SWI/SNF removes histone acetylated by SAGA from immobilized nucleosome arrays.
Our studies utilized immobilized nucleosome arrays. Once bound to magnetic beads, these nucleosomal arrays could be sequentially modified by chromatin modifying and/or chromatin remodeling complexes, which were added and removed, allowing for detailed mechanistic studies of chromatin dynamics. In this particular study, we used the pG5E4T template (31
), which contains five Gal4 binding sites and an E4 promoter. The pG5E4T template was linearized by restriction enzyme digestion and subsequently biotinylated at one end. Upon reconstitution with HeLa core histones, the nucleosome array was bound to streptavidin magnetic beads, and excess core histones were removed by washing. The divalent cation concentration never exceeded 2 mM, and no linker histone was present, so the arrays did not aggregate into higher-order chromatin structures (8
). The quality of the nucleosome array was confirmed by MNase digestion, and we observed that the array could be digested into mononucleosomes and dinucleosomes (57
). The amount of recovered array was quantified by silver staining (45
Since our system uses heterologous components, we tested whether HeLa nucleosomes and yeast nucleosomes were acetylated equivalently by yeast SAGA. As a loading control, we normalized for histone content by silver staining (Fig. ). While in parallel, immunoblots detected equivalent levels of H3 and H4 acetylation on yeast and HeLa nucleosomes, after incubation with SAGA and acetyl-CoA (Fig. , ii and iii). The immunoblots also revealed a low level of acetylation with the yeast nucleosomes when SAGA was not present, which was due to either preexisting acetylation or acetylation by substoichiometric amounts of nucleosomal HATs in the yeast nucleosome preparation. To distinguish between these two causes, we used fluorography and Coomassie blue staining as a loading control for histone content (Fig. , iv). After incubation with H3-acetyl-CoA and SAGA, a strong acetylation signal was detected on H3, H2B, and H4 for both yeast and HeLa nucleosomes (Fig. ). However, the fluorography also revealed that yeast nucleosomes were acetylated in the absence of SAGA, implying that the yeast nucleosomes contained HATs that modestly acetylated the nucleosomes.
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) (more ...)
Initially, we investigated whether SWI/SNF could affect the levels of SAGA acetylated histones on an immobilized nucleosome array. Interaction of SWI/SNF with an activator is important for the recruitment of this complex to promoters and subsequent transcription stimulation (37
). Therefore, we included the artificial activator, Gal4-VP16 in our assays. We previously demonstrated that Gal4-VP16 could target SAGA acetylation to promoter nucleosomes (57
). To track acetylated histones, the nucleosomal array was acetylated with H3
-acetyl-CoA by SAGA. After a washing step to remove the excess H3
-acetyl-CoA, the array was incubated with or without SWI/SNF and ATP. DNA was included in all reactions, which accepts transferred histones and simultaneously competes for SWI/SNF binding. Incubation of the array with SAGA and H3
-acetyl-CoA resulted in the incorporation of H3
-acetate into the nucleosome array, which could be measured by using a scintillation counter (Fig. ). After incubation with SWI/SNF and ATP, we observed that 40% of [H3
]acetate on the array was displaced into the supernatant. The loss of acetylation required the presence of both SWI/SNF and ATP, implying that SWI/SNF displaced SAGA-acetylated histones (Fig. and data not shown).
We confirmed the role of SWI/SNF in the loss of acetylated histones by using an in vitro chromatin immunoprecipitation (ChIP) assay (Fig. ). Immobilized arrays were bound by Gal4-VP16, and competitor chromatin was included prior to the addition of SAGA so acetylation would be targeted to the promoter region. The acetylated array was then incubated with or without SWI/SNF, ATP, and acceptor DNA. After being washed, the array was digested into mononucleosomes and dinucleosomes with MNase and then immunoprecipitated with an antibody against acetylated K9 on histone H3. Nucleosomes that bound the antibody were separated with protein A-agarose beads from the supernatant, and the beads were washed extensively. DNA was purified from the immunoprecipitated material, as well as the supernatant, and slot blotted onto a nylon membrane. After hybridization to a probe that spanned the length of the pG5E4T template, we observed a decrease in total signal (supernatant and beads) after SWI/SNF treatment, representing an overall loss of histones prior to MNase digestion. Immunoprecipitation experiments (i.e., %IP) revealed that a smaller fraction of nucleosomes remained on the array in the sample treated with SWI/SNF compared to that not treated with SWI/SNF (Fig. ). These data indicated that SAGA-acetylated histones were lost during SWI/SNF treatment. In fact, SWI/SNF treatment resulted in the loss of over half of the SAGA-acetylated histones (Fig. ).
SWI/SNF suppresses the histone acetylation peak generated by activator targeting of SAGA.
In previous studies, GCN5 was shown to produce a peak of acetylation in vivo at the HIS3 promoter. These results were recapitulated in vitro on a nucleosomal array, where the SAGA complex produced a peak of acetylation that surrounded activator binding sites (25
). We hypothesized that displacement of SAGA acetylated histones might suppress this activator-dependent SAGA acetylation peak on nucleosomal arrays.
We performed a scanning in vitro ChIP assay to detect the effects of SWI/SNF nucleosome displacement on the SAGA acetylation peak. The immobilized array was bound by Gal4-VP16. Competitor chromatin was added to the arrays, prior to the addition of acetyl-CoA and SAGA. The acetylation should be preferentially targeted at the promoter by Gal4-VP16 recruitment of SAGA in the presence of competitor chromatin, as previously described (57
). The acetylated array was then treated with SWI/SNF in the presence of acceptor DNA and ATP. After MNase digestion, immunoprecipitation (IP) with the acetyl H3 antibody, and DNA purification, the template was slot blotted onto a nylon membrane. The membrane was sequentially hybridized with a series of probes that spanned the length of the template (Fig. ). The IP efficiency, or %IP of each segment was normalized to the −A segment of the promoter region, where the SAGA acetylation peaked near the activator binding sites as expected (57
). After treatment with SWI/SNF, the overall levels of acetylation were decreased. Moreover, the loss of acetylation was most pronounced at the promoter where the SAGA acetylation peak was suppressed (Fig. ).
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. (more ...) SWI/SNF preferentially displaces nucleosomes acetylated by SAGA.
While the in vitro ChIP illustrated that SWI/SNF displaced acetylated promoter nucleosomes, we wanted to directly test whether SWI/SNF preferentially displaced acetylated nucleosomes. This was measured by immunoblotting the acetylated nucleosome arrays after SWI/SNF treatment with an antibody against acetylated H3 and an antibody against an unmodified patch of histone H4. The anti-acetyl H3 antibody recognized nucleosomes that are acetylated by SAGA at lysine 9 on histone H3, while the anti-histone H4 antibody recognized both acetylated and unmodified nucleosomes. At 50 mM KCl, the H3/H4 tetramer is stable and does not dissociate, so the anti-histone H4 antibody measured total nucleosome content on the array, including the acetylated nucleosomes that are also detected by the anti-acetyl H3 antibody. A comparison of acetylated H3 signal to histone H4 signal should indicate whether acetylated nucleosomes are lost to a greater extent than unmodified nucleosomes. SAGA acetylation was targeted to the promoter region of nucleosome arrays using Gal4-VP16 and competitor chromatin. After removal of the free acetyl-CoA, the arrays were then treated with SWI/SNF. The nucleosome arrays were Western blotted with the anti-acetyl H3 antibody and the anti-histone H4 antibody, which could detect all modified and unmodified nucleosomes. After SWI/SNF treatment, we observed a larger decrease of acetyl H3 signal relative to H4 signal (Fig. , compare lanes 1 and 2). The peaked acetylation profile from Fig. indicated that most SAGA acetylation occurred on the two promoter nucleosomes flanking the Gal4 binding sites. SWI/SNF specifically displaced the acetylated promoter nucleosomes, but the majority of nucleosomes remained on the array. The Western blot was not sensitive enough to detect the loss of a small fraction of the total nucleosome population. However, if SWI/SNF indiscriminately displaced nucleosomes from the array, we would expect to see a greater loss of total nucleosome content on the array. Using Western blot analysis, we detected a significant decrease in acetylation, and the Western blot did not show an appreciable decrease in total nucleosome content, as measured by the anti-histone H4 antibody.
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 (more ...)
To further examine the preferential displacement of acetylated nucleosomes by SWI/SNF, a ChIP was performed with a C-terminal H3 antibody that detects all modified an unmodified forms of histone H3 to measure of histone density on the array (28
). We sought to determine whether, compared to unacetylated arrays, SAGA acetylation stimulated the displacement of nucleosomes, specifically in the promoter region. After Gal4-VP16 binding and the addition of competitor chromatin, the immobilized array was either acetylated with SAGA or subjected to a mock acetylation reaction. SWI/SNF treatment was carried out in parallel on the acetylated array and the unacetylated array. After a washing step, the template was digested into mononucleosomes and dinucleosomes using MNase, followed by IP with the anti-histone H3 antibody. After SWI/SNF treatment, we observed a decrease in the %IP (Fig. ). After the probes were scanned at the promoter (−A) and distal segments (−C), we found that SWI/SNF nucleosome displacement was enhanced by SAGA acetylation at the promoter (Fig. ). When the array was not acetylated prior to SWI/SNF treatment, we observed that 55 and 60% of the nucleosomes remained on the array at the promoter and distal regions, respectively. However, if the array was acetylated prior to SWI/SNF treatment, 25% of the nucleosomes remained at the promoter, whereas 55% remained at the distal regions. Therefore, while SWI/SNF was able to target the activator-bound array in the absence of SAGA, acetylation significantly enhanced nucleosome displacement by SWI/SNF. Thus, SWI/SNF preferentially displaced SAGA-acetylated nucleosomes.
The Swi2/Snf2 bromodomain contributes to acetylated nucleosome displacement by SWI/SNF.
SWI/SNF is targeted to promoters by transcription activators in a manner similar to that of the SAGA complex (14
). Thus, SWI/SNF might preferentially remove histones acetylated by SAGA because it is targeted to the same location on the nucleosome array by Gal4-VP16. To test the importance of the activator in targeting SWI/SNF to the SAGA acetylation peak, we sought to determine whether SWI/SNF targeted acetylated histones, independent of activator. After Gal4-VP16 binding, the immobilized array was acetylated with SAGA, and then Gal4-VP16 was removed by using Gal4 oligonucleotide competition. SWI/SNF treatment was then carried out in the absence of activator. After IP with the anti-acetyl H3 antibody, we observed a decrease in the relative %IP after SWI/SNF treatment (Fig. ). After scanning with probes spanning the entire template, we found that SWI/SNF treatment did decrease the SAGA acetylation peak (Fig. ), but to a lesser extent than when activator was present (compare with Fig. ). When Gal4-VP16 was present during SWI/SNF treatment, 70 and 72% of the acetylated histones were displaced at positions −A and +A, respectively, whereas 51 and 44% of the acetylated histones were displaced at positions −A and +A, respectively, when GAL4-VP16 was removed prior to SWI/SNF treatment. With or without activator, SWI/SNF displacement of acetylated histones in these regions was significant, with P
values of <0.025, when analyzed by the Student t
test. Therefore, the presence of activator enhances SWI/SNF-mediated nucleosome displacement but is not required for this activity.
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 (more ...)
Preferential displacement of acetylated nucleosomes by SWI/SNF could result from acetylation enhancing nucleosome displacement and/or increased recognition of acetylated nucleosomes by SWI/SNF. The latter possibility was consistent with two previous observations. First, the Swi2/Snf2 bromodomain was shown to recognize and bind to acetylated nucleosomes (15
). Second, it was demonstrated that preferential displacement of acetylated nucleosomes did not require Gal4-VP16 targeting and that the bromodomain could provide acetylated nucleosome recognition. Since acetylated nucleosomes are the preferred substrate for SWI/SNF displacement, the Swi2/Snf2 bromodomain may play a role in the preferential action of SWI/SNF on these nucleosomes. To test this possibility, we used an in vitro ChIP assay to compare the acetylated nucleosome displacement activity of wild-type SWI/SNF complex to that of a complex lacking the Swi2/Snf2 bromodomain.
SAGA acetylation was targeted to the promoter by the artificial activator Gal4-VP16 and competitor chromatin. After the activator was removed with Gal4 oligonucleotide competition, we incubated the array with the Swi2 bromodomain mutant complex. When assayed with the scanning in vitro ChIP, the Swi2 bromodomain mutant complex showed reduced nucleosome displacement, compared to wild type as indicated by the lack of suppression of the SAGA nucleosome acetylation peak (compare Fig. ). The Swi2/Snf2 bromodomain mutant was not able to specifically target acetylation in the promoter region. Rather, hyperacetylated and hypoacetylated nucleosomes were equally displaced along the length of the array by the mutant complex. The wild-type SWI/SNF complex displaced 51% of acetylated nucleosomes at the −A probe and 11% at the −C probe, whereas the bromodomain mutant displaced only 16% at the −A probe, and 6% at the −C probe (Fig. ). Although some displacement occurred at the promoter, the mutant complex did not target acetylated nucleosomes as well as the wild type. Therefore, the Swi2/Snf2 bromodomain is important for SWI/SNF-mediated displacement of SAGA-acetylated histones.
RSC suppresses the histone acetylation peak generated by activator targeting of SAGA.
The Swi/Snf-related RSC complex also displaces nucleosomes in trans
). Indeed, Reinke et al. propose that other chromatin remodelers displace nucleosomes in the absence of SWI/SNF (50
). Although ChIP and microarray analysis localized RSC at the promoters of RNA polymerase III-transcribed genes, those authors acknowledge that RSC is difficult to immunoprecipitate and may bind at other promoters (40
). Moreover, RSC interacts genetically with SAGA and binds H3 peptides acetylated at lysine 14 (22
), while in higher eukaryotes, PBAP, the Drosophila
RSC homolog, localizes at hyperacetylated nucleosomes in polytene stains (36
). Thus, RSC may be functionally redundant with SWI/SNF and displaces promoter-acetylated nucleosomes in the absence of the latter remodeling complex.
We tested whether RSC displaced SAGA-acetylated nucleosomes by using the scanning in vitro ChIP experiment. The nucleosomal array was bound by the artificial activator Gal4-VP16 and acetylated by SAGA in the presence of competitor chromatin. After targeting acetylation at the promoter, the array was washed, and the activator was removed by Gal4 oligonucleotide competition. With the activator removed, the array was incubated with RSC, ATP, and acceptor DNA. After MNase digestion, IP with the acetyl H3 antibody, and DNA purification, the template was slot blotted onto a nylon membrane. The membrane was sequentially hybridized with a series of probes that spanned the length of the template (Fig. ). RSC decreased the level of acetylation at the promoter (Fig. ). Interestingly, we observed a slight increase in the level of acetylation at segments distal to the Gal4 binding sites, suggesting that acetylated nucleosomes were transferred from the promoter to the distal region. Thus, RSC nucleosome displacement suppressed the acetylation profile of SAGA.
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 (more ...)