DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.
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We thank all members of the Meissner laboratory, in particular C. Sindhu for helpful discussion and Z.D. Smith for critical feedback on the manuscript. We thank K. Musunuru (Harvard University) for providing the CRIPSR/Cas9 plasmids and thank Q. Ding from the Musunuru laboratory for technical support. J.L. was supported by a postdoctoral fellowship from the Human Frontiers Science Program. J.K.J. is supported by US NIH Director's Pioneer Award DP1 GM105378. A.M. is a New York Stem Cell Foundation Robertson Investigator. The work was funded by a US NIH (National Institute of General Medical Sciences) grant (P01GM099117) and the New York Stem Cell Foundation.
J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine and Transposagen Biopharmaceuticals. The interests of J.K.J. were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.
Integrated supplementary information
(a) Isoform information relevant to our targeting strategy. The targeted exon is shown in red. Ensembl transcript IDs are shown. (b) Experimental characterization of HUES64 cells. HUES64 cells showed a stable karyotype (46, XY) over the time we have maintained them in culture. Representative bright-field (BR) and OCT4 and NANOG immunostaining images are shown at 10× magnification. Teratoma results from HUES64 cells (P31) demonstrate pluripotency and contribution to all three germ layers: ectoderm (EC), mesoderm (ME) and endoderm (EN). A representative bright-field image of hematoxylin and eosin staining is shown at 4× magnification. Scale bars, 200 μm (4×), 100 μm (10×). (c) Summary of the CRISPR-based targeting efforts. Total refers to the number of total analyzed colonies after selection. Mutations include all validated heterozygous and homozygous mutations. The number of homozygous-mutant clones is shown in the right column for each. (d) Sanger sequencing information for the targeted region of representative clones of DNMT3A–/–, DNMT3B–/– and DNMT3A–/–; DNMT3B–/– cells. The colored lines under the sequence correspond to the lines under sequencing peaks.
Supplementary Figure 2 TaqMan hPSC Scorecard analysis for late-passage wild-type, DNMT3A–/–, DNMT3B–/– and DNMT3A–/–; DNMT3B–/– cells.
The gray boxes represent the score ranges of 11 reference human PSC lines. The wild-type and respective knockout cell lines are shown with different colored circles. Output is from our custom analysis.
Supplementary Figure 3 Global methylation trends in mouse Dnmt knockouts and fidelity of maintenance methylation, NANOG promoter methylation changes and satellite clusters in human DNMT knockouts.
(a) Global analysis of RRBS data from mouse Dnmt3a–/–, Dnmt3b–/–, Dnmt3a–/–; Dnmt3b–/–, and Dnmt1–/– and Dnmt3a–/–; Dnmt3b–/– with Dnmt1 knockdown (TKO). The fractions of CpGs with high (≥ 0.8; red), intermediate (inter, >0.2 and <0.8; green) and low (≤ 0.2; blue) methylation values are shown. Early- and late-passage cells were used for comparison. The “x” in the passage number indicates the passage when we received the cells. (b) Rate of methylation loss in DNMT3A–/–; DNMT3B–/– cells from passage 6 to passage 22 in different genomic features and imputed fidelity of DNMT1 in those regions (RRBS data). Fidelity was calculated by fitting an exponential model (Online Methods). (c) IGV genome browser snapshot of the NANOG promoter showing the complete absence of de novo methylation in DNMT3A–/–; DNMT3B–/– cells. In contrast, the wild-type and single-knockout cells show low levels of methylation that are consistent with the known heterogeneity of NANOG expression. The heat map below shows the DNA methylation values of individual CpGs within the highlighted region. The average DNA methylation value for the entire highlighted region is shown on the right. (d) Distribution of DNA methylation levels across various satellite clusters in the human DNMT knockouts.
Supplementary Figure 4 High-resolution view of CpG island methylation, methylation of imprinted regions and functional enrichment analysis of DMRs.
(a) Venn diagrams showing the overlap between the DMRs detected in each knockout cell line for non-repetitive 1-kb tiles and for CpG islands. (b) Aggregation plots for the Illingworth CpG island (CGI) set showing the loss of methylation in DNMT3A–/–; DNMT3B–/– cells relative to wild-type cells. (c) Methylation levels of known human imprinted control regions in wild-type, DNMT3A–/–, DNMT3B–/– and DNMT3A–/–; DNMT3B–/– cells. (d) Functional annotation enrichment for DNMT3A–/–; DNMT3B–/– DMRs. The test set was non-repetitive 1-kb tiles that were hypomethylated in the DNMT3A–/–; DNMT3B–/– cells, with the background being 1-kb tiles that had methylation of at least 0.4 in wild-type cells. Enrichments were calculated using GREAT, with DMRs being associated with genes within 20 kb.
Unsorted and sorted endoderm (dEN) cells are shown as different colored dots. The gray boxes represent the score ranges of 11 reference human PSC lines. Output was taken directly from the Life Technologies analysis website.
(a) Summary of the TALEN-based targeting efforts. Total refers to the number of colonies after selection. The mutations include all validated heterozygous and homozygous mutations. The number of homozygous clones is shown in the right column for each genotype. (b) RT-qPCR analysis of mRNA expression levels for DNMT1 in DNMT1-knockdown cells. DNMT1-knockdown lines were generated using HUES64 cells. Top, GIPZ DNMT1 shRNA sets from Thermo Scientific. Five individual shRNAs were tested. Bottom, TRIPZ DOX-inducible DNMT1 shRNA sets from Thermo Scientific. Four individual shRNAs were tested with and without DOX induction. (c) Schematic of the DNMT1 rescue constructs. (d) Designed mutations within the exogenous DNMT1* sequence do not alter the protein. The representative amino acid sequence representing the protein remains unchanged. The colored amino acid sequence corresponds to the DNA sequence in Figure 6a. (e) Targeting efficiency of DNMT1 knockout in the tTA+DNMT1* rescue line. The number of mutations includes the number of homozygous clones. (f) Representative sequencing information for DNMT1–/– cells. The colored lines under the sequence correspond to the lines under sequencing peaks. 3′ primer was used for sequencing. Therefore, the reverse-complement sequence is presented in the peaks. (g) RT-qPCR analysis of mRNA expression levels for DNMT3A and DNMT3B in clone 122 validated in f. Primer names and positions are shown in Figure 1d. Error bars were generated from technical triplicates and represent one standard error.
(a) Evidence of acute apoptosis after DNMT1* withdrawal. Representative apoptosis marker Annexin V immunostaining images are shown at 10× magnification. Scale bars, 100 μm. The cell line used here is clone 111. (b) Evidence of DNA damage after DNMT1* withdrawal. Representative DNA damage marker γ-H2A.X (phosphorylated at Ser139) images are shown at 10× magnification. Scale bars, 100 μm. The cell line used here is clone 111. (c) Cell cycle after DNMT1* withdrawal. The cell line used here is clone 111. (d) Schematic of the catalytically inactive DNMT1 rescue constructs. Clone 111 was separately transduced by plenti-ef1α-IRE-nlsEGFP as a non-rescue control, plenti-ef1α-DNMT1C1226W-IRE-nlsEGFP as a catalytically inactive DNMT1 rescue and plenti-ef1α-DNMT1WT-IRE-nlsEGFP as a wild-type DNMT1 rescue control. Stable rescue and control lines were established by sorting for GFP-positive cells. (e) Representative sequencing information for the catalytically inactive DNMT1C1226W construct. The bold letters within the sequence correspond to the lines under sequencing peaks. (f) Western blot analysis of expression of catalytically inactive DNMT1C1226W in 293 cells. There is no apparent degradation of the overexpressed proteins DNMT1WT or DNMT1C1226W. (g) Cell death of the catalytically inactive DNMT1C1226W rescue line after DNMT1* withdrawal. Cells were labeled with nucleus-localized EGFP as the schematic shows in d. Representative images of EGFP-positive cells are shown at 20× magnification. Scale bars, 100 μm. (h) Dot blot assay for DNMT1* withdrawal. The cell line used here is clone 111. (i) Dot blot assay for DNMT1* withdrawal in the catalytically inactive DNMT1C1226W rescue line.
Supplementary Figures 1–7 and Supplementary Tables 4 and 5 (PDF 5230 kb)
List of DMRs in knockout cell lines (1-kb tiles and CpG islands). (XLS 2098 kb)
GO analysis for enrichment of regions hypomethylated in DNMT3A–/–; DNMT3B–/– cells. (XLS 193 kb)
Cell cycle analysis after DNMT1* withdrawal. (XLS 27 kb)
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Liao, J., Karnik, R., Gu, H. et al. Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells. Nat Genet 47, 469–478 (2015). https://doi.org/10.1038/ng.3258
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