Fig. 6: Acetylation regulates the association of p300 with DNA and the nucleosome.

a Representative confocal images of phase-separated SIRT2-treated p300_BRPHZ condensates with FAM control (left), FAM-labeled H3_1–12 peptide (middle), and FAM-labeled DNA (right). A 50 µm × 50 µm square region is shown for each sample. b, c EMSA of 147-bp 601 DNA incubated with increasing amounts of SIRT2-treated (b) and untreated (c) p300_BRPHZ. DNA and protein concentrations are shown above the gel image. d, e EMSA of the reconstituted nucleosome incubated with increasing amounts of SIRT2-treated (d) and untreated (e) p300_BRPHZ. Nucleosome core particle (NCP) and protein concentrations are shown above the gel image. f Phase separation of SIRT2-treated p300_BRPHZ alone (left), p300_BRPHZ incubated with NCP (middle) and NCP alone (right) in a buffer containing 12% PEG. A 50 µm × 50 µm square region is shown for each sample. Experiments in a–f were performed in at least two replicates. g HAT activity of diluted (0.2 μM) (g) and concentrated (10 μM) (h) SIRT2-deacetylated p300_BRPHZ ± PEG was measured by a fluorometric assay. i HAT activity of SIRT2-deacetylated p300_BRPHZ in supernatant measured by a fluorometric assay. Droplets in the phase-separated p300_BRPHZ sample were spun down for 10 min, and the supernatant was transferred to another tube. For all experiments in (g–i), reactions were quenched by adding 6 M guanidine hydrochloride. The relative reaction rates (slope values) are 0.020 (−PEG) and 0.027 (+PEG) in (g), 0.27 (−PEG) and 0.09 (+PEG) in (h), and 0.035 in (i). Data in g, h are presented as mean values ± SD; error bars represent SD from triplicate measurements. j HAT activity of untreated p300_BRPHZ (blue), SIRT2-treated p300_BRPHZ (orange), and SIRT2-treated phase-separated p300_BRPHZ droplet suspension (gray) on NCP measured by a fluorometric assay. Data are presented as mean values from duplicate measurements.