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. Author manuscript; available in PMC: 2012 Dec 23.
Published in final edited form as: Cell. 2011 Dec 23;147(7):1498–1510. doi: 10.1016/j.cell.2011.11.054

Figure 7. Mbd3 is required for global hydroxymethylation in vivo.

Figure 7

(A) Dot blots of 5hmC. Top panel shows positive (5hmC) and negative (5mC) controls for antibody specificity. Bottom panels show dilution series of genomic DNA isolated from the indicated knockdown ES cells. Mbd3 and Brg1 KDs have similar effects to Tet1KD on bulk 5hmC levels.

(B) Thin layer chromatography of radioactively end-labeled bases from MspI-digested genomic DNA (Ficz et al., 2011) from the indicated knockdowns.

(C) Quantitation of 5hmC levels (normalized to levels of T) measured as in (B) for 5 independent replicate experiments. Columns show mean +/− sem.

(D) Tet1 and Mbd3 knockdowns have similar effects on hydroxymethylation of target gene promoters. Hydroxymethylated DNA was isolated by capture of biotin-glucosylated 5hmC-containing DNA fragments (Song et al., 2011), and fold enrichment over input was assessed by qPCR at the indicated loci and expressed as fold change relative to 5hmC levels in control (EGFP) KD cells. Data represent mean +/− sem.

(E) Immunofluorescence imaging of Mbd3 and 5hmC. Immunofluorescence images are pseudocolored blue (DAPI), green (Mbd3), and red (5hmC) for the indicated KDs – top panel shows 5hmC data only, bottom panel shows all 3 colors.

See also Figure S7.