A replication-dependent passive mechanism modulates DNA demethylation in mouse primordial germ cells

Abstract

SUMMARYGermline cells reprogramme extensive epigenetic modifications to ensure the cellular totipotency of subsequent generations and to prevent the accumulation of epimutations. Notably, primordial germ cells (PGCs) erase genome-wide DNA methylation and H3K9 dimethylation marks in a stepwise manner during migration and gonadal periods. In this study, we profiled DNA and histone methylation on transposable elements during PGC development, and examined the role of DNA replication in DNA demethylation in gonadal PGCs. CpGs in short interspersed nuclear elements (SINEs) B1 and B2 were substantially demethylated in migrating PGCs, whereas CpGs in long interspersed nuclear elements (LINEs), such as LINE-1, were resistant to early demethylation. By contrast, CpGs in both LINE-1 and SINEs were rapidly demethylated in gonadal PGCs. Four major modifiers of DNA and histone methylation, Dnmt3a, Dnmt3b, Glp and Uhrf1, were actively repressed at distinct stages of PGC development. DNMT1 was localised at replication foci in nascent PGCs, whereas the efficiency of recruitment of DNMT1 into replication foci was severely impaired in gonadal PGCs. Hairpin bisulphite sequencing analysis showed that strand-specific hemi-methylated CpGs on LINE-1 were predominant in gonadal PGCs. Furthermore, DNA demethylation in SINEs and LINE-1 was impaired in Cbx3-deficient PGCs, indicating abnormalities in G1 to S phase progression. We propose that PGCs employ active and passive mechanisms for efficient and widespread erasure of genomic DNA methylation.

Dynamics of epigenetic phenomena: intergenerational and intragenerational phenotype ‘washout’

“…Just as epigenetically modified phenotypes are variable across generations, so too is DNA methylation, the extent of which can occur across a continuum. In fact, with each subsequent generation, DNA methylation patterns are created, maintained, cleared (erased) and then re-established during the life cycle and subsequent inheritance of a phenotype (Alvarado et al, 2014a;Alvarado et al, 2014b;Golbabapour et al, 2011;Ohno et al, 2013). Thus, patterns of decline in DNA methylation or other epigenetic molecular agent could be the causal agent behind a decline in the epigenetically modified phenotype, until the effect had either disappeared or fallen below a threshold for detection of the modified phenotype.…”

PRDM14 promotes active DNA demethylation through the Ten-eleven translocation (TET)-mediated base excision repair pathway in embryonic stem cells

“…Protein complexes were washed with wash buffer (20 mM HEPES, pH 7.6, 1.5 mM MgCl 2 , 150 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT and 20% glycerol) and eluted by boiling with SDS sample buffer. ChIP analyses were performed as previously described (Ohno et al, 2013).…”

“…Recent whole-genome bisulphite sequencing has shown that global loss of DNA methylation occurs in migrating PGCs, whereas some resistant regions become demethylated only in gonadal PGCs (Seisenberger et al, 2012). Active repression of Dnmt3a, Dnmt3b and Uhrf1, coupled with rapid proliferation of PGCs, might trigger replication-dependent passive demethylation in developing PGCs (Kagiwada et al, 2013;Ohno et al, 2013).…”

How to make a primordial germ cell

“…The epigenetic programme induced primarily by BLIMP1 and PRDM14 leads to global DNA demethylation towards an epigenetic ground state in early PGCs Hajkova et al, 2008;Hajkova et al, 2010;Ohno et al, 2013;Seisenberger et al, 2012) (Fig. 4).…”

“…For example, the repression of Uhrf1 (Bostick et al, 2007) and Dnmt3b by BLIMP1 and PRDM14 promotes DNA replication-coupled DNA demethylation in PGCs. Additionally, TET1 and TET2, which are bound by PRDM14, catalyse hydroxylation of 5-methyl-cytosine, which provides an additional parallel redundant mechanism for DNA demethylation in PGCs Ma et al, 2011;Ohno et al, 2013;Seisenberger et al, 2012). There is potentially a role for a base excision repair mechanism (Hajkova et al, 2008) that could contribute to the erasure of imprints.…”