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. 2006 Oct;5(10):1738-47.
doi: 10.1128/EC.00165-06.

SWI/SNF displaces SAGA-acetylated nucleosomes

Mark Chandy  1 , José L Gutiérrez, Philippe Prochasson, Jerry L Workman
Affiliations

SWI/SNF displaces SAGA-acetylated nucleosomes

Mark Chandy et al. Eukaryot Cell. 2006 Oct.

Abstract

SWI/SNF is a well-characterized chromatin remodeling complex that remodels chromatin by sliding nucleosomes in cis and/or displacing nucleosomes in trans. The latter mechanism has the potential to remove promoter nucleosomes, allowing access to transcription factors and RNA polymerase. In vivo, histone acetylation often precedes apparent nucleosome loss; therefore, we sought to determine whether nucleosomes containing acetylated histones could be displaced by the SWI/SNF chromatin remodeling complex. We found that SAGA-acetylated histones were lost from an immobilized nucleosome array when treated with the SWI/SNF complex. When the nucleosome array was acetylated by SAGA in the presence of bound transcription activators, it generated a peak of acetylation surrounding the activator binding sites. Subsequent SWI/SNF treatment suppressed this acetylation peak. Immunoblots indicated that SWI/SNF preferentially displaced acetylated histones from the array relative to total histones. Moreover, the Swi2/Snf2 bromodomain, an acetyl-lysine binding domain, played a role in the displacement of acetylated histones. These data indicate that targeted histone acetylation by the SAGA complex predisposes promoter nucleosomes for displacement by the SWI/SNF complex.

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Figures

FIG. 1.
FIG. 1.
SWI/SNF reduces the amount of acetylation detected on immobilized nucleosomal arrays. (A) Yeast and HeLa nucleosomes are equivalent substrates for yeast SAGA acetylation. Yeast or HeLa nucleosomes were incubated with acetyl-CoA and/or yeast SAGA. (i) As a loading control, half the reaction was silver stained. (ii and iii) The nucleosomes were immunoblotted with antibodies directed against anti-acetyl H3 and anti-acetyl H4, respectively. For fluorography, yeast or HeLa nucleosomes were incubated with H3-acetyl-CoA and/or yeast SAGA. (iv and v) As a loading control, half the reaction was Coomassie blue stained (iv) prior to electrophoresis, ENHANCE treatment, and fluorography (v). (B) SWI/SNF treatment causes a decrease in acetylation on the nucleosome array. Using competitor chromatin, the artificial activator Gal4-VP16 targets SAGA acetylation, and the array is subjected to SWI/SNF treatment, with acceptor DNA and ATP. After nucleosome displacement, the supernatant is removed, and the array is MNase digested and immunoprecipitated with an anti-acetyl H3 antibody. The immunoprecipitated nucleosomes are subjected to DNA purification and slot blotted onto nylon membrane, along with the supernatant. The membrane is probed with end-labeled full-length pG5E4T template. The %IP is the immunoprecipitated fraction divided by the sum of the immunoprecipitated fraction and the supernatant. In this case, the %IP was normalized to the %IP without SWI/SNF treatment. (C) SWI/SNF targets and displaces activator-targeted SAGA acetylation. The artificial activator Gal4-VP16 is bound to the immobilized nucleosomal array. After the addition of competitor, the array is acetylated with SAGA. The array is subjected to SWI/SNF nucleosome displacement with ATP and acceptor DNA, and the beads are counted on a scintillation counter. The loss of acetylation on the beads corresponds to nucleosomes displaced by SWI/SNF from the array. The change in acetylation on the array is expressed as the relative percent acetylated histones on the beads.
FIG. 2.
FIG. 2.
Nucleosome displacement suppresses the SAGA acetylation peak. (A) Scanning in vitro ChIP assay, with the relative positions of the probes for the pG5E4T array indicated. The array was subjected to nucleosome displacement and ChIP as described in Fig. 1B. The membrane was hybridized to a series of labeled probes shown in the diagram. The positions of the probes relative to the HindIII restriction enzyme site are also indicated on the diagram. The blots are shown below each corresponding probe. (B) The SAGA acetylation peak is suppressed by SWI/SNF. The %IP was normalized to the %IP at the −A probe, which is upstream of the Gal4 binding sites and the E4 promoter. The solid line depicts the H3 acetylation profile along the template, while the dashed line corresponds to the effect of SWI/SNF nucleosome displacement on the H3 acetylation profile. A Student t test was used to determine the statistical significance of the difference in the acetylation profile before and after SWI/SNF treatment. The P values for the t test are given below each segment of the array.
FIG. 3.
FIG. 3.
Acetylated histones are preferentially lost after SWI/SNF nucleosome displacement. (A) The array is bound by activator Gal4-VP16 prior to the addition of competitor chromatin, followed by acetylation under competitive conditions with SAGA. After SWI/SNF nucleosome displacement, free histones are washed away, and the array is subjected to immunoblotting with an anti-VP16 antibody, an anti-acetyl H3 antibody, and an anti-histone H4 antibody. (B) Scanning in vitro ChIP assay with histone H3 antibody. The array is subjected to nucleosome displacement and ChIP as described in Fig. 1B. The membrane is hybridized to a promoter specific probe (−A) and a distal probe (−C) as shown in the diagram. The blots are shown below each corresponding probe, along with a bar graph for nucleosomes remaining on the array. The %IP was normalized to the %IP before nucleosome displacement.
FIG. 4.
FIG. 4.
The Swi2/Snf2 bromodomain is required for the transfer of acetylated histones. (A) SWI/SNF reduction of acetylated histone peak can occur without Gal4-VP16 targeting of this complex. Scanning in vitro ChIP analysis after SWI/SNF nucleosome displacement in the absence of activator Gal4-VP16. The experiment was previously described in Fig. 2. The template is bound to activator Gal4-VP16, acetylated by SAGA with competitor chromatin, and activator is removed by Gal4 oligonucleotide competition. The array was then subjected to SWI/SNF nucleosome displacement and immunoprecipitated with an anti-acetyl H3 antibody. The graph depicts the loss of promoter-acetylated nucleosomes after SWI/SNF nucleosome displacement without activator. The %IP was normalized to the %IP at the −A probe. The solid line depicts the H3 acetylation profile along the template, while the dashed line corresponds to the effect of SWI/SNF nucleosome displacement on the H3 acetylation profile. (B) Scanning in vitro ChIP analysis after activator is removed and SWI/SNF bromodomain mutant treatment. The experiment was as described in Fig. 2. Gal4-VP16 targeted SAGA acetylation at the promoter nucleosomes using competitor chromatin. The activator is removed by Gal4 oligonucleotide competition, and the array is subjected to nucleosome displacement by the bromodomain mutant complex, followed by IP with an anti-acetyl H3 antibody. The graph depicts the loss of promoter-acetylated nucleosomes after nucleosome displacement by the Swi2/Snf2 bromodomain mutant complex. The %IP was normalized to the %IP at the −A probe. The solid line depicts the H3 acetylation profile along the template, and the dashed line corresponds to the effect of Swi2/Snf2 bromodomain mutant on the H3 acetylation profile. A Student t test was used to determine the statistical significance of the difference in the acetylation profile before and after SWI/SNF treatment. The P values for the t test are given below each segment of the array.
FIG. 5.
FIG. 5.
RSC nucleosome displacement suppresses the SAGA acetylation peak. (A) RSC reduction of acetylated histone peak can occur without Gal4-VP16 targeting of this complex. Scanning in vitro ChIP analysis was performed after RSC nucleosome displacement in the absence of activator Gal4-VP16. The experiment was as described in Fig. 2. The template is bound to activator Gal4-VP16, acetylated by SAGA with competitor chromatin, and the activator is removed by Gal4 oligonucleotide competition. The array was then subjected to RSC nucleosome displacement and immunoprecipitated with an anti-acetyl H3 antibody. (B) The graph depicts the loss of promoter-acetylated nucleosomes after RSC nucleosome displacement without activator. The %IP was normalized to the %IP at the −A probe. The solid line depicts the H3 acetylation profile along the template, while the dashed line corresponds to the effect of RSC nucleosome displacement on the H3 acetylation profile. The graph shows the average of two independent experiments.
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