Fig 2: Dnmt1 and Tet1 are required for Mbd3 and Mbd2 binding in ES cells.(A) Genome browser tracks of replicate ChIP-seq experiments examining endogenous Mbd3 or Mbd2 occupancy in control (EGFP KD), Dnmt1 KD, and Tet1 KD ES cells over indicated loci (Hoxd cluster). (B) Overlap of Mbd3 and Mbd2 binding. Shown are Venn diagrams delineating the overlap between all genomic locations (top panel) or TSS-proximal locations (-1 kb to +100 bp; bottom panel) bound by Mbd3 and Mbd2. (C–D) Aggregation plots of Mbd3 (C) or Mbd2 (D) ChIP-seq data showing occupancy over ICPs (top left panel, from [Weber et al., 2007]), annotated TSSs (bottom left panel), the LMR subset (top middle panel, from [Stadler et al., 2011]), gene-distal DHSs (bottom middle panel, from GSM1014154 with TSSs removed) Mbd2 peaks (top right panel, called from EGFP KD ChIP-seq experiments), and Mbd3 peaks (bottom right panel, called from EGFP KD ChIP-seq experiments) ± 2 kb in control (EGFP KD), Dnmt1 KD, or Tet1 KD ES cells. (E–F) Heatmaps of Mbd3 enrichment over Mbd3 binding sites sorted by Mbd2 occupancy (E) and Mbd2 enrichment over Mbd2 binding sites sorted by Mbd3 occupancy (F) in control (EGFP KD), Dnmt1 KD, or Tet1 KD ES cells. The profiles shown in aggregation plots and heatmaps represent the average of two biological replicates.DOI: http://dx.doi.org/10.7554/eLife.21964.006

Fig 3: Mbd3 and Mbd2 are required for each other’s binding in ES cells.(A) Genome browser tracks of ChIP-seq experiments examining Mbd3 or Mbd2 occupancy in control (EGFP KD), Mbd2 KD, or Mbd3 KD ES cells over one example locus (Pitx1). (B) Aggregation plots of Mbd3 or Mbd2 ChIP-seq data showing occupancy over ICPs (top left panel), annotated TSSs (bottom left panel), LMR subset (top middle panel), gene-distal DHSs (bottom middle panel), Mbd2 peaks (top right panel), and Mbd3 peaks (bottom right panel) ± 2 kb in control (EGFP KD), Mbd3 KD, or Mbd2 KD ES cells. (C) Heatmaps of Mbd3 enrichment over Mbd3 binding sites ± 2 kb sorted by Mbd2 occupancy (top panel) and Mbd2 enrichment over Mbd2 binding sites ± 2 kb sorted by Mbd3 occupancy (bottom panel) in control (EGFP KD), Mbd2 KD, or Mbd3 KD ES cells. The profiles shown in the browser tracks, aggregation plots, and heatmaps represent the average of two biological replicates.DOI: http://dx.doi.org/10.7554/eLife.21964.015

Fig 4: Low doses of DOT1L inhibitor ablate bulk H3K79me2 and curtail MV4;11 proliferation without impacting expression of canonical target genes.(A) Conventional model depicting how DOT1L methyltransferase activity activates transcription of key proliferative oncogenic transcription factors (Okada et al., 2005; Bernt et al., 2011; Guenther et al., 2008; Armstrong et al., 2002; Zeisig et al., 2004; Kroon et al., 1998). (B) Proliferation assay of MV4;11 cells treated with the indicated concentrations of the DOT1L inhibitor pinometostat (EPZ5676). Cell viability was assayed every 2 days, starting 1 day after treatment commenced using the CellTiter Glo 2.0 reagent. Relative cell viability is presented as the mean fraction of pinometostat versus cells treated with the equivalent volume of DMSO from three independent experiments ± S.E.M. (C) Western blots for H3K79me2 with H4 or MBD3 loading controls in MV4;11 cells treated with 10–200 nM pinometostat for 5 or 7 days. (D) RT-qPCR analysis of HOXA9 and MEIS1 expression fold-change in MV4;11 cells treated with 100 nM or 1 µM pinometostat for 7 days. Results are shown as mean ± S.E.M. of three independent experiments. Student’s t-test (ns p > 0.05, ** p = 0.01, *** p = 0.001). (E) Western blot of HOXA9 and MEIS1 with H4 as a loading control from MV4;11 cells treated with 100 nM pinometostat for 7 days.

Fig 5: Dnmt1, Mbd3, and Mbd2 are required for 5mC and 5hmC in ES cells.(A–B) Aggregation plots of 5mC (A) or 5hmC (B) enrichment over ICPs (top left panel), annotated TSSs (bottom left panel), the LMR subset (top middle panel), gene-distal DHSs (bottom middle panel) Mbd2 peaks (top right panel), and Mbd3 peaks (bottom right panel) ± 2 kb in control (EGFP KD), Dnmt1 KD, Mbd3 KD, or Mbd2 KD ES cells. Aggregation plots represent the average of two biological replicates. (C–D) Dot blot of bulk 5mC (C) and 5hmC (D) levels in EGFP KD, Dnmt1 KD, Mbd3 KD, or Mbd2 KD ES cells. Left panel shows 5mC or 5hmC levels and right panel shows methylene blue staining, which serves as a loading control. (E) Thin layer chromatography separation of radioactively end-labeled bases from MspI digested genomic DNA. Quantification of 5mC or 5hmC is expressed relative to T in each KD. Levels in EGFP KD are set to 100%. (F) Restriction enzyme digest of genomic DNA from EGFP KD, Dnmt1 KD, Mbd3 KD, or Mbd2 KD ES cells using an enzyme blocked by CpG methylation/hydroxymethylation (HpaII) or a CpG methylation insensitive enzyme (MspI) that cuts the same site (CCGG).DOI: http://dx.doi.org/10.7554/eLife.21964.013