Abstract
DNA methylation is almost completely erased throughout the genome in primordial germ cells, and then reestablished during mammalian germ cell development. In this study, we demonstrate that in three primate species—marmosets, macaques, and humans–de novo methylation occurs postnatally in prospermatogonia in males and growing oocytes in females. In monkey prospermatogonia, de novo methylation is a prolonged process spanning 6 months to 1 year, primarily occurring within the first year after birth. In human testes, this process may occur more slowly over an extended period. Single-cell bisulfite sequencing analyses in spermatogonia of three species revealed that all genomic regions acquire DNA methylation gradually. However, DNA methylation levels increase faster in genic regions compared to intergenic regions. Unlike in mice, mitotic divisions occur during the establishment of methylation in prospermatogonia. The established methylation is likely maintained because maintenance methyltransferase DNMT1 is specifically expressed during the mitotic stage. Our findings show notable differences in the de novo DNA methylation processes in male germ cells between mice and primates.
Data availability
scRNA/scBS/scATAC-seq data have been deposited in the DDBJ database under accession numbers: DRA016143 and DRA016233 and in the NBDC Human database under accession numbers: JGAS000887 (https://humandbs.dbcls.jp/en/hum0542-v1) and JGAS000888 (https://humandbs.dbcls.jp/en/hum0544-v1). Source data are provided with this paper.
Code availability
Publicly available software and packages were mainly used to analyze scBS-seq data: for processing BAM files, Samtools version 1.21 (https://doi.org/10.1093/gigascience/giab008); for trimming adapter sequences, Trim Galore version 0.6.10 (https://zenodo.org/records/7598955), Cutadapt version 4.9 (https://doi.org/10.14806/ej.17.1.200), FastQC version 0.12.1; for mapping to the genome, Bismark version 0.24.2 (https://doi.org/10.1093/bioinformatics/btr167), Bowtie2 version 2.5.4 (https://doi.org/10.1109/SFCS.2000.892127); for annotation and analyzing data, bedtools version 2.31.0 (https://doi.org/10.1093/bioinformatics/btq033), Homer version 5.1 (https://doi.org/10.1016/j.molcel.2010.05.004); for displaying data on chromosomes, karyoploteR version 1.32.0 (https://doi.org/10.1093/bioinformatics/btx346), bismap version 1.2.1 (https://doi.org/10.1093/nar/gky677). Seurat version 5.1.0 (https://doi.org/10.1038/s41587-023-01767-y) and Cell Ranger were used to analyze scRNA-seq data. Cell Ranger and ArchR were used to analyze scATAC-seq data. The R and shell scripts used in the ATAC analyses can be obtained from GitHub (https://github.com/Hattyoriiiiiii/scatac-caljac-watanabe)33. Detailed information on analysis methods is provided in the respective sections of the Methods and Supplementary Methods.
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Acknowledgements
We thank Erika Sasaki and Akihiro Umezawa for support; Yoko Kuroki for mediation; Yoshiaki Kita, Erika Sasaki, and Tomomi Shimogori for the marmoset samples; Takayuki Mineshige, Keisuke Mukasa, Terumi Yurimoto, and Takashi Inoue for the help of surgery and veterinary care; Masatsugu Ema and Tomoyuki Tsukiyama for the cynomolgus monkey testes; Rui Wang and Fuchou Tang for information on generating scRNA-seq and scBS-seq libraries. Yoko Sato and Iwamoto Teruaki for samples. This work was supported by the following grants to T.W.: AMED PRIME (JP19gm6310010, JP20gm6310010, JP21gm6310010, and JP22gm6310010), KAKENHI (20H05764, 20H03177, 22K18356, 22H04923 (CoBiA) and 24KK0143), and JST (JPMJPR228B).
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K.K. and M.K.-I. prepared the scBS-seq and scRNA-seq libraries. Y.L., S.B.W., K.A. and L.B. analyzed the human samples. M.K. and T.S. collected the monkey samples. T.H. and S.M. performed the scATAC-seq analyses. S.T., Y.K., T.S., T.E. and R.N. performed the informatics analyses. T.Y., Y.H., Y.K., T.H., K.M. and H.K. provided the human testis samples. Y.M., S.S. and Y.D. provided the ovary samples. S.T. and T.W. wrote the paper. T.W. conceived the study.
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Kojima, K., Li, Y., Tomizawa, Si. et al. Establishment of DNA methylation during primate germ cell development. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71405-z
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DOI: https://doi.org/10.1038/s41467-026-71405-z
