Genome-wide erasure of DNA methylation in mouse primordial germ cells is affected by AID deficiency

Abstract

Epigenetic reprogramming including demethylation of DNA occurs in mammalian primordial germ cells (PGCs) and in early embryos, and is important for the erasure of imprints and epimutations, and the return to pluripotency. The extent of this reprogramming and its molecular mechanisms are poorly understood. We previously showed that the cytidine deaminases AID and APOBEC1 can deaminate 5-methylcytosine in vitro and in Escherichia coli, and in the mouse are expressed in tissues in which demethylation occurs. Here we profiled DNA methylation throughout the genome by unbiased bisulphite next generation sequencing in wild-type and AID-deficient mouse PGCs at embryonic day (E)13.5. Wild-type PGCs revealed marked genome-wide erasure of methylation to a level below that of methylation deficient (Np95(-/-), also called Uhrf1(-/-)) embryonic stem cells, with female PGCs being less methylated than male ones. By contrast, AID-deficient PGCs were up to three times more methylated than wild-type ones; this substantial difference occurred throughout the genome, with introns, intergenic regions and transposons being relatively more methylated than exons. Relative hypermethylation in AID-deficient PGCs was confirmed by analysis of individual loci in the genome. Our results reveal that erasure of DNA methylation in the germ line is a global process, hence limiting the potential for transgenerational epigenetic inheritance. AID deficiency interferes with genome-wide erasure of DNA methylation patterns, indicating that AID has a critical function in epigenetic reprogramming and potentially in restricting the inheritance of epimutations in mammals.

Figure 1. Genome-wide BS-Seq reveals global hypomethylation in PGCs dependent on Aid

Tissues and cells analysed by BS-Seq are shown in a gradient from red to yellow illustrating methylation levels from high to low. BS-Seq reads were analysed using windows of 250 kilobases across the whole genome. Yellow boxes show the range of the 25-75th quartiles of the data, the line in the middle the median value. Whiskers show either highest and lowest values (if there are no outliers) or upper and lower confidence intervals. Outliers are shown as circles. Placenta, fetal carcass and PGCs were collected at E13.5. Note the global hypomethylation in PGCs, and the substantially higher levels of methylation in Aid-/- PGCs.

Figure 2. Erasure of DNA methylation in different genomic elements in PGCs

Methylation levels in promoters, exons, introns and intergenic regions in ES cells, Np95-/- ES cells, and various tissues of C57BL/6J and Aid-/- knockout mice are shown based on ratios of methylated to unmethylated BS-Seq reads. Placenta, fetal carcass and PGCs were all collected at E13.5. Note the particularly pronounced effect of Aid deficiency on methylation of introns and intergenic regions.

Figure 3. Erasure of DNA methylation in different classes of transposable elements in PGCs

Methylation levels of different classes of transposons in ES cells, Np95-/- ES cells, and various tissues of C57BL/6J and Aid-/- knockout mice are shown based on ratios of methylated to unmethylated BS-Seq reads. Placenta, fetal carcass and PGCs were all collected at E13.5. Note that LTR-ERV1 and LTR-ERVK elements retain more methylation in PGCs than any other repeat family.

Figure 4. Analysis of DNA methylation of individual genomic loci in E13.5 PGCs by Sequenom MassArray

Methylation levels of individual genomic loci (R1-4, randomly selected sequences, located in intergenic regions, the first intron of Foxo1 and the seventh exon of Xirp2, respectively; the Dazl amplicon is located in the promoter region and the H19 and Lit1 amplicons are located in the differentially methylated regions), and of transposons and satellite repeats in wildtype and Aid-/- E13.5 PGCs are shown based on Sequenom Massarray analysis. The number of CpGs analysed for each region is stated in brackets. Un4, unmethylated control located in the Hoxc cluster. Note substantially increased levels of methylation in many genomic loci in Aid-/- PGCs.