Histone h3

Cell lysates were prepared in Co-IP buffer (10 mM HEPES, pH 7.5, 50 mM NaCl, 30 mM Na₄P₂O₇, 50 mM NaF, 0.2% (v/v) Triton X-100, 10% (v/v) glycerol, 5 μM ZnCl₂, 1 × PIC). Lysed cells were mixed vigorously for 45 sec and centrifuged at 16 000 × g and 4°C for 20 min. The protein concentrations of the supernatants were measured and adjusted accordingly. Aliquots were retained for direct Western blot analysis (Input) and otherwise subjected to immunoprecipitation (IP) with protein A-coupled Sepharose beads (Merck, 17-5280-02) pre-incubated with 2 μg anti-RbBP5 antibody or IgG at 4°C for 2 h. Protein–antibody-bead complexes were washed three times with Co-IP buffer and twice with SAM assay buffer (50 mM Tris–HCl (pH 8.0), 5 mM MgCl₂, 50 mM NaCl, 1 mM DTT, 1× PIC). Afterwards 2 μg of recombinant human Histone H3.1 (New England BioLabs, M2503), 100 μM S-(5′-adenosyl)-l-methionine chloride dihydrochloride (SAM, Merck, A7007) used as a primary methyl donor molecule, dissolved in 30 μl SAM assay buffer were added to the beads and incubated for 1 h at 31°C with agitation. Subsequently the beads were heated in sample buffer (40 mM Tris pH 6.8, 10% Glycerol, 2% SDS, bromophenol blue) at 95°C for 10 min and used for SDS-PAGE and immunoblotting.

Immunoprecipitation was performed as described above with the Aurora B antibody (H-75, Santa Cruz). Immunoprecipitates were resuspended in kinase buffer (20 mM Tris-HCl, 20 mM KCl, 20 mM MgCl₂, 0.4 μM ATP, 0.4 mM DTT) and incubated with 1 μg of purified Histone H3.1 (New England Biolabs) for 1 hour at 37 °C. The Ku70 WT and S155D vWA peptides were expressed in bacteria according to standard protocols. vWa domain-containing bacterial extracts were added to the immunoprecipitates and incubated for 30 min on ice before adding kinase buffer and Histone H3.1. Phosphorylation was detected by western blot with a phospho-H3S10 antibody (Cell Signaling).

Wild-type (WT) and non-phosphorylatable GST-CARM1 were incubated with JAK2 kinase, as described in the in vitro kinase assay, and used for in vitro assays. We also used MYC-tagged WT CARM1 and non-phosphorylatable mutant CARM1, which were purified from transfected 293 T cells by an anti-MYC immunoprecipitation. Two known CARM1 substrates, histone H3.1 (New England Biolabs, M2503S) and RUNX1b (MyBioSource, MBS1471613), were used in the methylation assay (Supplementary Table 5). The MYC-CARM1 protein or recombinant CARM1 protein was then incubated with substrates in the presence of 5 mM Tris [pH 8.5], 2 mM KCl, 1 mM MgCl2, 0.1 mM β-mercaptoethanol, 10 mM sucrose) and S-adenosyl-L-[methyl-3H] methionine (SAM)(15 Ci/mmol, 66 µM; Amersham Bioscience) at 30 °C for 4 h. The reactions were stopped by adding an SDS loading buffer, and the proteins were separated in 4-12% NuPAGE Bis-Tris Protein Gels (Invitrogen, NP0336BOX). After fixation in 45% methanol and 10% acetic acid for 30 min, the gels were treated with Gel Dry Drying Solution (Thermo Fisher Scientific, LC4025) for 15 min, and exposed to x-ray films.