Streptavidin beads

Bisulfite conversion of 1 μg genomic DNA was performed with the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol, and stored at −20 °C until use. Bisulfite converted DNA was eluted in 10 μl of elution buffer, diluted 1:3 with water and 2 μl of diluted DNA were used for PCR. Each 50 μl PCR reaction contained 2.5 mM MgCl₂ and 0.4 μM each of forward and reverse primers designed with PyroMark Assay Design Software 2.0 (Qiagen). Primers used to amplify CpG-rich regions of the mouse bace-1 promoter are detailed in Table S5. The reverse primer was biotinylated at its 5’ end to create a single-stranded DNA template. The PCR conditions were as follows: 98 °C for 15 s; 40 cycles of 98 °C for 10 s, 54 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min. Then, 25 μl of PCR product were immobilised on streptavidin beads (GE Healthcare Lifesciences, Baie d’Urfe QC) and pyrosequencing was performed using the PyroMark Q96 ID System (Qiagen)66 (link).

For peptide pulldown assays, 1 μg of biotinylated histone and ERα peptides with or without methylation were incubated with 1 μg GST-PHF20 Tudor in binding buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.1% NP-40 and 1 mM PMSF) for overnight. Streptavidin beads (GE Healthcare) were added into the mixture and incubated for 1 h with rotation. The beads were then washed three times, and analysed by SDS−PAGE and western blotting. For the protein domain array, the CADOR 5.0 protein domain array was probed with biotinylated ER peptides as described previously33 (link). The protein domains spotted on the CADOR 5.0 array are listed in the Supplementary Fig. 5.

Bisulfite-treated DNA was amplified using PyroMark PCR kit (QIAGEN, Germany) (Table 2). PCR and sequence primers for pyrosequencing were designed with PyroMark Assay Design 2.0 (QIAGEN, Germany). Primer sequences are listed in Table 3. CAV1 methylation was analyzed by pyrosequencing using Pyromark Q24 Advanced System with PyroMark Q24 CpG Advanced Reagents (QIAGEN, Germany) according to the manufacturer’s instructions. Briefly, in a skirted 24-well PCR plate 1 µl streptavidin beads (GE Healthcare, UK), 39 µl PyroMark binding buffer (QIAGEN, Germany), 20 µl high-purity water, and 20 µl PCR product were mixed and plate shook for a minimum of 10 min at 1400 rpm. By using the vacuum workstation, amplicons were denatured and transferred into a pyrosequencing plate containing sequencing primer. After heating the pyrosequencing plate for 3 min at 80 ^∘C, the plate was inserted into the pyrosequencer. Results were analyzed by the PyroMark Q24 Advanced Software 3.0.1. Analyzed sequence encompasses nine CpG sites (hg38; chr7:116,524,607-116,524,746) along EH38E2583972 ENCODE Candidate Cis-Regulatory Element (promoter). Average methylation of sample was calculated from nine CpG and also used in comparison. Which CpG from our study corresponds to which Illumina cg designations is listed in supplementary (Table S1).

Cells were collected and lysed with NETN lysis buffer (150 mM NaCl, 0.5 mM EDTA, 20 mM Tris–HCl at pH7.5, 0.5% Nonidet P-40) containing 10 mM MgCl₂, protease inhibitor (Roche), and TurboNuclease (Accelagen). For precipitation of GFP or SFB-tagged proteins, cell lysates were incubated with GFP-Trap magnetic agarose (Chromotek) or streptavidin beads (GE Healthcare) overnight at 4°C respectively. Immunocomplexes were washed with NETN buffer 3× at 4°C and eluted by boiling in 2× sample loading buffer (100 mM Tris–HCl at pH6.8, 4% SDS, 20% glycerol and 200 mM β-mercaptoethanol is added just before the buffer is used) at 95°C. Samples were resolved by SDS-PAGE and immunoblotted with antibodies as indicated.

Peptide pull-down assays have been described previously [3 (link)]. In brief, individual biotin-conjugated histone H3 peptide (amino acids 1–21; 2 μg) or biotin-conjugated acetyl-histone H3 (Lys9/14) peptide (amino acids 1–21; 2 μg) (Millipore) were incubated with nuclear extracts, precipitated using streptavidin beads (GE Healthcare). The co-precipitated protein was detected by immunoblots. Chromatin-enriched fractions were purified as described previously [3 (link)].