Fig. 4.

Add back of MLL-AF4 fusion reduces methylation of a portion of the protected CpG1 residues after 4 weeks and longer. Mll^−/− MEF cells were transfected with either plasmid to express MLL-AF4 or empty vector. Cell populations were analyzed after 4 weeks of selection (squares, MLL-AF4; diamonds, vector control) or individual clones were selected, replated, and expanded (crosses and circles, two different representative MLL-AF4 add-back clones). DNA was subjected to bisulfite treatment and nested PCR and sequenced. Area under the curve analysis was performed on DNA sequencing histograms and relative methylation percentage was determined for CpG residues. Regions protected from DNA methylation by MLL-AF4 and by MLL (data from Fig. 1) are highlighted under the graph.