Recruitment of SWI/SNF by Gcn4p does not require Snf2p or Gcn5p but depends strongly on SWI/SNF integrity, SRB mediator, and SAGA

Abstract

The nucleosome remodeling complex SWI/SNF is a coactivator for yeast transcriptional activator Gcn4p. We provide strong evidence that Gcn4p recruits the entire SWI/SNF complex to its target genes ARG1 and SNZ1 but that SWI/SNF is dispensable for Gcn4p binding to these promoters. It was shown previously that Snf2p/Swi2p, Snf5p, and Swi1p interact directly with Gcn4p in vitro. However, we found that Snf2p is not required for recruitment of SWI/SNF by Gcn4p nor can Snf2p be recruited independently of other SWI/SNF subunits in vivo. Snf5p was not recruited as an isolated subunit but was required with Snf6p and Swi3p for optimal recruitment of other SWI/SNF subunits. The results suggest that Snf2p, Snf5p, and Swi1p are recruited only as subunits of intact SWI/SNF, a model consistent with the idea that Gcn4p makes multiple contacts with SWI/SNF in vivo. Interestingly, Swp73p is necessary for efficient SWI/SNF recruitment at SNZ1 but not at ARG1, indicating distinct subunit requirements for SWI/SNF recruitment at different genes. Optimal recruitment of SWI/SNF by Gcn4p also requires specific subunits of SRB mediator (Gal11p, Med2p, and Rox3p) and SAGA (Ada1p and Ada5p) but is independent of the histone acetyltransferase in SAGA, Gcn5p. We suggest that SWI/SNF recruitment is enhanced by cooperative interactions with subunits of SRB mediator and SAGA recruited by Gcn4p to the same promoter but is insensitive to histone H3 acetylation by Gcn5p.

FIG. 1.

Gcn4p at native levels recruits the SWI/SNF complex to the ARG1 promoter. (A) WT (BY4741) and gcn4Δ (249) strains containing 13-myc tagged alleles of SNF2 (SY1 and SY127), SNF5 (SY162 and SY166), SNF6 (SY163 and SY167), SWI1 (SY160 and SY164), SWI3 (SY161 and SY165), and SWP73 (SY175 and SY176) were grown to an OD₆₀₀ of 0.8 to 1.0 in SC-ILV medium at 30°C and treated with 0.6 μg of SM/ml for 2 h. Cells were harvested, treated with formaldehyde, and broken by vortexing with glass beads, and the extracts were sonicated to produce chromatin fragments with an average length of ∼500 bp. Aliquots (5% of the total) were immunoprecipitated with anti-myc antibodies, and DNA was extracted from the immunoprecipitate (IP) after reversing the cross-links. DNA was extracted directly from another aliquot of the chromatin preparation (5% of the total) to serve as the input control. (A) A 600-fold dilution of the input control and the undiluted IP DNA were amplified by PCR by using primers specific for the ARG1 UAS or POL1 ORF in the presence of [³³P]dATP. The PCR products were resolved by PAGE and quantified by phosphorimaging analysis, and the ratio of the ARG1 UAS signals in the IP-to-input samples was calculated and normalized for the corresponding ratio calculated for the POL1 signals to yield the normalized percentage of IP (Normalized %IP). The ratio of the normalized percentage of IP values for the GCN4 to gcn4Δ strains was calculated to yield the ratio percentage of IP (Ratio %IP) (GCN4/gcn4Δ). (B) As a negative control, the same IP and input samples described in panel A were analyzed using primers to amplify a sequence located 4.5-kb upstream of the SER3 gene.

FIG. 2.

Overexpression of Gcn4p increases the recruitment of SWI/SNF at ARG1 and allows detection of SWI/SNF recruitment at SNZ1. (A) High-copy-number 2μm plasmid pHQ1239 harboring the GCN4-HA allele was introduced into the GCN4 strains containing myc-tagged SWI/SNF subunits, and empty vector YCp50 was introduced into the gcn4Δ myc-tagged strains that were described in the legend to Fig. 1. The strains were cultured in SC-ILV-Ura (SC-ILV also lacking uracil) and induced with SM and then subjected to ChIP analysis as described in the legend to Fig. 1. The results of a typical experiment are depicted at the top, while the histograms below summarize the averages and standard errors of the ratio percent IP (GCN4/gcn4Δ) (as defined in the legend to Fig. 1) that were derived from multiple determinations for each strain. Two independent cultures and two to four independent immunoprecipitations were analyzed for each strain to provide the data used to calculate the mean values and standard errors shown in the histogram. (B) The same process was used as for panel A except that the SWI/SNF subunit binding to the SNZ1 promoter was analyzed.

FIG. 3.

Hydrophobic clusters in the Gcn4p activation domain are required for recruitment of Snf2p and Snf5p to ARG1 and SNZ1, but Gcn4p binding at these promoters is independent of SWI/SNF. (A and B) The gcn4Δ SNF2-myc (SY127) and gcn4Δ SNF5-myc (SY166) strains described in the legend to Fig. 1 were transformed with single-copy (s.c) plasmid p2382 or pSY285, harboring GCN4-HA or gcn4-14Ala-HA, respectively, or high-copy-number (h.c.) plasmid pHQ1303 or pHQ1304, harboring GCN4 or gcn4-14Ala, respectively, and subjected to ChIP analysis as described in the legend to Fig. 1 to measure binding of myc-tagged Snf2p or Snf5p to ARG1 (A) or SNZ1 (B). (C and D) ChIP analysis was conducted as described above to measure binding of myc-Gcn4p to ARG1 (C) or SNZ1 (D) in the following strains: transformants of the gcn4Δ strains containing either all WT SWI/SNF genes (249), snf2Δ (SY169), swi3Δ (SY294), snf6Δ (SY295), snf11Δ (SY296), snf5Δ (SY327), or swp73Δ (SY298), all bearing single-copy plasmid pSK1 containing GCN4-myc, lanes 2 through 8; gcn4Δ strain (249) transformed with empty vector YCp50 or single-copy plasmid pSY284 containing gcn4-14Ala-myc, respectively, lanes 1 and 9.

FIG. 4.

Recruitment of SWI/SNF subunits by Gcn4p to ARG1 is impaired in snf6Δ, snf5Δ, and swi3Δ mutants. (A through F) We generated a panel of strains harboring the chromosomal myc-tagged SWI/SNF alleles designated in the upper left of each histogram from GCN4 strains containing all WT SWI/SNF subunits or the indicated swi/snf deletion alleles (shown at the top) and from the gcn4Δ strain containing all WT SWI/SNF subunits (gcn4Δ). (See Table 1 for a list of all strains employed.) High-copy-number plasmid pHQ1239 was introduced into the GCN4 strains, and empty vector YCp50 was introduced into the gcn4Δ strains. The resulting transformants were subjected to ChIP analysis as described in the legend to Fig. 1. NA, not applicable; ND, not determined.

FIG. 5.

Recruitment of SWI/SNF subunits by Gcn4p to SNZ1 is impaired in snf6Δ, snf5Δ, swi3Δ, and swp73Δ mutants. The selected transformants described in the legend to Fig. 4 were subjected to ChIP analysis to measure binding of the indicated myc-tagged SWI/SNF subunits to SNZ1 in GCN4 or gcn4Δ strains containing all WT SWI/SNF subunits (WT and gcn4Δ, respectively) and in GCN4 strains containing the indicated swi/snf deletion alleles.

FIG. 6.

Western blot analysis of SWI/SNF subunits in swi/snf mutants. (A) The GCN4 strains containing SNF2-myc and either WT SWI/SNF subunits or the indicated swi/snf deletions were grown in SC-ILV, and total proteins were extracted as described in Materials and Methods. Aliqouts with equal amounts of total proteins were separated by SDS-PAGE by using 10% gels, transferred to a nitrocellulose membrane, and probed with antibodies against myc, Snf5p, or Swp73p, as indicated on the left of the blot. Probing with Gcd6p antibodies provided a loading control. The Western blotting signals were quantified by video densitometry by using NIH Image software, normalized for the Gcd6p signals, expressed relative to the normalized value measured in WT cells, and listed below the corresponding blots for myc-Snf2p, Snf5p, and Swp73p (Rel. amount). (B) The swp73Δ and swi3Δ strains exhibit similar reductions in SWI/SNF subunit levels. The analysis described for panel A was repeated for the WT, swi3Δ, swp73Δ, and snf5Δ strains harboring SNF2-myc, loading two different amounts of extract in adjacent lanes; thus, the sizes of samples in lanes 1, 3, 5, 7, and 9 were 50% of those loaded in lanes 2, 4, 6, 8, and 10.

FIG. 7.

Examination of SWI/SNF complex integrity in swi/snf mutants by coimmunoprecipitation analysis. (A) SNF2-myc strains containing WT SWI/SNF subunits or the indicated swi/snf deletions were grown in YPD, and WCEs were prepared as described in Materials and Methods. Aliqouts containing 0.3 to 0.5 mg of protein were immunoprecipitated with mouse monoclonal myc antibodies. Ten percent of the input samples (Input), 100% of the immunoprecipitates (Ppt), and 10% of the supernatant (Sup) fractions were resolved by 4 to 20% SDS-PAGE and subjected to Western blot analysis using monoclonal mouse antibodies against myc or rabbit antibodies against Snf5p. The upper and lower portions of each membrane were probed separately with different antibodies. (B) The same kind of analysis as that described for panel A was carried out by using the WT or snf2Δ strains harboring SWI3-myc.

FIG. 8.

Overexpression of myc-Snf2p or myc-Snf5p does not increase their recruitment to ARG1. (A) The SNF2-myc (SY1), SNF5-myc (SY162), and snf6Δ SNF2-myc (SY3) strains described in the legends to Fig. 1 and 4 were transformed with high-copy-number plasmid pSY286 or pSY287, harboring SNF2-myc or SNF5-myc, respectively, or with empty vector YCp50 and subjected to Western blot analysis as described in the legend to Fig. 6. Lanes 1 through 3, SNF2-myc strain transformed with the plasmids indicated at the top; lanes 4 and 5, snf6Δ SNF2-myc strain transformed with pSY286; lanes 6 through 8, SNF5-myc strain transformed with the plasmids indicated at the top. The samples in lanes 2, 5, and 7 contained 50% of those loaded in lanes 1, 4, and 6, respectively. (B) ChIP analysis was performed on the transformants described for panel A. Lanes 1 through 4, transformants of the SNF2-myc and snf6Δ SNF2-myc strains harboring the plasmids indicated at the top; lanes 6 and 7, transformants of the SNF5-myc strain harboring the plasmids indicated at the top; lane 5, transformant of the gcn4Δ SNF2-myc strain containing YCp50.

FIG. 9.

Subunits of mediator and SAGA are required for optimal recruitment of SWI/SNF by Gcn4p. (A through C and E through G) We generated a panel of strains harboring the chromosomal myc-tagged SWI/SNF alleles designated in the upper left of each histogram from GCN4 strains containing all WT coactivator subunits (WT) or the indicated deletion alleles (shown at the top) that remove subunits of mediator (A through C) or SAGA (E through G) and from the gcn4Δ strain containing all WT coactivator subunits (gcn4Δ) (see Table 1 for a list of strains). High-copy-number plasmid pHQ1239 was introduced into the GCN4 strains, and empty vector YCp50 was introduced into the gcn4Δ strains. The resulting transformants were subjected to ChIP analysis to measure binding of the myc-tagged SWI/SNF subunit to ARG1, as described in the legend to Fig. 1. (D and H) The strains described for panels A and E that harbor the SNF5-myc allele were subjected to ChIP analysis to measure binding of myc-Snf5p to SNZ1. ND, not determined.