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. 2016 Nov 16;5:e21964. doi: 10.7554/eLife.21964

Figure 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

Figure 2.

Figure 2—figure supplement 1. Loss of Dnmt1 and Tet1 results in reduced occupancy of both Mbd3 and Mbd2 in ES cells.

Figure 2—figure supplement 1.

(A) Efficient KD of Dnmt1, Tet1, Mbd3, Mbd2, and Chd4 is confirmed by RT-qPCR with expression levels normalized to GAPDH and shown relative to control (EGFP) KD. Shown are the mean ± SD values of three biological replicates after acute (48 hr) KD. (B) Efficient KD of Dnmt1, Tet1, Mbd3, Mbd2, and Chd4 was confirmed by Western blotting, where β-actin serves as a loading control. (C–D) Genome browser tracks of replicate ChIP-seq experiments examining Mbd3 or Mbd2 occupancy in control (EGFP KD), Dnmt1 KD, and Tet1 KD ES cells over two example loci (Tfap2a (C) and Pitx1 (D)) show reduced occupancy of Mbd2 and Mbd3 in Dnmt1 KD and Tet1 KD cells over the promoter-proximal regions of each gene. (E) Western blotting of Mbd3 and Mbd2, where β-actin serves as a loading control, in control (EGFP KD), Dnmt1 KD, Tet1 KD, Mbd3 KD, and Mbd2 KD ES cells demonstrates that levels of Mbd3 and Mbd2 are only altered upon KD of Mbd3 or Mbd2, respectively.
DOI: http://dx.doi.org/10.7554/eLife.21964.007
Figure 2—figure supplement 2. Validation of endogenous Mbd3 and Mbd2 ChIP-seq experiments.

Figure 2—figure supplement 2.

(A–B) Biological replicates of Mbd3 (A) and Mbd2 (B) ChIP-seq experiments demonstrate similar results. Shown are aggregation plots of Mbd3 (A) or Mbd2 (B) ChIP-seq datasets over Mbd3 peaks (A) or Mbd2 peaks (B) ± 2 kb in control (EGFP) KD, Tet1 KD, and Dnmt1 KD ES cells. (C–D) Heatmaps of biological replicates of Mbd3 (C) and Mbd2 (D) ChIP-seq experiments demonstrate similar results when compared gene-by-gene. Shown are heatmaps of Mbd3 (C) or Mbd2 (D) ChIP-seq dataset over Mbd3 peaks ranked by Mbd2 occupancy (C) or Mbd2 peaks ranked by Mbd3 occupancy (D) ± 2 kb. (E) Validation of changes in occupancy of Mbd3 and Mbd2. ChIP-qPCR was performed on Mbd3 or Mbd2 in control (EGFP) KD, Tet1 KD, Dnmt1 KD, Mbd3 KD, and Mbd2 KD ES cells. Mbd3 or Mbd2 levels are expressed as a fraction of input. Shown are the mean ± SD values of three biological replicates. (F) Western blot showing loss of Dnmt1 protein expression in Dnmt1 KO ES cells. β-actin serves as a loading control. (G) Validation of changes in occupancy of Mbd3 and Mbd2 in Dnmt1 KO cells. ChIP-qPCR was performed as indicated in control (WT) and Dnmt1 KO ES cells.
DOI: http://dx.doi.org/10.7554/eLife.21964.008
Figure 2—figure supplement 3. FLAG ChIPs confirm Dnmt1 and Tet1 are required for Mbd3 and Mbd2 occupancies.

Figure 2—figure supplement 3.

(A) Western blots showing expression in Mbd3abc-3XFLAG, Mbd3a-3XFLAG, and Mbd2-3XFLAG ES cell lines. β-actin serves as a loading control. (B) Aggregation plots of Mbd3abc-3XFLAG ChIP-seq 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 Untagged, EGFP KD, Dnmt1 KD, and Tet1 KD ES cells. (C) Validation of changes in occupancy of Mbd3. ChIP-qPCR was performed on Mbd3a-3XFLAG and Mbd3abc-3XFLAG in Untagged, EGFP KD, Tet1 KD, Dnmt1 KD, Mbd3 KD, and Mbd2 KD ES cells. FLAG levels are expressed as a fraction of input. Shown are the mean ± SD values of three biological replicates. (D) Validation of changes in occupancy of Mbd2. ChIP-qPCR was performed on Mbd2-3XFLAG in control (EGFP) KD, Tet1 KD, Dnmt1 KD, Mbd3 KD, and Mbd2 KD ES cells. Shown as in (C).
DOI: http://dx.doi.org/10.7554/eLife.21964.009
Figure 2—figure supplement 4. ChIP-seq experiments using MBD-FLAG fusions.

Figure 2—figure supplement 4.

(A–B) Aggregation plots of Mbd3abc-3XFLAG (A) or Mbd2-3XFLAG (B) ChIP-seq 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 Untagged, EGFP KD, Mbd3 KD, and Mbd2 KD ES cells. (C) Aggregation plot of Mbd3 ChIP-seq from (Yildirim et al., 2011) over gene-distal DHSs.
DOI: http://dx.doi.org/10.7554/eLife.21964.010