Fluorescence-based Histone H3/H4-Asf1 Interaction Assay

The X. laevis H3^C110A mutant (xH3^C110A) was used to mimic the yeast histone dimerization surface and to avoid any unwanted cysteine / dye reaction (46 (link)). X. laevis histone H4^T71C mutant (xH4^T71C) was fluorescently labeled (26 (link),46 (link)) (Supplementary Figure S1) with the Qsy9 fluorescence quencher or fluorescein (FM) (Invitrogen) as previously described (46 (link)). The xH3^C110A and Qsy9 or FM labeled xH4^T71C were then prepared as tetramers yielding either H3/H4*^Q or H3/H4*^FM. yAsf1 was labeled with Alexa Fluor 532 (Invitrogen) yielding yAsf1*. The position of the labeled cysteine is shown in Supplementary Figure S1, while the other two cysteines in yAsf1 (Cys³⁰ and Cys⁹⁹) are not near the histone-binding interface and are not labeled efficiently in this procedure (as verified by mass spectrometry, data not shown). Measurements were performed at a yAsf1 concentration of 1 nM in buffer [10 mM Tris-HCl (pH 7.5), 150 mM KCl, 2 mM MgCl₂, 1% glycerol and 0.05% Brij-35]. Double measurements were made with an integration time of 0.5 s on a Horiba Fluorolog-3 spectrometer at 20°C, using a 0.5 cm path-length cuvette. The excitation wavelength was 528 nm, with a slit width of 7 mm; emission was recorded at 547 nm with a slit width of 7 mm. H3/H4 labeled with Qsy9 was titrated to the cuvette containing yAsf1* and the decrease of yAsf1* fluorescence was monitored. Control samples with buffer or unlabeled histones were analyzed likewise to discount buffer effects on fluorescence. The buffer was scanned in the same range used in all experiments for background contributions to the readings and was corrected for in each spectrum. Varying incubation times (0–30 min) confirmed that the fluorescence signal had reached equilibrium by 5 min. The reactions were allowed to equilibrate at 20°C for at least 5 min prior to measurement, and at least three independent experiments were performed for each sample. Data were fitted with the ligand-depleted binding model [Equation (1 (link))] in cases where the concentration of yAsf1* was within 10-fold of the K_d value.

with the variable i indicating the varying concentrations of H3/H4 that were titrated into the yAsf1*.