A SILAC-Based Screen for Methyl-CpG Binding Proteins Identifies RBP-J as a DNA Methylation and Sequence-Specific Binding Protein
Figure 2
RBP-J preferentially binds a methylated CpG-island sequence in vitro.
(A) Results of a methyl-CpG pull-down/SILAC experiment with U937 nuclear extracts. Forward and reverse pull-downs were performed, and forward heavy/light ratios of identified proteins were plotted against their reverse heavy/light ratios in a scatter-plot. Proteins that bind specifically to the methylated DNA show high ratios in the forward experiment and low ratios in the reverse experiment and therefore cluster in the lower right quadrant. Background binders appear around the centre of the axes with ratios close to one in both experiments. (B) RBP-J specifically binds to the methylated GSTP1 CpG-island DNA. Shown are MS signals of peptides from MBD2 (upper panel) and RBP-J (lower panel) from both forward (left) and reverse (right) experiments. L, light; H, heavy. (C) RBP-J directly binds to the methylated GSTP1 CpG-island DNA. Methyl-CpG pull-down experiments were performed, using western blotting as a read-out. Upper and middle panel: pull-down with U937 nuclear extract and probing for MBD2 (positive control) and RBP-J. Lower panel: pull-down with cleared lysate of E. coli expressing GST-tagged human RBP-J and probing for RBP-J.