Journal: Nature genetics

Article Title: TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers

doi: 10.1038/s41588-020-0639-9

Figure Lengend Snippet: a) Schematic overview. Knockouts are shown under each arrow. 3A = DNMT3A, 3B = DNMT3B. Barplot shows the global mean methylation for each sample using matched CpGs ( n = 6,708,067) generated by WGBS. DKO and PKO ESCs were passaged six times. b) Schematic as in a. Barplot shows the global mean methylation for each sample (CpGs match panel a). DKO-A and DKO-B denote clone A and B (passage 3), respectively. 3AKO and 3BKO are from published datasets . c) Genome browser tracks displaying WT and DKO methylation levels, sample-specific DKO-DMRs (boxes) and shared cDKO-DMRs (shaded vertical bars). d) The overlap between DKO-DMRs hypomethylated in each sample. e) Genome browser track of selected DKO-DMRs shown in panel c. Class 1 (light shading) border already hypomethylated regions and class 2 (dark shading) are flanked by highly methylated DNA. f) Heatmap of cDKO-DMRs including 2 kb on either side, separated by class. g) Methylation composite plot of class 1 and class 2 cDKO-DMRs with 5 kb on either side. h) The percentage of cDKO-DMRs that overlap with the shown genomic features. Categories are not exclusive. CGI = CpG island, TSS = transcription start site and IG = intergenic. Enhancers are defined from H1 ESCs . i) Violin plots for CpGs located within cDKO-DMRs ( n = 214,732, 180,019, 138,424, 215,263, 203,504, 182,322, 202,028, 212,215, 145,869 CpGs respectively). Violin plots extend from the data minima to the maxima with the white dot indicating median, thick bar showing the interquartile range and thin bar showing 1.5× interquartile range.

Article Snippet: To create Dnmt3a and Dnmt3b double knockout mouse ESCs and EpiSCs, we targeted both genes simultaneously by transfecting the cells with px458 (Addgene plasmid #48138) containing guides targeting the highly conserved PC motif in the catalytic domains in ESCs, and guides targeting both the PC motif as well as early coding exons in EpiSCs ( ).

Techniques: Methylation, Generated

Journal: Nature genetics

Article Title: TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers

doi: 10.1038/s41588-020-0639-9

Figure Lengend Snippet: a) Sequencing data showing the mutations introduced into the DKO and PKO ESCs for DNMT3A . Green box = PAM sequence, grey box = sgRNA sequence. Codons are colored black or blue, grey text denotes non-coding DNA. The respective amino acid (aa) single letter name and sequence number is displayed below the DNA. b) Sequencing data for DNMT3B , as described in a. A different sgRNA was used to target DNMT3B in HUES8 ESCs compared to HUES64. c) Western blot for DNMT3A in HUES8 WT, HUES64 DKO clone A, HUES8 TKO, QKO and PKO cells. GAPDH is shown as the loading control. Full blots in . PKO cells were assessed for DNMT3A expression by Western blot six times with consistent results. d) HUES8 lines at 10X magnification showing that all KO cell lines appear morphologically normal. These images are representative of a full 10 cm culture dish over weeks of passaging. e) Heatmap showing RNA sequencing data as log 2 TPM expression values for a set of pluripotency and self-renewal associated genes (highly expressed), as well as a subset of differentiation associated genes (low expression). DKO-A was collected at passage 6 and PKO at passage 12. f) Heatmap showing RNA sequencing data as log 2 TPM expression values DNMTs and TETs, grey boxes depict genes that are knocked out in each sample. g) Comparison of global methylation levels between HUES64 and HUES8 wildtype cells where intensity of blue shading indicates density of data points. The Pearson correlation coefficient (cor.) is displayed.

Article Snippet: To create Dnmt3a and Dnmt3b double knockout mouse ESCs and EpiSCs, we targeted both genes simultaneously by transfecting the cells with px458 (Addgene plasmid #48138) containing guides targeting the highly conserved PC motif in the catalytic domains in ESCs, and guides targeting both the PC motif as well as early coding exons in EpiSCs ( ).

Techniques: Sequencing, Western Blot, Control, Expressing, Passaging, RNA Sequencing, Comparison, Methylation

Journal: Nature genetics

Article Title: TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers

doi: 10.1038/s41588-020-0639-9

Figure Lengend Snippet: a) Bar chart showing the proportion of CpGs with varying methylation loss in DKO cells compared to WT. CpGs that lost >60% methylation were generally located in closer proximity compared to CpGs that lost 10–20% methylation. b) The proportion of cDKO-DMRs with respective mean methylation levels in single DNMT3A −/− (3AKO) or DNMT3B −/− (3BKO) knockout ESCs. c) Composite plot showing methylation difference between passage (P) 20 and 6 in PKO cells as distance from cDKO-DMR. There is a small background loss (−0.045) with a slightly greater focal decrease (−0.07), but methylation levels stay generally high. d) The methylation level in HUES64 DKO-A, HUES64 WT and HUES8 TKO cells including the 2 kb either side is displayed. For regions where the neighboring 2 kb is hypomethylated in WT cells (class 1), almost every region shows an increase in methylation following TET loss. e) Violin plots showing methylation at TKO-DMRs across different HUES8 lines. We used stringent parameters to define TKO-DMRs that aberrantly gain methylation upon loss of TET expression (also described later in ). Methylation levels were rescued by re-expression of TET. Violin plots extend from the data minima to the maxima, white dot indicates median, thick bar shows the interquartile range and thin bar shows 1.5x interquartile range. f) Methylation levels across 1 kb tiles for PKO ESCs rescued with either TET1s or TET2. Intensity of blue shading indicates density of data points. Pearson correlation coefficient (cor) is displayed. g) ChIP-seq enrichment (from Ref ) for TET1 and DNMT3B over CpG islands (CGI), H1-specific enhancers and cDKO-DMRs. h) Representative browser tracks displaying methylation levels in WT and DKO cells as well as 5hmC levels in WT ESCs. Increased 5hmC is observed over the cDKO-DMR and at the border of the hypomethylated CGI, where TETs are known to localize.

Article Snippet: To create Dnmt3a and Dnmt3b double knockout mouse ESCs and EpiSCs, we targeted both genes simultaneously by transfecting the cells with px458 (Addgene plasmid #48138) containing guides targeting the highly conserved PC motif in the catalytic domains in ESCs, and guides targeting both the PC motif as well as early coding exons in EpiSCs ( ).

Techniques: Methylation, Knock-Out, Expressing, ChIP-sequencing

Journal: Nature genetics

Article Title: TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers

doi: 10.1038/s41588-020-0639-9

Figure Lengend Snippet: a) Sequencing data showing the Dnmt3a and Dnmt3b mutations introduced into mouse DKO EpiSCs. Two different sgRNAs were used for each gene. Green box = PAM sequence, grey box = sgRNA sequence. Codons are colored black or blue, grey text denotes non-coding DNA. The respective single letter amino acid (aa) and protein sequence number is displayed above the DNA. b) Western blots showing protein levels for DNMT1, DNMT3A and DNMT3B in WT and DKO EpiSCs. PCNA is used as a loading control. Full blots in . Western blots were performed twice with consistent results. c) Violin plots display methylation levels for cDKO-DMRs and EpiSC DKO-DMRs in WT and DKO cells for each species. The UCSC LiftOver tool was used to map DKO-DMRs between species. Violin plots extend from the data minima to the maxima with the white dot indicating median, thick bar showing the interquartile range and thin bar showing 1.5x interquartile range. d) Schematic depicting how orthologous genes shared between human and mouse were assigned to the nearest DKO-DMR (left). Each gene was given a number (in kb) denoting the distance to the nearest DKO-DMR either up- or down-stream. Box plots for genes that were binned based in their distance to the nearest DKO-DMR in EpiSCs (shown in legend key), displaying the distance to the nearest DKO-DMR in human ESCs (right). A matched trend between distances in mouse and human cells in evident. For the box plots: the median is shown in bold, the box displays interquartile range and whiskers extend to 1.5 times the interquartile range. e) Venn diagram showing the overlap of orthologous genes that have a DKO-DMR within the gene body (i.e. 0 kb away, as shown in panel d) in mouse EpiSCs and human ESCs. This overlap was highly significant (hypergeometric test).

Article Snippet: To create Dnmt3a and Dnmt3b double knockout mouse ESCs and EpiSCs, we targeted both genes simultaneously by transfecting the cells with px458 (Addgene plasmid #48138) containing guides targeting the highly conserved PC motif in the catalytic domains in ESCs, and guides targeting both the PC motif as well as early coding exons in EpiSCs ( ).

Techniques: Sequencing, Western Blot, Control, Methylation

Journal: Nature genetics

Article Title: TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers

doi: 10.1038/s41588-020-0639-9

Figure Lengend Snippet: a) Sequence mutations introduced into the DKO ESCs. Green box = PAM sequence, grey box = sgRNA. Codons are colored black or blue, grey text denotes non-coding DNA. Amino acid (aa) and protein sequence number are displayed above the DNA. b) Western blots for WT, DNMT3A −/− (3AKO), DNMT3B −/− (3BKO) and double knockout (DKO) mouse ESCs. GAPDH or H3 were used as loading controls. Full blots in . Western blots were performed three times with consistent results. c) Overlap between DKO-DMRs called in each Dnmt3a −/− tissue. d) The difference in methylation between WT and Dnmt3a −/− tissues, ESCs or EpiSCs. e) Methylation levels for EpiSC DKO-DMRs in EpiSCs and ESCs. Violin plots extend from the data minima to the maxima, white dot indicates median, thick bar shows the interquartile range and thin bar shows 1.5x interquartile range. f) Methylation levels for tissue-specific DKO-DMRs in WT and Dnmt3a −/− E6.5 epiblast and 8 day old tissues. Somatic DKO-DMRs were fully methylated in Dnmt3a −/− embryos following implantation due to DNMT3B expression hence must have lost methylation at a later stage. Violin plots extend from the data minima to the maxima, white dot indicates median, thick bar shows the interquartile range and thin bar shows 1.5x interquartile range. g) The number of DKO-DMRs identified when using a reduced stringency of 0.25 differential methylation instead of 0.6 to compensate for mixed cell types. We identified more DKO-DMRs (1,186–2,182) but still many less than in ESCs, and this may include false positives. h) The percentage of DKO-DMRs that fall into class 1 or 2 (described in ). i) The overlap of DKO-DMRs with genomic features. Categories are not exclusive. CGI = CpG island, TSS = transcription start site. j) Schematic showing the differentiation of human ESCs to motor neurons (MNs) via neuronal progenitor cells (NPCs). For WT and 3AKO ESCs expression of DNMT3A and DNMT3B is displayed across the time course. After day two, 3AKO cells are “DKO-like” as they do not express either DNMT3. k) The percentage of cDKO-DMRs with mean methylation <0.2 in each sample during differentiation. Only 1.3% and 2.4% of cDKO-DMRs lost methylation for WT and 3AKO MNs respectively. WGBS is from Ref . l) Expression of TET1–3 in WT and 3AKO ESCs during differentiation. RNA-seq is from Ref .

Article Snippet: To create Dnmt3a and Dnmt3b double knockout mouse ESCs and EpiSCs, we targeted both genes simultaneously by transfecting the cells with px458 (Addgene plasmid #48138) containing guides targeting the highly conserved PC motif in the catalytic domains in ESCs, and guides targeting both the PC motif as well as early coding exons in EpiSCs ( ).

Techniques: Sequencing, Western Blot, Double Knockout, Methylation, Expressing, RNA Sequencing

Journal: Nature genetics

Article Title: TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers

doi: 10.1038/s41588-020-0639-9

Figure Lengend Snippet: a) Schematic illustrating how DMR calling was performed between each pair of samples. The number of DMRs that gain methylation (Incr.) in the cell type shown to the right is located above the arrow, and the number of DMRs that lose methylation (Decr.) is shown below the arrow. As we identified only few regions for other comparisons that met the same criteria as for DKO-DMRs (stringent DMRs; black) we also used less stringent criteria (Δ > 0.25) to define DMRs (blue). P = passage. b) CpG methylation of TKO-, QKO- and PKO-DMRs detected with lower stringency as defined in panel a. The n is lower than shown in panel a due to drop-out from requiring 10x coverage per region. c) Location of the DMRs shown in b. Features are not exclusive. Enh. = enhancer, CGI = CpG island, TSS = transcription start site, IG = intergenic. As expected and previously reported in Ref , the majority of DMRs that occur in the absence of TETs were CGI promoters or shores of developmental genes that may be more susceptible to hypermethylation due to lack of transcriptional activity. d) Low stringency TKO-DMRs are grouped by their methylation status in QKO and PKO cells. Of the TKO-DMRs, 23% lost the aberrantly gained methylation upon KO of DNMT3B (in QKO), 21% only upon further loss of DNMT3A (PKO) and 56% remained highly methylated despite loss of both DNMT3s pointing to the importance of TET activity in keeping these loci demethylated. e) The difference in methylation over passaging across each genomic feature. f) The location of DMRs identified between PKO at P6 and P20. Categories are not exclusive. g) CpG methylation levels for a representative early-replicating region, where methylation in DKO cells decreases the most, (left) and a representative late-replicating region, where PKO cells show the strongest methylation loss (right).

Article Snippet: To create Dnmt3a and Dnmt3b double knockout mouse ESCs and EpiSCs, we targeted both genes simultaneously by transfecting the cells with px458 (Addgene plasmid #48138) containing guides targeting the highly conserved PC motif in the catalytic domains in ESCs, and guides targeting both the PC motif as well as early coding exons in EpiSCs ( ).

Techniques: Methylation, CpG Methylation Assay, Activity Assay, Passaging