and TET2 are nuclear and expressed at significantly higher levels in PGCs than neighbouring somatic cells between E9.5-E11.5 (Fig. 1B & S1-S2). This suggests that erasure of 5mC in PGCs could occur through conversion to 5-hydroxymethylcytosine (5hmC) by TET1/TET2 (4, 5).We pursued this possibility by IF and found a progressive reduction of 5mC in PGCs between E9.5-E10.5, until it became undetectable by E11.5 (Fig. 1C). The loss of 5mC occurs concurrently with a global enrichment of 5hmC in PGCs between E9.5-E10.5, † To whom correspondence should be addressed. a.surani@gurdon.cam.ac.uk.

Modulating Epigenetic Memory through Vitamins and TET: Implications for Regenerative Medicine and Cancer Treatment

“…Given that loss of imprinting is a known driver of developmental disease and cancer [64,65], its occurrence in naive cells would render them unsafe for clinical and transgenics applications. ICR erasure in primordial germ cells is thought to involve TET protein action [66][67][68][69], and as such, it is not unreasonable to expect that loss of imprinting in naive ESCs could be exacerbated by supplementation of vitamins A and C. This means that in order to strike a balance between imprinting fidelity in naive pluripotent stem cells and the efficiency at which they are derived, fine-tuning of vitamin levels and TET activity may be required …”

The crucial role of Activin A on the formation of primordial germ cell-like cells from skin-derived stem cells in vitro

“…Recent studies have demonstrated that TET family proteins have potential roles in epigenetic regulation during germ cell reprogramming and meiotic entry of early germ cells through modification of 5mC to 5hmC, [37][38][39][40] an important member of the TET family is TET1 which catalyzes the conversion of 5mC to 5hmC during PGC reprogramming. 41 We thus evaluated the expression levels of TET1 at different culture stages.…”

A Life Course Perspective on Body Size and Cardio-metabolic Health

“…In this scenario, direct and simultaneous exposure of three generations (i.e., the pregnant mother, her fetus, and the fetuses' primordial germ cells) might be responsible for the increased disease risk seen in each generation. Alternatively, and if more than three generations are affected, research is revealing how inheritance of epigenetic modifications to the genome may be responsible (Hackett et al 2013). …”