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. 2008 May 15;105(21):7517–7522. doi: 10.1073/pnas.0800090105

Fig. 1.

Fig. 1.

MLL protects specific CpG residues in the Hoxa9 locus from methylation. (A) Schematic representation of the murine Hoxa9 region. Eight kilobases of the genomic region of Hoxa9 is represented. CpG Islands are numbered beneath. Upstream alternative first exon is labeled AB, the canonical Hoxa9 first exon is labeled CD and the canonical second exon, containing the homeodomain, is labeled II. Large asterisks indicate Mll binding as determined by ChIP. (B) MLL-dependent protection of CpG Island 1 from methylation. Direct bisulfite sequencing reveals a difference in methylation status in Mll+/+ MEF cells (circles) and Mll−/− MEF cells (X). Area under the curve analysis was performed on DNA sequence histograms and relative methylation percentage was determined for CpG residues. Each point on the graph represents a single CpG residue from the sequence below the graph. Beneath the graph are sequencing results from individual clones (10 clones each from Mll+/+ and Mll−/− MEFs) of PCR products from bisulfite treated template. Empty circles, unmethylated CpG residues; filled circles, methylated CpG residues. (C) Upon shRNA knockdown of Mll in Mll+/+ MEF cells, protection from methylation is lost. Triangles, shRNA Mll knockdown; circles, shRNA pSuper vector alone. Knockdowns were selected for 2 weeks. Four-week selection showed similar results (data not shown). (D) Upon add-back of MLL in Mll−/− MEF cells, protection from methylation returns. Filled circles, MLL add-back; x's, vector-only control. Data shown are from cells kept under selection for one week.