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. 2011;6(10):e25884.
doi: 10.1371/journal.pone.0025884. Epub 2011 Oct 3.

A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific binding protein

Stefanie J J Bartels  1 , Cornelia G Spruijt, Arie B Brinkman, Pascal W T C Jansen, Michiel Vermeulen, Hendrik G Stunnenberg
Affiliations

A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific binding protein

Stefanie J J Bartels et al. PLoS One. 2011.

Abstract

Background: DNA methylation is an epigenetic modification that plays a crucial role in a variety of biological processes. Methylated DNA is specifically bound by Methyl-CpG Binding Proteins (MBPs). Three different types of MBPs have been identified so far: the Methyl-CpG Binding Domain (MBD) family proteins, three BTB/POZ-Zn-finger proteins, and UHRF1. Most of the known MBPs have been identified via homology with the MBD and Zn-finger domains as present in MeCP2 and Kaiso, respectively. It is conceivable that other proteins are capable of recognizing methylated DNA.

Methodology/principal findings: For the purpose of identifying novel 'readers' we set up a methyl-CpG pull-down assay combined with stable-isotope labeling by amino acids in cell culture (SILAC). In a methyl-CpG pull-down with U937 nuclear extracts, we recovered several known MBPs and almost all subunits of the MBD2/NuRD complex as methylation specific binders, providing proof-of-principle. Interestingly, RBP-J, the transcription factor downstream of Notch receptors, also bound the DNA in a methylation dependent manner. Follow-up pull-downs and electrophoretic mobility shift assays (EMSAs) showed that RBP-J binds methylated DNA in the context of a mutated RBP-J consensus motif.

Conclusions/significance: The here described SILAC/methyl-CpG pull-down constitutes a new approach to identify potential novel DNAme readers and will advance unraveling of the complete methyl-DNA interactome.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. A SILAC-based approach to identify novel methyl-CpG binding proteins.
(A) Schematic depiction of the methyl-CpG pull-down assay combined with SILAC. Synthetic DNA is coupled to beads to capture binding proteins from a nuclear extract. Shown is a forward experiment, in which fully methylated DNA is used in a heavy-labeled extract and unmethylated DNA in an unlabeled extract. In a reverse experiment unmethylated DNA is used in a heavy-labeled extract, whereas methylated DNA is used in an unlabeled extract. After several washing steps and elution, both pull-down fractions are combined and analysed by mass spectrometry. Proteins that directly or indirectly specifically bind to the methylated DNA are identified by the quantitative ratios between heavy and light form. (B) DNA used in the methyl-CpG pull-down. The DNA fragment contains part of the sequence of the GSTP1 CpG-island, sites for primer annealing and a methylation-sensitive restriction site. After ligation a mixture of fragments with different lengths is obtained, which is subsequently biotinylated and methylated. Methylation is checked by a methylation-sensitive digestion followed by quantitative PCR.
Figure 2
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.
Figure 3
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.
Figure 4
Figure 4. RBP-J binding to a mutated RBP-J consensus site is restored by methylation in vitro.
RBP-J binding to the perfect RBP-J consensus motif and various altered sites in unmethylated and fully methylated states. EMSAs were performed with recombinant GST-tagged human RBP-J, and DNA fragments as indicated. Altered motifs were designed by substituting residues in the normal consensus (5′-AGCGTGGGAACTT-3′) upstream of a guanine with a cytosine, and residues downstream of a cytosine with a guanine. When required, the single CpG site in the normal consensus was replaced by a TpG to maintain only one CpG per sequence. The resulting 6 altered sites with one CpG per sequence and the normal consensus (lane 7–8) sequence are indicated above lanes.
Figure 5
Figure 5. Schematic overview of RBP-J binding motifs in vitro.
RBP-J is known to bind to the RBP-J consensus motif. Binding is maintained when replacing thymine by a methyl-cytosine, whereas replacement by cytosine results in very weak binding only.
See this image and copyright information in PMC

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