Abstract
De novo DNA methylation (DNAme) in mammalian germ cells is dependent on DNMT3A and DNMT3L. However, oocytes and spermatozoa show distinct patterns of DNAme. In mouse oocytes, de novo DNAme requires the lysine methyltransferase (KMTase) SETD2, which deposits H3K36me3. We show here that SETD2 is dispensable for de novo DNAme in the male germline. Instead, the lysine methyltransferase NSD1, which broadly deposits H3K36me2 in euchromatic regions, plays a critical role in de novo DNAme in prospermatogonia, including at imprinted genes. However, males deficient in germline NSD1 show a more severe defect in spermatogenesis than Dnmt3lā/ā males. Notably, unlike DNMT3L, NSD1 safeguards a subset of genes against H3K27me3-associated transcriptional silencing. In contrast, H3K36me2 in oocytes is predominantly dependent on SETD2 and coincides with H3K36me3. Furthermore, females with NSD1-deficient oocytes are fertile. Thus, the sexually dimorphic pattern of DNAme in mature mouse gametes is orchestrated by distinct profiles of H3K36 methylation.
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Acknowledgements
We thank P. Chambon (University of Strasbourg) for the Nsd1 conditional mouse line, C. L. Walker (Baylor College of Medicine) for the Setd2 conditional mouse line and J. Trasler (McGill University) for the Dnmt3l KO mouse line; A. Bogutz, J. BrindāAmour, J. Richard Albert, K. Mochizuki and S. Janssen for helpful discussions and technical assistance; W. Xie (Tsinghua University) and L. Lefebvre (UBC) for critical reading of the manuscript; and R. Vander Werff and T. Stach (BRC-seq, UBC) for deep sequencing and A. Johnson and J. Wong (ubcFLOW, UBC) for cell sorting. This research was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED (Japan Agency for Medical Research and Development) under grant number JP20am0101103 (support number 1080). M.C.L. is supported by CIHR grants PJT-153049 and PJT-166170. K.S. was a recipient of a Uehara Memorial Foundation postdoctoral fellowship and a CIHR Banting postdoctoral fellowship, BPF-156568.
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K.S. and M.C.L. conceived and designed the project and wrote the manuscript and all authors reviewed the manuscript. F.M. and T.I. performed the WGBS experiments. K.S. performed all other experiments and analysis.
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Extended data
Extended Data Fig. 1 Setd2 deletion in male PGCs leads to hypogonadism in adults.
a, Schematic representation of the dynamics of DNA methylation (DNAme) during female (red) and male (blue) germline development. Developing germ cells analyzed in this study are shown in bold. Key events for germline development are also shown. Note that in both sexes, de novo DNAme occurs in non-dividing quiescent cells. PGC: primordial germ cell; SG: spermatogonium; E: embryonic day; P: postnatal day. b, Dynamics of Dnmt3l and Dnmt3a expression across key stages of male germ cell development. FPKM (fragments per kilobase of exon model per million reads mapped) values were extracted from RNA-seq data from a previous study34. The window of de novo DNAme during prenatal male germline development is shaded in yellow. c, Breeding scheme to obtain germline-specific Setd2 knockout (KO) prospermatogonia (PSG) and sorting strategy for purification of PSG using OCT3/4-EGFP36. Potential genotypes of embryos derived from this cross are indicated and a microscopy image of GFP expression in E16.5 testis is shown (scale bar, 500āĪ¼m). Note that OCT3/4-EGFP is expressed exclusively in PSG in the testis. Dead cells were excluded by gating out PI positive cells during FACS. Sorted PSG (3.9% of total testis cells) were processed as previously described for ULI-N-ChIP-seq35 and PBAT41,42. d, Gross morphology of 12-week-old testis isolated from WT control (left) or Setd2 KO mice (right). Image is representative of three litters. Scale bar, 5āmm. e, Hematoxylin and eosin (HE) staining of 12-week-old testis (top) and epididymis (bottom) isolated from WT control (left) or Setd2 KO mice (right). Histological analyses shown are representative of multiple imaged sections from a single experiment. Scale bar, 50āĪ¼m.
Extended Data Fig. 2 Genome-wide relationship between de novo DNAme in prenatal male germ cells and histone H3 methylation states.
a, Contour scatter plots with outliers showing the relationship between enrichment (RPKM) of the indicated histone marks over 10-kb genomic bins and the difference in the % of DNAme between E16.5 PSG and E13.5 mPGCs33 in the same regions. The % of genomic coverage for each histone mark in E16.5 PSG is shown in each plot. R indicates Spearmanās rank correlation coefficient. b, Genome browser representation of a region on chromosome 3 showing % DNAme in E13.5 mPGCs and E16.5 PSG33 as well as RPM values of H3K4me3, H3K27me3, H3K9me3, H3K36me2 and H3K36me3 in E16.5 PSG. c, Violin plots showing % DNAme over H3K9me3 domains of <10ākb (total DNAme genomic coverage, 0.1āGb) or >20ākb (total DNAme genomic coverage, 0.2āGb) in E13.5 mPGCs, E16.5 PSG33 and P0 PSG43 (nā=ā1). The % of the genome in E16.5 PSG showing H3K9me3 enrichment in domains of <10ākb or >20ākb is shown in each plot. Box (25-75th percentile), median (inside the box) and whisker (1.5x interquartile range from the box) are included in the violin plots. d, Violin plots showing the distribution of enrichment of H3K36me2 over 10-kb genomic bins (left) or H3K9me3 domains of <10ākb (middle) or >20ākb (right) in E16.5 PSG (nā=ā1). Note that H3K36me2 is particularly depleted in regions of broad H3K9me3. Box, median and whisker are included in the violin plots as shown in c. e, Stacked bar chart showing % total of unique or repeat regions in the genome (total) versus H3K9me3 domains of <10ākb or >20ākb. Repeats were identified by RepeatMasker annotation.
Extended Data Fig. 3 Expression of H3K36 KMTases and the role of H3K36me2 during male germline development.
a, Dynamics of expression of H3K36 KMTases across prenatal male germ cell development. FPKM values were extracted from RNA-seq data from a previous study34. The window of global de novo DNAme is shaded in yellow. b, Breeding scheme to obtain germline-specific Nsd1 KO PSG and sorting strategy for PSG purification, as in Extended Data Fig. 1c. A microscopy image of GFP expression in E16.5 testis is shown (scale bar, 500āĪ¼m) as well as a representative FACS plot for control testis, with PSG representing 3.1% of total testis cells in this experiment. c, Tracks showing coverages and junctions of RNA-seq reads over the Nsd1 locus in Nsd1 HET and KO E16.5 PSG. Note that the exon between the two loxP sites (shaded in yellow) is efficiently excised (>99%) in the KO. d, Bar graph showing the % genomic coverage of H3K36me2 and H3K36me3 in Nsd1 HET versus KO E16.5 PSG. e, Scatterplot showing the correlation of H3K9me3 (RPKM) over 10-kb genomic bins in Nsd1 HET versus KO E16.5 PSG. f, Scatterplot showing H3K9me3 versus H3K36me2 enrichment (RPKM over 10-kb genomic bins) in Nsd1 KO E16.5 PSG.
Extended Data Fig. 4 NSD1 is required for de novo DNAme of a subset of CpG islands hypermethylated in sperm.
a, Histogram showing the frequency distribution of CpG island (CGI) % DNAme (mean across each annotated CGI) in mature sperm. WGBS data from sperm43 was re-processed for this analysis. b, Stacked bar chart showing relative % of CGIs in promoter, gene body or intergenic regions for all annotated CGIs52 in the genome (total) versus CGIs hypermethylated (>70%) in sperm. c, Parallel coordinate plot showing % DNAme over all CGIs in the indicated cell types during male germline development. Color coded according to methylation state in sperm. d, Scatterplot showing the relationship between % DNAme in Nsd1 HET versus KO P0 PSG for 50,000 randomly selected 10ākb genomic bins versus at CGIs hypermethylated in WT sperm. e, Heatmaps showing the levels of DNAme in Nsd1 HET or KO P0 PSG and sperm43 as well as H3K4me3 and H3K36me2 in Nsd1 HET or KO P0 PSG over CGIs and flanking (+/-0.5ākb) regions. The data is sorted based on H3K4me3 in Nsd1 KO P0 PSG. f, Genome browser representation of % DNAme and RPM values of H3K4me3 and H3K36me2 (spike-in normalized) in the indicated cells at representative CGIs (hatched boxes) and their flanking regions (left: non-promoter CGI, right: promoter CGI). WGBS data from E13.5 mPGCs33 was re-processed. g, Scatterplot showing the relationship between fold-change of gene expression and the difference in % DNAme between Nsd1 HET and KO P0 PSG at hypermethylated CGI promoters (as defined in panel b). Genes showing aā>ā20% difference in DNAme and a 2-fold difference in expression are labeled.
Extended Data Fig. 5 Comparison of germ cell phenotypes in Nsd1 KO versus Dnmt3l KO male mice.
a, Gross morphology of testes isolated from Nsd1 HET and KO (top) as well as Dnmt3l HET and KO (bottom) mice at P10, P14, P21 and 3 months. Images shown for 3 month testes are representative of three independent experiments. Scale bar, 5āmm. b, HE staining of sections of testis and epididymis isolated from 3-month-old Nsd1 HET and KO as well as Dnmt3l HET and KO mice. Histological analyses shown are representative of multiple imaged sections from a single experiment. Note the complete absence of spermatogonia in Nsd1 KO testis. Scale bar, 50āĪ¼m. c, Gross morphology of perinatal testes isolated from Oct3/4-EGFP Nsd1 HET and KO (top) as well as Dnmt3l HET and KO mice (bottom). Note that OCT3/4-EGFP is expressed exclusively in PSG in the testis. Scale bar, 500āĪ¼m.
Extended Data Fig. 6 Silencing of TEs in male germ cells is dependent upon DNMT3L but not NSD1.
a, Scatterplots showing the expression (agglomerated RNA-seq reads) of LTR (top, nā=ā539) or L1 (bottom, nā=ā145) families in Nsd1 HET versus KO P0 PSG (left panels) as well as Dnmt3l HET versus KO (right panels). A color gradient of H3K4me3 RPKM values in Nsd1 HET E16.5 PSG is also shown. Only TE families with >100 genomic copies are plotted and TE families showing a 2-fold increase in expression in the Dnmt3l KO are labelled. b, Immunofluorescence (IF) analysis of LINE1 ORF1 expression in 3-week testis isolated from HET control and Nsd1 KO or Dnmt3l KO mice. Testis were co-stained with the germ cell specific marker TRA98. IF analyses shown are representative of multiple imaged sections from a single experiment. Scale bar, 25āĪ¼m. c, Scatterplots showing the correlation of mean % DNAme of LTR (top) or L1 families (bottom) in Nsd1 HET versus KO P0 PSG. A color gradient of H3K36me2 RPKM values in Nsd1 HET E16.5 PSG is also shown. Only TE families with >100 genomic copies are plotted. d, Scatterplots showing the correlation of mean % DNAme of LTR or L1 families in Nsd1 HET versus KO P0 PSG as well as Dnmt3l or Dnmt3c HET versus KO P10 male germ cells24. RPKM values for H3K4me3 in Nsd1 HET E16.5 PSG is shown via color gradient in all plots. Only TEs with >100 genomic copies are plotted.
Extended Data Fig. 7 Effects of Nsd1 or Dnmt3l deletion on H3K36me2, H3K27me3 and gene expression in E16.5 PSG.
a, Pie chart showing the relative prevalence of promoter, intergenic or gene body regions enriched for both H3K36me2 and H3K36me3 in E16.5 PSG. Peaks were annotated using ChIPseeker96. b, Scatterplot showing H3K36me2 versus H3K27me3 enrichment (RPKM) over 10-kb genomic bins in Nsd1 HET E16.5 PSG. 50,000 randomly selected data points are plotted. The color gradient shows the difference in % DNAme between Nsd1 control HET versus KO E16.5 PSG. c, Scatterplot showing the correlation of H3K36me2 (RPKM) over 10-kb genomic bins in Dnmt3l HET versus KO E16.5 PSG. d, Scatterplot showing the correlation of H3K27me3 (RPKM) over 10-kb genomic bins in Dnmt3l HET versus KO E16.5 PSG. e, Contour scatter plot and the outliers showing the relationship between log2 fold-change of H3K27me3 over 10-kb genomic bins in Nsd1 HET versus KO and Dnmt3l HET versus KO E16.5 PSG. f, Scatterplot showing the fold-change of gene expression in E16.5 Nsd1 HET versus KO PSG. Only genes showing total count >10 in Nsd1 HET and KO are plotted. Genes showing aā>ā2-fold difference in expression with pā<ā0.05 (Wald test by DESeq284) in HET and KO groups (nā=ā2) were scored as up- or down-regulated and are highlighted as indicated. g, Scatterplot showing the fold-change of gene expression in E16.5 Dnmt3l HET versus KO PSG. Only genes showing total count >10 in Dnmt3l HET and KO are plotted. Genes showing aā>ā2-fold difference in expression with pā<ā0.05 (Wald test by DESeq284) in HET and KO groups (nā=ā2) were scored as up- or down-regulated and are highlighted as indicated.
Extended Data Fig. 8 Aberrant repression of PRC2 target genes in Nsd1 KO PSG.
a, Violin plots showing the distribution of expression levels (log2 RPKMā+ā0.01) (nā=ā2) of all genes in Nsd1 HET E16.5 PSG (left) versus genes downregulated in the Nsd1 KO. b, Scatterplot showing the relationship between H3K36me2 (RPKM) and H3K36me3 over gene body regions of all expressed (FPKMā>ā1) genes in control E16.5 PSG (grey) or genes downregulated in Nsd1 KO E16.5 PSG (blue). c, Scatterplot showing the relationship between the enrichment of H3K4me3 and H3K27me3 at all genic TSSs (Ā±1ākb) in E16.5 PSG. The expression levels of associated genes in control E16.5 PSG is indicated via color gradient. Genes with H3K4me3 RPKMā>ā2 and H3K27me3 RPKMā>ā0.5 in E16.5 PSG were defined as bivalent (hatched boxes). d, Venn diagram showing the overlap of genes downregulated in Nsd1 KO E16.5 PSG with bivalent expressed genes in Nsd1 HET E16.5 PSG. e, Genome browser representation of % DNAme and RPM values of H3K36me2, H3K4me3 and H3K27me3 at the Gfra1 locus in the indicated cells. H3K36me2 and H3K27me3 levels are normalized by spike-in. Published WGBS data from E13.5 mPGCs33 was reprocessed. f, Bar graph showing the expression of Gfra1 in Nsd1 KO and Dnmt3l KO E16.5 and P0 PSG relative to their matching controls. g, Bar graph showing the expression of key transcription factors acting downstream of the GDNF/GFRa1 signaling pathway in Nsd1 KO and Dnmt3l KO E16.5 and P0 PSG relative to their matching controls.
Extended Data Fig. 9 SETD2 is required for intragenic H3K36me2 and H3K36me3 in oocytes.
a, Violin plots showing % DNAme over 10-kb genomic bins (left, total genomic coverage, 2.5āGb), H3K36me2 enriched regions (middle, genomic coverage, 0.8āGb) or H3K36me3 enriched regions (right, genomic coverage, 0.6āGb) in P0-3 NGOs, P12 GOs and FGOs (nā=ā1). The % genomic coverage of H3K36me2 (middle) and H3K36me3 (right) in P12 GOs is shown in each plot. Box (25-75th percentile), median (inside the box) and whisker (1.5x interquartile range from the box) are included in the violin plots. NGO: non-growing oocyte; GO: growing oocyte; FGO: fully-grown oocyte. Publicly available WGBS data from P0-3 NGOs, FGOs19 and P12 GOs69 were reprocessed. b, Scatterplot showing H3K36me2 versus H3K36me3 enrichment (RPKM) over 10-kb genomic bins in P12 GOs. 50,000 randomly selected data points are plotted and the color gradient shows the difference in % DNAme between P12 GOs and NGOs. c, Heatmaps showing the levels of DNAme, H3K36me2, H3K36me3, H3K4me3 and H3K27me3 in gene bodies and their flanking 3ākb regions in control or Setd2 KO FGOs. Genes with expression RPKM values >1 in E16.5 PSGs and/or FGOs26,27 were defined as expressed. Only forward strand genes are plotted and the heatmaps are sorted by the level of H3K36me3 in Setd2 control FGOs. TSS: transcription start site; TTS: transcription termination site. Publicly available ChIP-seq data from H3K4me3 and H3K27me394 as well as WGBS data from Setd2 control and KO FGOs28 were reprocessed. d, Bar graph showing the % genomic coverage of H3K36me2 and H3K36me3 in control or Setd2 KO FGOs. Note that the majority of H3K36me2 is dependent upon SETD2. e, Pie chart showing the relative prevalence of promoter, intergenic or gene body regions with residual H3K36me2 enrichment in Setd2 KO FGOs. f, Tracks showing coverages and junctions of RNA-seq reads over the Nsd1 locus in oocytes26 and PSG. Note the predominant form of Nsd1 initiates from an LTR in oocytes. g, Relative junction read count of the canonical exon 8-exon 9 junction versus the alternative LTR-exon 9 junction in GOs and FGOs. h, Domain structure of canonical NSD1 versus the predicted oocyte-specific, LTR-initiated isoform. Note that the oocyte-specific NSD1 lacks the amino-terminal PWWP domain of canonical NSD1.
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Shirane, K., Miura, F., Ito, T. et al. NSD1-deposited H3K36me2 directs de novo methylation in the mouse male germline and counteracts Polycomb-associated silencing. Nat Genet 52, 1088ā1098 (2020). https://doi.org/10.1038/s41588-020-0689-z
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DOI: https://doi.org/10.1038/s41588-020-0689-z
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