Fluorescence polarization assay. Fluorescence polarization (FP) competitive binding assays were run as described previously with a final volume of 15µL, with each well solution containing 15 nM VCB protein, 10nM FAM-labeled HIF-1α peptide (FAM-DEALAHypYIPMDDDFQLRSF, "JC9") and decreasing concentrations of SIM1 (15-point 2-fold serial dilution starting from 10µM) or SIM1:BET tandem bromodomain protein (15-point 2-fold serial dilutions starting from 10µM SIM1:20µM BET protein). Assays were prepared in triplicate on 384-well plates (Corning 3575) and all measurements taken using a PHERAstar FS (BMG LABTECH) with fluorescence excitation and emission wavelengths (λ) of 485 and 520nm, respectively. Components were dissolved from stock solutions using 100mM Bis-Tris propane, 100mM NaCl, 1mM TCEP, pH 7.0, and DMSO was added as appropriate to ensure a final concentration of 1%. Control wells containing VCB and JC9 with no compound (zero displacement), or JC9 in the absence of protein (maximum displacement) were also included to allow for normalization. Normalized (%) displacement values were plotted against log[SIM1] and curves were fitted by nonlinear regression using GraphPad Prism to determine the IC50 values for each titration. Ki values were backcalculated from the Kd for JC9 (~2nM, determined from direct binding) and fitted IC50 values, as described previously . Cooperativity (α) values were calculated from the ratio of binary Ki and ternary Ki values determined for JC9 displacement by SIM1 alone or SIM1+BET protein, respectively. SPR binding studies. SPR experiments were performed on Biacore T200 instruments (GE Healthcare) as described previously . Immobilization of biotinylated VCB was carried out at 22 °C on a Series S SA chip in an immobilisation buffer containing 20 mM HEPES, 150 mM NaCl, 1 mM TCEP, 0.05 % TWEEN 20, pH 7.0. For binary studies (binding of SIM1 only) the final surface density of biotinylated VHL was approximately 1600-1800 RU; for ternary studies (binding of pre-formed SIM1:target protein complex) multiple lower surface densities of biotinylated VHL used (30-50 RU) to minimize mass transfer effects. Biotinylated VCB was prepared as previously described . All interaction experiments were performed at 22 °C in a running buffer containing 20mM TRIS, 250mM NaCl, 0.2% (w/v) PEG 3350, 0.2% (w/v) BSA, 1mM TCEP, 0.05% TWEEN 20, 1% DMSO; pH 7.5. For binary interaction experiments, SIM1 were initially prepared at 1.5µM solution in the running buffer containing 1% DMSO. This stock solution was then serially diluted in the running buffer containing 1% DMSO (two-fold serial dilution). Solutions were injected individually (duplicate wells) in multi-cycle kinetic format (contact time 60s, flow rate 50 μL/min, dissociation time 150s) using a stabilization period of 30s and syringe wash (50 % DMSO) between injections. For ternary interaction experiments, SIM1 were initially prepared at 500nM in the running buffer containing 2% DMSO. This solution was mixed 1:1 with a solution of 1µM of the BET tandem protein in the running buffer without DMSO, to prepare a final solution of 250nM SIM1 and 500nM BET in running buffer containing 1% DMSO. This complex was then serially diluted in the running buffer containing 500nM BET and 1% DMSO (5-point three-fold serial dilution). All ternary experiments were run in both single-cycle and multi-cycle kinetic modes (two replicate series per experimental repeat, contact time 60s, flow rate 100µL/min, dissociation time 600s) using a stabilization period of 30s and syringe wash (50% DMSO) between injections. Two series of blank injections were performed for all experiments. Sensorgrams from reference surfaces and blank injections were subtracted from the raw data before data analysis using Biacore Insight Evaluation Software. To calculate the association rate (kon), dissociation rate (koff), and dissociation constant (KD), experiments were fitted using a 1:1 Langmuir interaction model, with a term for mass-transport included.