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. 2019 Apr 9;8:e39380. doi: 10.7554/eLife.39380

Figure 4. Persistent DMRs are associated with altered H3K27me3 and enhancer regions.

(A) Left, differentially methylated regions (DMRs) in sperm. Right, DMRs in F1 bone marrow. Red, false discovery rate (FDR) < 0.05. (B) Sperm volcano plot from (A); DMRs with FDR < 0.05 in both sperm and F1 bone marrow are in red. (C) Magnitude of DNA methylation difference (Kdm6a cKO vs. control or Kdm6a F1 vs. control F1) for the 299 DMRs shared between sperm and F1 bone marrow. Box, persistent DMRs. (D) Distribution of persistent DMRs in the mouse genome. (E) Distance from persistent DMRs to the 25% of regions with greatest change in H3K27me3 in sperm. ‘All’ refers to the complete set of tiles covered by RRBS. ***p<0.001, Mann-Whitney U test. (F) Left, distance to CpG islands. Right, distance to transcription start sites (TSS). (G) Fraction of DMRs overlapping repetitive elements. (H) Distance to poised enhancers in sorted bone marrow macrophages. ***p<0.001, Mann-Whitney U test. (I) Top 10 mouse phenotypes associated with persistent DMRs. (J) Representative persistent DMR in the enhancer of a cancer-associated gene (Etv6). Error bars, SEM of three replicates. See Figure 4—source data 1.

Figure 4—source data 1. DNA methylation in Kdm6a cKO sperm and Kdm6a F1 bone marrow.
elife-39380-fig4-data1.xlsx (53.5KB, xlsx)
DOI: 10.7554/eLife.39380.037
Figure 4—source data 2. RRBS libraries.
elife-39380-fig4-data2.xlsx (49.7KB, xlsx)
DOI: 10.7554/eLife.39380.038
Figure 4—source data 3. DMRs shared between Kdm6a cKO sperm and Kdm6a F1 bone marrow.
elife-39380-fig4-data3.xlsx (82.3KB, xlsx)
DOI: 10.7554/eLife.39380.039
Figure 4—source data 4. Genes within 1 kilobase of persistent DMRs.
elife-39380-fig4-data4.xlsx (52.1KB, xlsx)
DOI: 10.7554/eLife.39380.040

Figure 4.

Figure 4—figure supplement 1. Representative pyrosequencing data at three persistent DMRs, including two tumor-associated enhancers (Foxa2 and Lmo2) and one promoter (Lama3).

Figure 4—figure supplement 1.

Figure 4—figure supplement 2. Distance relationships between persistent DMRs and various genomic features.

Figure 4—figure supplement 2.

(A) Distance to maternally- or paternally-expressed imprinted regions, as defined in Babak et al. (2015). (B) Distance to nearest transcription start site (TSS) of genes expressed in wild type round spermatids (TPM >5) (left) or with transcripts present in sperm (TPM >5) (right). (C) Distance to nearest poised enhancer in wild type bone marrow (ENCODE data) and in spermatids. (D) Distance to nearest active enhancer in bone marrow macrophages, bone marrow, and spermatids. *p<0.05, **p<0.01, ***p<0.001, Mann-Whitney U test.
Figure 4—figure supplement 3. Sorting of round spermatids by flow cytometry.

Figure 4—figure supplement 3.

(A) Green (530 nm) fluorescence profiles of dissociated testis cells stained with DyeCycle Green, a vital nucleic acid dye. 1C (haploid, spermatids), 2C (diploid, stem cells and secondary spermatocytes) and 4C (tetraploid, primary spermatocytes and mitotic stem cells) populations are marked. Profile for all cells is in orange, 1C 'small' (elongating spermatids, back-gated from plot in B) in green, and 1C 'large' (round spermatids, back-gated from plot in B) in blue. (B) Forward scatter profile for 1C cells gated in A. ‘1C large’ population represents round spermatids. (C) RT-qPCR for sorted round spermatids compared to whole testes. Sycp2 marks spermatocytes, Prm2 marks round and elongating spermatids, Aqp8 marks elongating spermatids only, Lin28a marks spermtogonial stem cells, and Gata4 marks testis somatic cells. Expression is shown relative to whole testis, with Actb as an internal control. Error bars represent SD for three technical replicates. (D) Representatitve 100x images of spermatids stained with DyeCycle Green and sorted directly onto a slide. Scale bars, 5 um.
Figure 4—figure supplement 4. Additional examples of DNA methylation gains in sperm and F1 bone marrow.

Figure 4—figure supplement 4.

H3K27me3 ChIP-seq and RRBS data at representative persistent DMRs overlapping enhancers of cancer-associated genes. Error bars, SEM of three replicates.
Figure 4—figure supplement 5. Additional examples of DNA methylation gains in sperm and F1 bone marrow in enhancers associated with tumorigenesis.

Figure 4—figure supplement 5.

RRBS data at additional persistent DMRs overlapping enhancers of cancer-associated genes. Error bars, SEM of three replicates.