A SILAC-Based Screen for Methyl-CpG Binding Proteins Identifies RBP-J as a DNA Methylation and Sequence-Specific Binding Protein
Figure 3
Preferential binding of RBP-J to methylated DNA is sequence specific.
(A) RBP-J binds methylated GSTP1 CpG-island DNA but not methylated GAM12. EMSAs were performed with recombinant GST-tagged human RBP-J, and DNA fragments as indicated. Recombinant MBD2 was used as a positive control. Anti-RBP-J and anti-MBD2 antibodies were added to supershift the DNA. Anti-RBP-J(1): ab25949 (Abcam); anti-RBP-J(2): ab33065 (Abcam); anti-MBD2: 07-198 (Millipore). (B) RBP-J binds to the first part of the methylated GSTP1 CpG-island DNA and not to methylated GAM4. Methyl-CpG pull-downs were performed with U937 nuclear extract and different DNA fragments as indicated. In GSTP1-del the double CpG is replaced by a single CpG. Western blotting was used as a read-out, and MBD2 was probed as a positive control.