. SEC experiments were carried out in a ÄKTA pure system (GE Healthcare) at room temperature. The oligomeric state of the BRD4 BD1-BD2 tandem protein in solution was analyzed by gel filtration in a buffer containing 20mM HEPES (pH 7.5), 100mM NaCl and 1mM TCEP using a Superdex 200 Increase 10/300 GL column (GE Healthcare) calibrated with globular proteins of known molecular weight (GE Healthcare, 28-4038-41/42). BRD4 tandem (25 µM) was incubated with SIM1 (25µM), MZ1 (25µM), MT1 (25µM) or DMSO (0.5 %) for 30 min at room temperature prior to injection. Sample volume for each injection was 200µl, and the flow rate was 0.8 ml/min. Peak elution was monitored using ultraviolet absorbance at 280nm. ITC. Titrations were performed as reverse titration on an ITC200 micro-calorimeter (Malvern). SIM1 was not soluble enough to be loaded at the required concentrations in the syringe (normal direct titration), therefore reverse titrations were performed. The titrations consisted of 19 injections of 2µl tandem BRD4 BD1-BD2 construct (WT or N140F or N433F) solution in 20mM Bis-Tris propane, 100mM NaCl, 1mM TCEP, 1.6% DMSO, pH 7.5, at a rate of 0.5µl/s at 120 s time intervals. An initial injection of protein (0.4µl) was made and discarded during data analysis. All experiments were performed at 25°C, whilst stirring at 750 r.p.m. SIM1 from 10mM DMSO stock solution and VCB were diluted in buffer containing 20mM Bis-Tris propane, 100mM NaCl, 1mM TCEP, pH 7.5. The final DMSO concentration was 1.6% v/v. BRD4 protein (200µM, in the syringe) was titrated into the SIM1-VCB complex (SIM1 16µM, VCB 32µM, in the cell). Data were fitted to a single-binding site model for each BRD4 mutant to obtain the stoichiometry (n), the dissociation constant (Kd) and the enthalpy of binding (ΔH). Data for WT BRD4 was fitted to a two sets of sites model to account for the reverse titration set-up whereby a two-site protein is titrated into a bivalent ligand (see Malvern MicroCal ITC analysis software using Origin™ User Manual, pg. 102). Data fitting was performed using Microcal LLC ITC200 Origin software provided by the manufacturer.