Active DNA demethylation is required for complete imprint erasure in primordial germ cells

Abstract

In mammalian primordial germ cells (PGCs), DNA demethylation is indispensible for parental imprint erasure, which is a reprogramming process essential for normal developmental potential. Thus, it is important to elucidate how DNA demethylation occurs in each imprinted region in PGCs and to determine which DNA demethylation pathway, passive or active, essentially contributes to the erasure of the imprint. Here, we report that active DNA demethylation via a putative Poly(ADP-ribose) polymerase (PARP) pathway is involved in H19-DMR imprint erasure in PGCs, as shown by an in vivo small molecule inhibitor assay. To the best of our knowledge, this is the first direct demonstration of a DNA replication-independent active DNA demethylation pathway in the erasure process of genomic imprinting in PGCs in vivo. The data also suggest that active DNA demethylation plays a significant role in the complete erasure of paternal imprinting in the female germ line.

Figure 1. DNA methylation analysis of imprinted DMRs in PGCs.

(a), (b) DNA methylation level and status of DMRs in E10.5 PGCs and somatic cells. The gray and red bars represent each litter's and the average DNA methylation level of PGCs, respectively and the blue bars represent the DNA methylation level of somatic cells (a). Open circles indicate the unmethylated sites and closed circles indicate the methylated sites (b). (c) Dynamic DNA demethylation of H19-DMR in PGCs. PGCs were isolated from each stage in a manner precisely determined based on the number of tail somites (left). The bar represents DNA methylation level of the paternal allele (right, top) based on the result of bisulfite sequencing (right, bottom). Photograph by Kawasaki et al.

Figure 2. Small molecule inhibitor assay in the fetus during embryonic development.

(a) Experimental scheme of the inhibitor assay (drawings by Kawasaki et al.). (b) EdU detection in PGCs and somatic cells in the genital ridges from an inhibitor-treated fetus. The incorporated EdU (red) was detected in PGCs and somatic cells. DAPI was used for nuclear staining (blue). For the identification of PGCs, gonad cells were stained with Dazl (green; arrows indicate PGCs). Scale bar = 20 μm. EdU was incorporated in the 3-AB-treated or MOCK fetal genomic DNA, but not in the aphidicolin-treated fetal genomic DNA. (c) Percentage of EdU (−) and EdU (+) cells of each group of PGCs and somatic cells was presented.

Figure 3. DNA methylation analysis of H19-DMRs in inhibitor-treated PGCs in vivo.

(a) Experimental scheme (drawings by Kawasaki et al.). After treatment of inhibitors, PGCs in the E11.25 fetus were isolated by sorting EGFP-positive cells and used for DNA methylation analysis. (b) E11.25 fetus and the dissected gonads after inhibitor treatment (top; * EGFP positive fetus). The body size of each fetus was measured (red bars) and the number of EGFP positive PGCs was counted by FACS (blue bars), as shown in the graph (bottom). Scale bar = 2mm. (c) DNA methylation level (top) and status (bottom) of H19-DMR in the inhibitor-treated PGCs, as shown by bisulfite sequencing. p value by t-test.