Mbp magnetic beads

His-MBP-ZBTB43 recombinant protein was purified from E. coli and stored in dialysis buffer (50 mM HEPES pH 7.4, 50% glycerol and 5 mM 2-mercaptoethanol) at −80 °C. Unmethylated genomic DNA was purified from mouse TKO ES cells (Dnmt1^−/−Dnmt3a^−/−Dnmt3b^−/−) ES (clone19) RBRC no. AES0146 (ref. ^{16 (link)}) with phenol–chloroform extraction and ethanol precipitation, and was sonicated to an average fragment size of 200 bp using a Covaris E220 Evolution ultrasound sonicator. When indicated, M.SssI (NEB, cat. no. M0226M) was used to methylate the CpGs in fragmented mouse lung genomic DNA. His-MBP-ZBTB43 protein was pre-incubated with MBP-magnetic beads (NEB, cat. no. E8037S) at 4 °C for 90 min. To capture the ZBTB43 fraction, the fragmented mouse genomic DNA was mixed with the pre-incubated beads in a 200 µl reaction volume of binding buffer (10 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl₂, 0.05% NP40, 25 ng/µl BSA, 1 mM DTT, 0.05 mM ZnCl₂) at 37 °C for 2 h. After incubation, the beads were washed with binding buffer 3 times to remove unbound DNA. The bound DNA was eluted in elution buffer (10 mM Tris–HCl pH 8.0, 10 mM EDTA, 500 mM NaCl, 1% SDS and 10 mM maltose) and collected by ethanol precipitation. Precipitated DNA was stored at −80 °C before library preparation.

Infiltrated leaves expressing the protein of interest were powdered in a mortar under liquid N₂. About 2 g of tissue was weighed, and to it, 3 volumes of lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM KCL, 1% Triton X100, Protease inhibitor 1 X [Roche], NEM 20uM) were added. The supernatant was collected after a spin at 16000g for 30 min and incubated with GFP-Trap (Chromtek) or MBP magnetic beads (NEB) for 3 h at 4 °C. Beads were magnetically separated from the lysate and washed 5 times in wash buffer (50 mM Tris-Cl, pH 7.4; 150 mM KCL, 1 mM PMSF) until the green colour completely disappeared. The final pull-down beads were transferred to a 1.5-ml tube and again washed twice with wash buffer. The 3X SDS sample dye was added to the beads, and the sample was heated at 70 °C for 10 min. The pull-down products were resolved in 4–20% Tris-Glycine SDS gradient gels (Bio-Rad). IP beads used are listed in Additional file 2: Table S4.

His-MBP-tagged UHRF1 SRA-RING (amino acids 405–793) was produced in E. coli as described above. GST-tagged UHRF1 TTD-PHD (amino acids 123–366) and BPTF PHD-Bromo (gift from Dr. Alex Ruthenburg [Ruthenburg et al., 2011 (link)]) were produced as previously described (Rothbart et al., 2013 (link)). Proteins (each at 1 μM) were incubated overnight at 4°C with MBP magnetic beads (NEB, Ipswich, MA) in binding buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1% NP-40, 0.5% BSA, and, where indicated, 25 μM DNA oligonucleotides or histone peptides. Pulldown experiments with the SRA-RING DNA^mut were performed with 5 μM DNA. Samples were washed extensively with binding buffer, eluted in SDS sample buffer, resolved by SDS-PAGE, transferred to PVDF membrane (Thermo), and probed with GST antibody (Sigma #G7781, 1:2,000). Pull-down assays were performed in triplicate.