Hek293f suspension grown cells

Wild type and mutant MTA1 constructs were cloned into pcDNA3 vectors containing an amino terminal His₁₀-Flag₃ purification tag and a TEV protease cleavage site. Full length HDAC1 (residues 1–482), RBBP4 (residues 1–425) and MBD2 (residues 145–411) were cloned without affinity tags into the same vectors. Protein was expressed in HEK293F suspension-grown cells (Invitrogen) using polyethylenimine (PEI; Sigma) as a transfection reagent and harvested after 48 h as previously described (15 (link),30 ). Cells were lysed in 50 mM Tris–HCl (pH 7.5), 100 mM potassium acetate, 10% (v/v) glycerol, 0.3% (v/v) Triton X-100 and Roche Protease Inhibitor (buffer A). The lysate was clarified by centrifugation and applied to FLAG resin (Sigma) for 30 min and washed three times with 50 mM Tris–HCl (pH 7.5), 100 mM potassium acetate and 5% (v/v) glycerol (buffer B). The protein was treated with RNase A in buffer B for 1 h, washed twice more with 25 mM Tris–HCl (pH 7.5), 75 mM potassium acetate and 0.5 mM TCEP (buffer C), before being eluted with TEV protease overnight. The protein complexes were purified further by gel filtration using a Superose 6 column (GE Healthcare, UK).

MIER1 constructs were cloned into pcDNA3.0 expression vectors containing an amino-terminal 10xHis-3xFLAG tag followed by a TEV protease cleavage site. Full length HDAC1(aa:1–482) and BAHD1(aa:525–780) were cloned without affinity tags into the same vectors. Constructs were co-transfected in HEK293F suspension grown cells (Invitrogen) using polyethylenimine (PEI; Sigma) as a transfection reagent and harvested after 48 h. Cells were lysed in buffer containing 50 mM Tris/HCl pH 7.5, 50 mM potassium acetate, 5% v/v glycerol, 0.4% v/v Triton X-100 and Complete EDTA-free protease inhibitor (Roche). The lysate was clarified by centrifugation and applied to Anti-FLAG M2 affinity resin (Sigma Aldrich) for 30 min and washed three times with 50 mM Tris/HCl pH 7.5, 50 mM potassium acetate and 5% v/v glycerol and three times more with 50 mM Tris/HCl pH 7.5, 50 mM potassium acetate and 0.5 mM TCEP before being cleaved by TEV protease overnight. The protein complexes were purified further by gel filtration using a Superdex S200 column (Cytiva) with buffer containing 25 mM Tris/HCl pH 7.5, 50 mM potassium acetate and 0.5 mM TCEP.