Swi/Snf dynamics on stress-responsive genes is governed by competitive bromodomain interactions

Abstract

The Swi/Snf chromatin remodeling complex functions to alter nucleosome positions by either sliding nucleosomes on DNA or the eviction of histones. The presence of histone acetylation and activator-dependent recruitment and retention of Swi/Snf is important for its efficient function. It is not understood, however, why such mechanisms are required to enhance Swi/Snf activity on nucleosomes. Snf2, the catalytic subunit of the Swi/Snf remodeling complex, has been shown to be a target of the Gcn5 acetyltransferase. Our study found that acetylation of Snf2 regulates both recruitment and release of Swi/Snf from stress-responsive genes. Also, the intramolecular interaction of the Snf2 bromodomain with the acetylated lysine residues on Snf2 negatively regulates binding and remodeling of acetylated nucleosomes by Swi/Snf. Interestingly, the presence of transcription activators mitigates the effects of the reduced affinity of acetylated Snf2 for acetylated nucleosomes. Supporting our in vitro results, we found that activator-bound genes regulating metabolic processes showed greater retention of the Swi/Snf complex even when Snf2 was acetylated. Our studies demonstrate that competing effects of (1) Swi/Snf retention by activators or high levels of histone acetylation and (2) Snf2 acetylation-mediated release regulate dynamics of Swi/Snf occupancy at target genes.

Keywords: Swi/Snf; activator; histone acetylation; stress.

Figure 1.

Swi/Snf occupancy at genes under stressed and unstressed conditions. (A) Occupancy of Snf2 under unstressed conditions. (B) Occupancy of Snf2 under stressed conditions. ChIP experiments were performed using Snf2 antibody in wild-type (WT) and Snf2 K1493R K1497R (2R) strains. Log₂ ratios of normalized enrichment (immunoprecipitation [IP]/input) for each gene region, including 500 bp upstream of and downstream from the gene, were used for average gene analysis. Genome average data of genes that showed enrichment of at least 1.7-fold of immunoprecipitation/input in both replicates were calculated and plotted. The TSS and termination site (TES) are indicated. Transparent shading around the line indicates the standard error of the mean. (C) Genes were grouped based on changes in Snf2 occupancy during stress. The heat map of Snf2 occupancy for wild type and Snf2 K1493R K1497R (2R) in each group under stressed and unstressed conditions was generated by hierarchically row-clustering normalized log₂ (immunoprecipitation/input) values for regions −700/+500 around the TSS of genes.

Figure 2.

Acetylation of Snf2 regulates recruitment and release of Swi/Snf from genes during stress. (A,B) Group 1: Genes (391) at which Swi/Snf is occupied under unstressed conditions but reduced during stress. (D,E) Group 2: Genes (529) that are not occupied by Swi/Snf during unstressed conditions, but their occupancy increases during stress. (G,H) Group 3: Genes (452) that are occupied by Swi/Snf under both unstressed and stress conditions. Average log₂ ratios of normalized enrichment (immunoprecipitation [IP]/input) for each gene region, including 500 bp upstream of and downstream from the gene, were calculated and plotted for both the wild type (WT) and Snf2 K1493R K1497R (2R) under unstressed and stressed conditions. The TSS and TES are indicated. Transparent shading around the line indicates the standard error of the mean. P-values are based on mean values per gene in the TSS region (−700/+500) and are from a two-sided Student’s t-test between samples. (C,F,I) Average log₂ ratios of changes in Snf2 occupancy during stressed conditions for each gene region, including 500 bp upstream of and downstream from the gene, were calculated and plotted for both the wild type and Snf2 K1493R K1497R (2R) for each group.

Figure 3.

Snf2 acetylation regulates Swi/Snf binding and remodeling of acetylated nucleosomes. Twenty femtomoles of 5′ Cy5-labeled unacetylated 25N47 mononucleosomes (A) and 20 fmol of 5′ Cy5-labeled acetylated 25N47 mononucleosomes (B) were incubated with increasing concentrations of Swi/Snf complexes (0, 10, 20, 40, 60, and 80 fmol) and analyzed by native PAGE. Bands indicating unbound mononucleosome and Swi/Snf-bound mononucleosomes are indicated. Plots for Swi/Snf binding represent mean values ± SEM for at least three independent experiments. Remodeling of unacetylated (C) versus acetylated (D) nucleosomes as a function of Swi/Snf concentration. In each case, 10 fmol of mononucleosomes was incubated with increasing concentrations of Swi/Snf complexes (0, 10, 15, 20, 30, and 40 fmol) in the presence of 1.5 ng of competitor salmon sperm DNA. Reactions were carried out for 30 min. Plots for remodeling are shown.

Figure 4.

Histone acetylation levels determine differential recruitment of Swi/Snf between the wild type (WT) and mutant. Histone acetylation was assayed by ChIP experiments using H3K9Ac antibody in the wild-type and Snf2 K1493R K1497R (2R) strains. Log₂ ratios of normalized enrichment (immunoprecipitation [IP]/input) for each gene region, including 500 bp upstream of and downstream from the gene, were used for average gene analysis. The TSS and TES are indicated. Transparent shading around the line indicates the standard error of the mean. Genes were grouped based on changes in Snf2 occupancy as described earlier. H3K9Ac at group 1 genes in unstressed (A) and stressed (B) conditions, group 2 genes in unstressed (D) and stressed (E) conditions, and group 3 genes in unstressed (G) and stressed (H) conditions are shown. (C,F,I) Average log₂ ratios of changes in H3K9Ac occupancy during stressed conditions for each gene region, including 500 bp upstream of and downstream from the gene, were calculated and plotted for both the wild type and Snf2 K1493R K1497R (2R) for each group. (J) Competition assay was carried out by binding 25 fmol of Swi/Snf complexes (purified from wild-type, Snf2 K1493R K1497R [2R], and Δ gcn5 strains) to 100 fmol of unlabeled unacetylated 25N47 nucleosomes followed by competition with increasing concentrations (0, 10, 20, 40, and 60 fmol) of acetylated 5′ Cy5-labeled 25N47 nucleosomes. Swi/Snf-bound complexes were resolved by native PAGE. Bands indicating unbound mononucleosomes and Swi/Snf-bound mononucleosomes are indicated. Plots for Swi/Snf binding are normalized to wild-type Swi/Snf binding.

Figure 5.

Differential remodeling due to Snf2 acetylation affects nucleosome occupancy in genes. Nucleosome positioning was determined by MNase-seq. The average nucleosome profile in wild-type (WT) and Snf2 K1493R K1497R (2R) strains for group 1 (A,B), group 2 (C,D), and group 3 (E,F) genes during unstressed and stressed conditions was calculated and plotted 500 bp upstream of and downstream from the TSS using ngsplot. Transparent shading around the line indicates the standard error of the mean.

Figure 6.

Acetylation of Snf2 prevents aberrant engagement of Swi/Snf in stress-dependent genes. Snf2 occupancy (A,B) and nucleosome occupancy (C) in genes in which Swi/Snf is slow to release from the promoters and ORF in Snf2 K1493R K1497R (2R) compared with the wild type (WT). This set of genes was defined as a subset of group 1 in which the sum of normalized log₂ immunoprecipitation (IP)/input TSS (−700/+500) coverage values was <0 in the wild type (stressed) and >0 in 2R (stressed) and was plotted. Snf2 occupancy (D,E) and nucleosome occupancy (F) in genes in which Swi/Snf is preoccupied at the promoters and ORF in Snf2 K1493R K1497R (2R) but not in the wild type. Genes in group 2 in which the sum of normalized log₂ immunoprecipitation/input TSS (−700/+500) coverage values was <0 in wild type (unstressed) and >0 in 2R (unstressed) were used for the genome average plot. Transparent shading around the line indicates the standard error of the mean. P-values are based on mean values per gene in the TSS region (−700/+500) and are from a two-sided Student’s t-test between samples.

Figure 7.

Bromodomain of Snf2 regulates Swi/Snf occupancy during stress. (A) Genes were grouped based on changes in Snf2 occupancy during stress as in Figure 1C. The heat map of Snf2 occupancy for the wild type (WT) and the Snf2 ∆bromodomain in each group under stressed and unstressed conditions was generated by hierarchically row-clustering normalized log₂ (immunoprecipitation [IP]/input) values for regions −700/+500 around the TSS of genes. (B) Group 2 genes were clustered based on the highest to lowest levels of H3K9Ac and divided into quartiles. Average gene analysis was done as described in the Materials and Methods. Difference profiles for changes in Snf2 occupancy in mutants compared with the wild type for each quartile are shown. The TSS and TES are indicated.

Figure 8.

The presence of activator increases binding and remodeling of the wild-type (WT) Swi/Snf complex to levels similar to that of the mutant with unacetylated Snf2. To assay for binding in the presence of Gal4-VP16, 20 fmol of 5′ Cy5-labeled unacetylated N72 mononucleosomes (A) or 20 fmol of 5′ Cy5-labeled acetylated N72 mononucleosomes (B) containing Gal4-binding sequence was incubated with/without 200 fmol of Gal4-VP16 followed by binding with increasing concentrations of Swi/Snf complexes (0, 20, 40, and 60 fmol) in the presence of 2 ng of competitor salmon sperm DNA and was analyzed by native PAGE. Bands indicating unbound mononucleosome, Gal4-VP16-bound DNA/mononucleosome, and Swi/Snf-bound mononucleosomes or Swi/Snf-bound Gal4-VP16 + mononucleosome are indicated. Remodeling of Gal4-VP16-bound unacetylated (C) or acetylated (D) mononucleosomes was assayed using 20 fmol of 5′ Cy5-labeled mononucleosomes with 20 fmol of Swi/Snf complex (from wild-type, Snf2 K1493R K1497R [2R], and Δ gcn5 strains) and 200 fmol of Gal4-VP16 in the presence of 2 ng of competitor salmon sperm DNA as a function of time (5, 15, and 30 min). (E) Genome browser screenshots of Snf2 occupancy for wild type, 2R, Snf2 Δbromodomain, and 2R Δbromodomain strains at HXT6 and HXT7 that are regulated by Adr1 and YPL270W that were not found to be bound by activator using online tool YEASTRACT. The top two rows in each genome browser screenshot show wild-type H3K9Ac occupancy during unstressed and stressed conditions.

Figure 9.

Model for regulation of Swi/Snf recruitment and release from genes. Swi/Snf can be recruited to genes by activators and histone acetylation. High levels of histone acetylation help compete against Snf2 acetylation, allowing for interaction with the bromodomain (group 2 genes). Acting simultaneously or in parallel, activator-dependent recruitment of Swi/Snf can further stabilize Swi/Snf occupancy due to additional contacts with activator by subunits of the Swi/Snf complex that allow for retention of Swi/Snf, keeping genes in an ON state (groups 1, 2, and 3). When genes are turned off, activators are quickly released from promoters of genes (group 1). In the absence of positive regulation by activators and reduction in levels of histone acetylation, decreased competition from acetylated histones allows the bromodomain to bind acetylated residues of Snf2 for limited retention and quick release of Swi/Snf from promoters.