Chatterjee N , Sinha D , Lemma-Dechassa M , Tan S , Shogren-Knaak MA , Bartholomew B .

GM 48413 NIGMS NIH HHS , GM 79663 NIGMS NIH HHS , GM060489 NIGMS NIH HHS , R01 GM060489 NIGMS NIH HHS , R01 GM048413 NIGMS NIH HHS

	Figure 1. H3 tail acetylation enhances the affinity of RSC and SWI/SNF for nucleosomes. Gel shift assays were performed with 80 nM RSC (A), 16 nM SWI/SNF (C) or 13 nM ΔBr SWI/SNF (E) and 10 nM mononucleosomes with (H3 Ac) or without (H3) tetra-acetylated H3 tails. Samples in (A and C) contained increasing amounts of competitor DNA (0.25, 0.45, 0.9, 1.8, 3.6 and 7.2 ng/µl in lanes 3–8 and 11–16, respectively) or no competitor DNA (lanes 2 and 10). Lanes 1 and 9 are nucleosomes only. In (E) the competitor DNA used was 0.45, 0.9, 1.8, 3.6 ng/µl, respectively, in lanes 3–6 and 9–12. Quantification of the gel shift assays in (A, C and E) are shown in (B, D and F), respectively. Numbers above the bars indicate the ratio of H3 Ac versus H3 nucleosomes binding for that particular concentration of competitor DNA. The binding ratios are included for only those gel shift lanes in which we have high confidence in the quantification of each species.
	Figure 2. H4 tail acetylation does not increase the affinity of RSC or SWI/SNF for nucleosomes. (A–F) The sections in this figure are the same as shown in Figure 1, except that 10 nM of tetra-acetylated H4 mononucleosomes (H4 Ac) are used instead of tetra-acetylated H3 mononucleosomes. Un-acetylated nucleosomes are represented as H4.
	Figure 3. Acetylation changes the interactions of SWI/SNF with the H3 histone tail. (A) Mononucleosomes coupled to photoreactive I125-PEAS at residues 3, 7, 15 or 22 in the H3 tail were acetylated by yGcn5/Ada2/Ada3 SAGA subcomplex (lanes 1–8). Other nucleosomes had I125-PEAS coupled to residues 15 or 22 in the H4 tail and were acetylated by Piccolo NuA4 (lanes 9–14). Nucleosomes were analyzed before (odd lanes) and after acetylation (even lanes) on a 4% native PAGE. (B) SWI/SNF subunits labeled by crosslinking at positions 3, 7, 15 and 22 of H3 were separated on 4–12% Bis–Tris SDS–PAGE and visualized by phosphorimaging. The quantification of these profiles are overlaid using Image Quant software (Molecular Dynamics) for non-acetylated and yGcn5/Ada2.Ada3 acetylated nucleosomes as shown along with the positions of the Swi2/Snf2, Snf5, Swp82, SWP73, Arp7/9 and Snf6 subunits of SWI/SNF. (C) The same approach as in (B) was used to determine which SWI/SNF subunits were crosslinked at positions 15 and 22 of H4 in non-acetylated and Piccolo NuA4 acetylated nucleosomes.
	Figure 4. SWI/SNF recruitment by the transcription activator Gal4-VP16 and H3 acetylation. (A) The order of addition of nucleosomes, competitor DNA and SWI/SNF is shown for the recruitment assays in (B and D). Nucleosomes contain one Gal4 site in extranucleosomal DNA, 27 bp from the entry site. Gel shift assays are shown with 15 nM SWI/SNF (B) or 10 nm ΔBr SWI/SNF (D) that had 10 nM nucleosomes and 25 nM Gal4-VP16 where indicated. H3 acetylated nucleosomes (H3 Ac) are in lanes 12–25 and nucleosomes without acetylation (H3) are in lanes 1–11. Increasing amounts of competitor DNA were added ranging from 0.45 to 35 ng/µl for lanes 7–11 and 21–25 or from 0.45 to 1.3 ng/µl for lanes 3–4 and 14–18. Species I, II, III and IV refer to nucleosome-Gal4-SWI/SNF, nucleosome-SWI/SNF, nucleosome-Gal4 and DNA-Gal4, respectively. Quantification of (B and D) are shown in (C and E), respectively, for SWI/SNF and ΔBr SWI/SNF binding to H3 Ac versus H3 nucleosomes with and without Gal4-VP16. The numbers above the bars is the ratio of H3 Ac versus H3 nucleosomes binding with or without Gal4-VP16 added. The binding ratios are included for those particular gel shift lanes in which each species could be accurately quantified.
	Figure 5. H3 tail acetylation stimulates nucleosome mobilization in a bromodomain dependent manner. The rate of nucleosome movement by 80 nm RSC (A and B), 20 nM SWI/SNF (C and D), and 20 nm ΔBr SWI/SNF (E and F) was determined under the same conditions as in Supplementary Figure S5 by gel shift assay using 10 nM 601–603 dinucleosomes. (A, C and E) The concentration of mobilized dinucleosomes moved versus time was plotted and fitted non-linearly to the Michaelis–Menten equation using Graph Pad Prism. (B, D and F) The rate at which the first H2A/H2B dimer is displaced from dinucleosomes was estimated by tracking the appearance of the remodeled species I in two independent experiments. This rate was determined in the same way as the rate of nucleosome movement.
	Figure 6. Nucleosome disassembly by RSC and SWI/SNF is not enhanced by H3 tail acetylation. The rate at which one nucleosome is removed from the 601–603 dinucleosome was determined using conditions similar to that in Figure 5, except that the ATP concentration was increased to 55 µM and incubated at 25°C. The appearance of the second remodeled species (II) was followed by gel shift on a 4% native polyacrylamide gel for RSC (A), SWI/SNF (C), and ΔBr SWI/SNF (E). The concentration of remodeled species II formed by RSC (B), SWI/SNF (D), and ΔBr SWI/SNF (F) was plotted against time and fitted non-linearly to the Michaelis–Menten equation when using non-acetylated (H3) or H3 acetylated (H3Ac) dinucleosomes.
	Figure 7. H3 tail acetylation modulates SWI/SNF and RSC function by distinct mechanisms. RSC and SWI/SNF recruitment occurs by two independent pathways mediated by some DNA sequence specific transcription factors (TF). Transcription activators recruit via direct interactions with SWI/SNF. Alternatively, transcription factors mediate recruitment of histone acetyl transferases (HATs) that catalyzes site-specific histone H3 tail acetylation and leads to SWI/SNF and RSC recruitment via bromodomain-acetyl lysine interaction. Recognition of acetyl marks on H3 tails via their bromodomains also modulates the nucleosome remodeling function of these complexes apart from recruitment. Specifically, H3 acetylation facilitates nucleosome movement in nucleosomal arrays and enhances H2A/H2B displacement from neighboring nucleosomes.

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