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Imprecise DNMT1 activity coupled with neighbor-guided correction enables robust yet flexible epigenetic inheritance

Abstract

The epigenome, including DNA methylation, is stably propagated during mitotic division. However, single-cell clonal expansion produces heterogeneous methylomes, thus raising the question of how the DNA methylome remains stable despite constant epigenetic drift. Here, we report that a clonal population of DNA (cytosine-5)-methyltransferase 1 (DNMT1)-only cells produces a heterogeneous methylome, which is robustly propagated on cell expansion and differentiation. Our data show that DNMT1 has imprecise maintenance activity and possibly possesses weak de novo activity, leading to spontaneous ‘epimutations’. However, these epimutations tend to be corrected through a neighbor-guided mechanism, which is likely to be enabled by the environment-sensitive de novo activity (‘tuner’) and maintenance activity (‘stabilizer’) of DNMT1. By generating base-resolution maps of de novo and maintenance activities, we find that H3K9me2/3-marked regions show enhanced de novo activity, and CpG islands have both poor maintenance and de novo activities. The imprecise epigenetic machinery coupled with neighbor-guided correction may be a fundamental mechanism underlying robust yet flexible epigenetic inheritance.

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Fig. 1: Generation and characterization of PKO mESCs.
Fig. 2: DNMT1 stably maintains the methylome of PKO mESCs.
Fig. 3: PKO mESCs show severe differentiation defects.
Fig. 4: DNMT1 has imprecise methylation maintenance activity.
Fig. 5: Epimutation correction and methylome convergence during clonal expansion through neighbor-guided correction.
Fig. 6: Stable propagation of DNA methylome requires the de novo and maintenance activities of DNMT1.
Fig. 7: Mapping the maintenance and de novo methylation activities of DNMT1.
Fig. 8: A model illustrating robust yet flexible inheritance of DNA methylation mediated by neighbor-guided correction.

Data availability

All data have been deposited with the Gene Expression Omnibus under accession no. GSE116482.

Code availability

The software and code used to analyze the study data are listed in the Nature Research Reporting Summary and are publicly available.

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Acknowledgements

We thank G. Xu for providing the anti-TET1 antibody and members of the Xie laboratory for helpful comments during preparation of the manuscript. We also thank the Sequencing Core Facility, Biocomputing Facility and Center of Biomedical Analysis at Tsinghua University. This work was supported by the National Key R&D Program of China (grant no. 2019YFA0508901 to W. Xie), the National Natural Science Foundation of China (grant nos. 31988101 and 31725018 to W. Xie), the Chinese Academy of Sciences (grant nos. XDB39000000 and QYZDY-SSW-SMC031 to B.Z.), the THU-PKU Center for Life Sciences (W. Xie), the Beijing Municipal Commission of Science and Technology (grant no. Z181100001318006 to W. Xie). Q.W. is supported by postdoctoral fellowships from the Tsinghua-Peking Joint Center for Life Sciences. W. Xie is an HHMI International Research Scholar.

Author information

Authors and Affiliations

Authors

Contributions

Q.W. and W. Xie conceived and designed the experiments. Q.W. performed most of the experiments. G.Y. and Q.W. performed the bioinformatics analyses. X.M. performed the hairpin BS-seq experiments. W. Xia participated in the early phase of the project. X.X. performed the pilot hairpin BS-seq experiments. Y.Z. helped with various methylome profiling. W.Z. and W. Xia helped generate the Tet TKO mESCs. Y.L. helped generate the Eed knockout mESCs. C.H. helped with various experiments. Q.W., G.Y. and W. Xie wrote the manuscript. Q.W. and Y.L. conducted the high-throughput sequencing. H.X. and B.Z. advised with the hairpin BS-seq analyses and manuscript preparation. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Bing Zhu or Wei Xie.

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Extended data

Extended Data Fig. 1 Validation and characterization of PKO mESC.

a, Schematic of Tet1/2/3 and Dnmt3a/3b knockout strategies. b, Western blot of TET1/2 and DNMT3A/3B proteins (left) and dot blot results of 5mC and 5hmC (right) in WT, TKO and PKO mESCs. TET3 is not expressed in mESCs. Western blot images were cropped (see Extended Data Fig. 10). c, The Sanger sequencing results for Tet mutations. d, Bar charts showing numbers of TKO-WT, PKO-TKO and PKO-WT DMRs. e, Bar charts showing the enriched gene ontology (GO) terms for genes associated with either promoter or distal TKO-WT DMRs (left). Similar analysis results are shown for PKO-TKO DMRs (right). P-values (Fisher’s exact test, two-sided, not adjusted by multiple comparisons) are transformed by log10. f, Boxplots showing the gene expression changes (log2 ratio of RPKM) for those associated with TKO-WT or PKO-WT DMRs (n = 2 biologically independent experiments). The median is indicated by the center lines. The bottom, top edges and whiskers represent the 25th/75th percentiles and 1.5 times of the IQR, respectively. g, Bar charts showing cell numbers of WT, TKO and PKO mESCs upon cell propagation (n = 3 biologically independent experiments. Data are presented as mean values + /- SD). h, Bar charts showing the pluripotency gene expression in WT, TKO and PKO mESCs.

Extended Data Fig. 2 DNMT1 maintains the PKO methylome.

a, Schematic showing Dnmt1, Uhrf1 and Dnmt3c knockout strategies. b, Venn diagrams showing the overlap among RMRs identified in PKO;Dnmt1-/-, PKO;Uhrf1-/- and PKO;Dnmt1-/-;[OE] cells. Only regions with minimal 5× read depth in all three cell lines are included. Overlap among pairwise comparison of the three samples are all significant. Two-sided P values are calculated by Fisher’s Exact Test. c, A heatmap showing the enrichment of repeats at RMRs. The enrichment was calculated as a log2 ratio for the numbers of RMRs that overlap with repeats divided by the numbers of a random set of regions (with lengths matching RMRs) that overlap with repeats. d, Scatter plots comparing methylomes of PKO;Dnmt1-/-;[OE] with WT, PGCs30 and Dnmt3a/3b DKO31 (genome wide, 10-kb bin). e, Venn diagrams showing the overlap of RMRs between PKO;Dnmt1-/- and PGCs or Dnmt3a/3b DKO cells. Two-sided P values were calculated by Fisher’s Exact Test. f, Bar charts showing the numbers of WT-TKO, PKO rep1-PKO rep2 and PKO-SKO DMRs.

Extended Data Fig. 3 PKO mESCs have severe differentiation defects.

a, Bright field images of embryonic bodies at day 4 upon neuron differentiation for WT, TKO and PKO. One representative image from three independent experiments is shown. Scale bar, 50 μm. b, Teratoma assays and HE staining results for WT, TKO and PKO. N.D., not detected. One representative image from three independent experiments is shown. Scale bar, 100 μm. c, Heatmaps showing expression of Dnmt1, Uhrf1, Dnmt3a, Dnmt3b, Tet in WT, TKO and PKO cells. *, knock-out. d, Scatter plots showing DNA methylation levels for distal DMRs (defined in WT) between 13D vs. 0D cells. e, Bar charts showing DMR number (top) and genome coverage (bottom) during differentiation under different methylation-difference cutoffs. f, Boxplots showing the expression levels of “repressed” genes (2-fold change, FPKM > 10 before differentiation and FPKM < 5 after differentiation), “activated” genes (2-fold change, FPKM < 5 before differentiation and FPKM > 10 after differentiation) (defined in WT) or all genes before and after differentiation (n = 2 biologically independent experiments). The median is indicated by the center lines. The bottom, top edges and whiskers represent the 25th/75th percentiles and 1.5 times of the IQR, respectively. g, Boxplots showing DNA methylation (n = 2 biologically independent experiments) and histone modification enrichment (n = 2 biologically independent experiments) at promoters of differentially expressed genes (defined in WT) before and after differentiation. The median is indicated by the center lines. The bottom, top edges and whiskers represent the 25th/75th percentiles and 1.5 times of the IQR, respectively.

Extended Data Fig. 4 Ectopic H3K27me3 occurs at pluripotency gene promoters and enhancers in differentiated PKO cells.

a, Boxplots showing the DNA methylation (left) and H3K27me3 (right) enrichment at H3K27me3 failed to gain methylation regions (FMRs) in WT, TKO and PKO mESCs (0D) and differentiated cells (13D) (n = 2 biologically independent experiments). The median is indicated by the center lines. The bottom, top edges and whiskers represent the 25th/75th percentiles and 1.5 times the IQR, respectively. b, Boxplots showing the expression of genes near distal H3K27me3 FMRs before and after differentiation (n = 2 biologically independent experiments). All gene expression is shown as the control. The median is indicated by the center line. The bottom, top edges and whiskers represent the 25th/75th percentiles and 1.5 times the IQR, respectively. c, Bar charts showing H3K27me3 enrichment at Nanog, Pou5f1, Hoxa11, and Actb promoter regions during differentiation induced by RA in WT and TKO.

Extended Data Fig. 5 Comparison between H3K27me3 and non-H3K27me3 FMRs.

a, UCSC genome browser snapshots showing DNA methylation and H3K27me3 enrichment at H3K27me3 (pale blue) and non-H3K27me3 FMRs (light pink). b, Bar charts showing the enriched gene ontology (GO) terms for genes associated with H3K27me3 (top) and non-H3K27me3 FMRs (bottom). P-values (Fisher’s exact test, two-sided, not adjusted by multiple comparisons) are transformed by log10. c, Boxplots showing DNA methylation (left), H3K27me3 enrichment (middle) and nearest gene expression (right) at H3K27me3 and non-H3K27me3 FMRs (n = 2 biologically independent experiments). One-sided P values were calculated by Mann-Whitney U test. The median is indicated by the center lines. The bottom, top edges and whiskers represent the 25th/75th percentiles and 1.5 times of the IQR, respectively. d, Bright field images of cells at day 9 (D9) and day 11 (D11) upon neuron differentiation for WT, TKO, PKO, Eed-/- and PKO;Eed-/- cells (n = 3 biologically independent experiments; one representative image is shown). Red arrows show the cell clones that successfully attach to the plate, and black arrow shows embryonic bodies of PKO;Eed-/- cells that fail to attach to the plate. Scale bar, 50 μm. e, Bar charts showing Pou5f1 expression during differentiation induced by retinoic acid (RA) in various cell lines (n = 2 biologically independent experiments).

Extended Data Fig. 6 DNMT1 has imprecise methylation replication activity.

a, Methylation fidelity is as defined by the percentages of symmetrically methylated or un-methylated CG dyads among all sequenced reads at a given CG position. 100% fidelity indicates all CG dyads are symmetrically methylated or un-methylated, while 0% fidelity indicates all CG dyads are asymmetrically methylated. b, Bar charts showing DNA methylation fidelity of WT, PKO, and SKO mESCs when treated with or without nocodazole. c, Bar charts showing the CG coverages with different sequence depths for SKO clonal samples. d, Bar charts showing DMR number (left) and genome coverage (right) under different methylation cutoffs comparing D60 to D15 samples. e, Heatmaps showing the various DNA elements and repeats enrichment at D60 vs. D15 DMRs. The enrichment was calculated as a log2 ratio for the numbers of DMRs that overlap with repeats divided by the numbers of a random set of CGs that overlap with repeats. f, Bar charts showing the enriched gene ontology (GO) terms for genes associated with D60 vs. D15 DMRs. P-values (Fisher’s exact test, two-sided, not adjusted by multiple comparisons) are transformed by log10. g, UCSC genome browser snapshots showing D60 vs. D15 DMRs.

Extended Data Fig. 7 Convergence of methylome during clonal expansion through “neighbor-guided correction”.

a, Boxplots showing DNA methylation levels, CG densities and neighbor mCG densities (100-bp bin) for retained, intermediate and reverted expansion-DMCs in C2 (n = 1) and C3 (n = 1) samples. The median is indicated by the center line. The bottom, top edges and whiskers represent the 25th and 75th percentiles and 1.5 times the IQR, respectively. One-sided P values (Mann-Whitney U test) are also shown. b, Boxplots showing DNA methylation levels, CG and neighbor mCG densities (100-bp bin) of retained, intermediate and reverted interclone-DMCs for D15 samples (C2 vs. C1) (n = 1). The median is indicated by the center line. The bottom, top edges and whiskers represent the 25th and 75th percentiles and 1.5 times the IQR, respectively. One-sided P values (Mann-Whitney U test) are also shown.

Extended Data Fig. 8 Analyses of maintenance and de novo methylation activities of DNMT1.

a, Schematic showing the dynamics of DNA methylation states after each cell division. For each CG, (0,0), (1,0), (0,1), and (1,1) refer to un-methylated, two types of hemi-methylated, and fully-methylated states. Red arrows refer to the conversion of four methylation states during DNA replication. Blue arrows refer to the conversion of four methylation states by maintenance probability (M) and de novo methylation probability (D). When reaching methylation equilibrium, the same methylation states would be re-generated after each cell cycle with a proper M-D pair (Methods). b, Line graphs showing the average maintenance probability (M) and de novo probability (D) in the whole genome, or in regions with or without H3K9me2 (ref. 56), according to different neighbor mCG densities.

Extended Data Fig. 9 Distinct DNA methylation and gene expression patterns in PKO and R-PKO mESCs.

a, A UCSC genome browser snapshot showing the DNA methylation levels in PKO, Dnmt3a/3b DKO, R-PKO and R-PKO;Dnmt3bTOE mESCs. b, Violin plots showing the global DNA methylation levels of PKO, Dnmt3a/3b DKO, R-PKO and R-PKO;Dnmt3bTOE mESCs (n = 1). The median is indicated by the white dots. The edges of thick and thin black lines represent the 25th/75th percentiles and 1.5 times of the IQR, respectively. c, Bar charts showing the average DNA methylation levels at various types of DNA elements in WT, TKO, PKO, and R-PKO. DMV, DNA methylation valley18. d, Boxplots showing the gene expression and promoter DNA methylation levels for differentially expressed genes (R-PKO vs. PKO) in WT, TKO, PKO, R-PKO, and Dnmt TKO mESCs52. The median is indicated by the center lines. The bottom, top edges and whiskers represent the 25th/75th percentiles and 1.5 times of the IQR, respectively. e, Schematic showing generation of R-PKO with transient expression of Dnmt3b (R-PKO;Dnmt3bTOE) (Methods). f, Bar charts showing the relative ratios of fully and hemi-methylated CGs in SKO, PKO and R-PKO cells at hypomethylated and hypermethylated CGI. For R-PKO, the ratios for RMRs and non-RMRs are also shown. Un-methylated CG dyads are excluded from this analysis which would otherwise dominate the bar charts due to their large numbers.

Extended Data Fig. 10 Original uncropped Western blot gel for Extended Data Fig. 1b.

a–e, Original uncropped Western blot gel for TET1, TET2, DNMT3A, DNMT3B and TUBULIN. Boxes indicate the cropped regions in Extended Data Fig. 1b. f, A short-exposure blot gel showing TUBULIN control (second run using the same cell samples as e).

Supplementary information

Reporting Summary

Supplementary Tables 1–4

Supplementary Table 1 Sequencing information. Supplementary Table 2 GOs for DMR associated genes. Supplementary Table 3 Fail-to-be-methylated regions (FMRs) with ectopic H3K27me3 in differentiated PKO cells. Supplementary Table 4 Primers used in this study.

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Wang, Q., Yu, G., Ming, X. et al. Imprecise DNMT1 activity coupled with neighbor-guided correction enables robust yet flexible epigenetic inheritance. Nat Genet 52, 828–839 (2020). https://doi.org/10.1038/s41588-020-0661-y

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  • DOI: https://doi.org/10.1038/s41588-020-0661-y

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