1. Volume 123, Issue 3, Pages 423-436 (November 2005)
The Proteasome Regulatory Particle Alters the SAGA Coactivator to Enhance Its Interactions with Transcriptional Activators 
Daeyoup Lee, Elena Ezhkova, Bing Li, Samantha G. Pattenden, William P. Tansey, Jerry L. Workman 
Cell 
Volume 123, Issue 3, Pages (November 2005)
DOI: /j.cell
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
The 19S RP Stimulates SAGA Targeting by Gal4-VP16 in a Dose-Dependent Manner
(A) Targeting of the SAGA complex to the Gal4-VP16 bound nucleosome is stimulated by the 19S RP. A 183 bp DNA fragment containing one Gal4 site was bound by no Gal4 derivative (lanes 1–4), Gal4-VP16 (lanes 5–8), and Gal4 alone (lanes 9–12). SAGA was subsequently added to the reactions with or without the 19S RP and resolved on a native acrylamide gel (3.5% 79:1 acrylamide:bisacrylamide) in 0.25× TBE buffer.
(B–D) The 19S RP increased targeting efficiency of SAGA to Gal4-VP16 (B), Gal4-Gcn4 (C), and Gal4-Gal4AD (D) bound DNA template. Left panel: gel shift analysis to assess Gal4-activator binding alone (lane 1) or in combination with purified SAGA complex (lane 2) to a 32P-end-labeled DNA template containing a single Gal4 binding site. Increasing amounts (2.5 to 40 nM) of the 19S RP were added with (lanes 3–7) or without (lanes 8–12) purified SAGA complex. Arrows indicate free DNA probe (DNA), Gal4 activator bound to DNA (G4-VP16-DNA, G4-Gcn4-DNA, or G4-G4AD-DNA), and the supershifted band containing both Gal4 activator and SAGA (*). In the absence of SAGA, increasing amounts of the 19S RP failed to produce a defined supershifted band (lanes 8–12). Addition of both SAGA and the 19S RP, however, increased SAGA targeting in a dose-dependent manner (lanes 3–7) compared to SAGA alone (lane 2). Right panel: the supershifted band from raw data was quantified by ImageQuant software (Amersham TYPHOON). Values represent an average of three independent experiments where SAGA binding in the absence of the 19S RP was represented as 1-fold (lane 2). Error bars indicate standard deviation. Fold targeting efficiency of SAGA increased in a dose-dependent manner in the presence of the 19S RP.
(E) Gel shift Western assay. In place of radiolabeled probe, Cy5 probe was utilized. After separation (Cy5 fluorography, left), the native gel was incubated in the transfer buffer for 15 min followed by transfer onto nitrocellulose membrane. Western blot was performed with HA monoclonal antibody (Roche) (right). Signal was detected on the TYPHOON (ECL Plus, Amersham Biosciences).
(F) ATPase components play a significant role in SAGA-targeting stimulation. Gel shift assays were performed using GUB DNA template. After adding Gal4-VP16 for 15 min, the indicated amount of the 19S RP or lid or base with or without SAGA was added to the reaction for 30 min at 30°C.
(G) ATPγS inhibits SAGA targeting by the 19S RP. Gel shift assay was carried out as described above, except 1 mM ATP or ATPγS was added.
Cell  , DOI: ( /j.cell )
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3. Figure 2
SAGA/SLIK Targeting Is Stimulated by the 19S RP, and SAGA Targeting Is Not Augmented by sug S RP
(A and B) Densitometry analysis of gel shift assays using Gal4-VP16 and 32P-end-labeled DNA template with a single Gal4 binding site. Percentage targeting stimulation of either SAGA or SLIK was defined as the ratio of Gal4-VP16-DNA binding signal (α) versus the supershifted signal (β). Fold targeting stimulation by the 19S RP (10 nM) was determined by relative ratio between lane 2 (1-fold) and lanes 3–13. Error bars represent the standard deviation of three independent repeats. Fold targeting stimulation is represented in the graph to the right of each gel shift on a log scale versus SAGA concentration.
(C) sug1-25 ATPase mutant 19S RP (10 nM) was unable to efficiently stimulate SAGA targeting to Gal4-VP16 bound DNA template. Left panel: assays were performed and evaluated as described in (A) and (B). Right panel: fold targeting stimulation is plotted versus SAGA concentration. Error bars represent the standard deviation of three independent repeats.
Cell  , DOI: ( /j.cell )
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4. Figure 3
The 19S RP Enhanced SAGA Targeting to Gal4-VP16 Bound, Immobilized DNA Template
(A) Targeting-assay schematic. Biotinylated reconstituted polynucleosome array containing a minimal promoter with five Gal4 binding sites was immobilized to streptavidin-coupled paramagnetic beads. Gal4-VP16 was incubated with the template in the presence or absence of competitor polynucleosome (LON) followed by addition of SAGA and/or the 19S RP. Reactions were washed and loaded onto an SDS-PAGE gel for Western blotting.
(B) Strong SAGA interaction with the activator bound nucleosomes was observed in the presence of the 19S RP and competitor. Top: Western blotting was used to detect the 19S RP (anti-CBP), SAGA (anti-Ada1), and Gal4-VP16 (anti-VP16 activation domain) in the supernatant (S) and bound (B) fractions. Neither the 19S RP nor SAGA bound to the beads in the absence of nucleosomal template (compare lanes 1 and 2). Without Gal4-VP16, the 19S RP and SAGA bound the template nonspecifically (lane 4) as binding was competed away by addition of LON (lane 6). SAGA bound weakly to Gal4-VP16 bound template in the presence of competitor (lane 8). The 19S RP alone interacted with the Gal4-VP16 bound template in the presence of competitor (lane 10). Addition of Gal4-VP16 and the 19S RP in the presence of competitor strongly enhanced SAGA targeting to the polynucleosome template (compare lanes 12 and 8). Bottom: quantitation of lane 8 (1-fold) and lane 12 (1.9-fold) using a TYPHOON phosphorimager (Amersham). Error bars represent the standard error of the mean of two independent repeats.
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5. Figure 4 The 19S RP Modulates the SAGA Complex Specifically
(A) The 19S RP can not stimulate NuA4 targeting to the activator-anchored DNA. Shown is a gel shift analysis to assess Gal4-VP16 binding alone (lane 2) or in combination with purified NuA4 complex using Epl1-TAP (lane 3) to a 32P-end-labeled DNA template (GUB). Increasing amounts (5 to 100 mM) of the 19S RP were added without (lanes 4–8) or with (lanes 9–13) purified SAGA complex. Arrows indicate free DNA probe (DNA), Gal4-VP16 bound to DNA (DNA-G4-VP16), and the supershifted band containing both Gal4-VP16 and SAGA or NuA4 (*). In the absence of SAGA or NuA4, increasing amounts of the 19S RP failed to produce a defined supershifted band (lanes 4–8). Right panel: comparative quantitation of SAGA- and NuA4-targeting efficiency under the same condition. Values are an average of three independent experiments where SAGA or NuA4 binding in the absence of the 19S RP is represented as 100% targeting efficiency. Error bars indicate standard deviation.
(B) The 19S RP stimulates DNA binding activity of SAGA. Gel shift analysis was carried out with radiolabeled GUB DNA using the conditions in (A) and addition of sonicated genomic DNA. Since DNA binding activity of SAGA preferred normal DNA sequence over poly dI:dC, DNA binding activity of SAGA was detected even in the presence of 2 μg of poly dI:dC. In contrast, DNA binding activity of NuA4 did not show any DNA sequence preference (lanes 9 and 10); thus, poly dI:dC could compete with NuA4.
(C) HAT activity of SAGA was increased by the 19S RP, while HAT activity of NuA4 was not augmented by the 19S RP. Filter binding assay was performed as described previously (Eberharter et al., 1998). HAT assays were carried out using HeLa oligonucleosomes in the presence of [3H]acetyl-CoA. Shown are counts/min (cpm) from liquid HAT assays. Data are averaged from two independent experiments. Standard error of the mean is shown.
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6. Figure 5 The 19S RP and SAGA Directly Interact
(A) The 19S RP and SAGA directly interacted in vivo. Left panel: 5% of total extracts from wild-type (lane 1), Spt7-TAP (lane 2), and Epl1-TAP (lane 3) were used as input. Right panel: calmodulin resin was used to pull down SAGA from the Spt7-TAP yeast strain and NuA4 from the Epl1-TAP yeast strain. Both input and IP fractions were Western blotted to detect the 19S RP (anti-Sug1), SAGA (anti-CBP to detect protein A portion of TAP tag), or NuA4 (anti-CBP to detect protein A portion of TAP tag). No nonspecific binding to the calmodulin resin was detected in lysates from a control wild-type (wt) yeast strain (lanes 2 and 3). Both the 19S RP and SAGA were detected in the Spt7-TAP pull-down, indicating that these two complexes interacted in vivo, while the 19S RP was not detected in the Epl1-TAP pull-down.
(B) The 19S RP and SAGA directly interacted in vitro. Purified 19S RP (800 ng) and HA-tagged Spt7 SAGA complexes (400 ng) were mixed, and immunoprecipitation was performed with anti-HA antibody. Supernatant (S) and bound (B) fractions were immunoblotted to detect the 19S RP (anti-Sug1) and SAGA (anti-Ada1). The 19S RP alone was not precipitated with anti-HA (compare lanes 1 and 2), while the SAGA complex containing HA-tagged Spt7 was bound by the anti-HA antibody (compare lanes 5 and 6). When mixed, the 19S RP and SAGA were both immunoprecipitated (compare lanes 3 and 4), indicating that these two complexes interacted in vitro.
Cell  , DOI: ( /j.cell )
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7. Figure 6
19S RP ATPase Function Is Important for SAGA Targeting In Vivo
(A) 19S RP ATPase activity was required to maintain in vivo global H3 acetylation levels. Whole-cell lysates from Sug1 mutant yeast strains were Western blotted for H3 acetylation, H3 dimethylated lysine 4, and histone H3. sug1-25 was mutated in the ATPase domain, while sug1-1 and sug1-3 were mutated outside the ATPase domain. The wild-type control lysates were positive for H3 acetylation and dimethylation of lysine 4 (lane 1). sug1-1 (lane 2) and sug1-3 (lane 3) mutants produced results similar to that of the wild-type strain. The sug1-25 ATPase mutant, however, showed a marked decrease in both H3 dimethylated lysine 4 and global H3 acetylation levels (lane 4, compare to lanes 1–3).
(B) SAGA binding to the induced GAL1-10 promoter was decreased in sug1-25 ATPase mutant in vivo. ChIP assays were performed with anti-Gcn5 antibody to detect SAGA binding as described in Experimental Procedures using precipitated DNA from wt, sug1-3 19S RP mutant (from [A]), and sug2-1 and sug S RP ATPase domain mutants. Relative Gcn5 binding was determined by quantitative real-time PCR; cycle thresholds for target genes were calculated relative to those of the intragenic region on chromosome V. Yeast strains were grown to mid-log phase in YP + 2% glucose at 30°C, and GAL1-10 was induced by shift to YP + 2% galactose for 1 hr at 30°C. Relative Gcn5 binding to GAL1-10 was low in all strains prior to induction (left panel). Following induction, wild-type and sug1-3 showed similar patterns of increase in relative Gcn5 binding, especially around the UAS (right panel). sug2-1 and sug1-25, however, showed a significant decrease in relative Gcn5 binding at GAL1-10 when compared to either the wild-type or sug1-3 strains (right panel). Error bars represent the standard deviation of three independent repeats.
(C) Mutation of the 19S RP sug1 ATPase domain disrupted SAGA targeting and H3 acetylation at the ADH1 promoter in vivo. ChIP assays were performed as described in (B) on the ADH1 promoter and promoter-proximal regions with either anti-Gcn5 antibody to detect SAGA binding or anti-pan H3 acetylation antibody to determine relative H3 acetylation levels. wt was compared to the sug S RP ATPase mutant strain. Relative Gcn5 binding was significantly reduced in the sug1-25 mutant compared to the wild-type strain at the ADH1 promoter (left panel). Error bars represent the standard deviation of three independent repeats. Relative levels of H3 acetylation were also reduced in the sug1-25 strain compared to wild-type at both the promoter and promoter-proximal regions (right panel).
Cell  , DOI: ( /j.cell )
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8. Figure 7
SAGA Targeting and 19S RP ATPase Activity Are Genetically Linked
(A) sug1-25Δgcn5 double-mutant strain was synthetically sick at 37°C, establishing a genetic link between 19S RP ATPase and SAGA catalytic activity. Yeast strains were serially diluted 10-fold and grown on YPD at 30°C or 37°C for 2 days. At 30°C, growth of the mutant strains was similar to that of wild-type (bottom panel). sug1-25Δgcn5 double-mutant strain growth at 37°C was impaired compared to either deletion alone or wild-type (top panel).
(B) NuA3 histone-acetyltransferase activity was not genetically linked to 19S RP ATPase function. Sas3, the catalytic subunit of the NuA3 H3 acetylation complex, was deleted in the sug1-25 mutant strain background. At both 30°C (bottom panel) and 37°C (top panel), growth of both the single- and double-mutant strains was comparable to wild-type, indicating a lack of synthetic phenotype.
(C) SAGA integrity was genetically linked to 19S RP ATPase function. Deletion of the SPT20 subunit leads to dissociation of the SAGA complex. SPT20 was deleted in the sug1-25 background strain to assess the genetic link between intact SAGA and 19S RP ATPase function. At 30°C, growth of the single-mutant strains was comparable to that of wild-type, while sug1-25Δspt20 showed a slight decrease in growth (bottom panel). At 37°C, however, the double-mutant strain was synthetically sick compared to either deletion alone or wild-type (top panel), pointing to a genetic link between SAGA-complex integrity and 19S RP ATPase function.
(D) Ubp8 deubiquitylation enzyme in SAGA was not genetically linked to 19S RP ATPase function. UBP8 was deleted in the sug1-25 mutant strain background. At both 30°C (bottom panel) and 37°C (top panel), growth of both the single- and double-mutant strains was comparable to wild-type, indicating a lack of synthetic phenotype.
Cell  , DOI: ( /j.cell )
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