Gal4-VP16 Binding Dynamics within Nucleosomes

FRET experiments were used to determine Gal4-VP16 binding to its target site within the nucleosome as previously described.^{21 (link), 61 (link)} Gal4-VP16 titrations were done with 5 nM Cy3-Cy5-labeled nucleosomes in 50 μl volume of buffer containing 49 mM Tris-HCl pH 8.0, 200 mM NaCl, 1.08 mM MgCl₂, and 0.1mM EDTA using a FluoroMax4 fluorescence spectrometer (Horiba). The FRET efficiency was calculated using the (ratio)_A method^{15 (link)} using the fluorescence emission spectra of Cy3 and Cy5 fluorophores. The apparent binding affinity S_1/2 was determined by fitting the FRET efficiency with a sigmoidal binding curve with a Hill coefficient of 1. Gal4-VP16 titrations were performed using nucleosomes with and without neutravidin pre-bound and the S_1/2 is not significantly altered (Figure 6). Control experiments were done using FRET nucleosomes that do not contain a Gal4 binding site (Figure S14). These FRET efficiency data were also fit to a binding curve where the results show extremely similar initial and final FRET efficiency, implying that the observed reduction in FRET is due to Gal4-VP16 binding to the target sequence within the nucleosome.