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Figure 1 In vivo binding of Swi/Snf to the GAL1 and GAL7 UASG elements. (A) Top: Representation of the GAL7 and GAL1-10 loci. Transcriptional initiation sites (arrows with +1), Gal4 binding sites (UASG) (black crossbars), and open reading frames (open shaded rectangles) are represented. Regions amplified by PCR in the ChIP experiments are identified by a line. Bottom: PCR titration of input and immunoprecipitated material for Snf5-Myc at the GAL1 UASG. (B) ChIP analysis of the binding of Swi/Snf to the GAL1 and GAL7 promoters. Binding of Swi1-Myc and Snf5-Myc over time after galactose induction is shown for both the WT (KLY012 and KLY014) and gal4 strains (KLY015 and KLY016). (C, D) Binding of Swi/Snf to the GAL1 and GAL7 promoters, respectively. (E) Ability of swi1 cells to induce the GAL1 and GAL7 genes. GAL1 primer extension analyses from WT (CY258) and swi1 cells (CY340) grown either in the presence of glucose (Glu) or induced by galactose for 3 h (Gal).
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 | Figure 2 Distribution of Swi/Snf and SAGA at the GAL1-10 locus. (A) ChIP analysis of the binding of Gcn5-Myc (KLY031) to the GAL1 and GAL7 UASG during galactose induction. (B) Quantification of experiments as illustrated in (A). (C) Top: Representation of the GAL1-10 locus. Regions amplified in the GAL10 open reading frame (10 O-A), the GAL1-10 promoter region (10 P-A, UASG1-10, and 1 P-A) and in the GAL1 open reading frame (1 O-A to 1 O-C) are represented. Bottom: Distribution of Snf5-Myc and Gcn5-Myc over the GAL1-10 locus. ChIP analysis of the binding of Snf5-Myc and Gcn5-Myc to the GAL1-10 locus after 30 min addition of galactose. Data of input and immunoprecipitated material are shown for each region investigated. (D) Quantification of experiments as illustrated in (C).
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Figure 3 SAGA is not required for Swi/Snf recruitment at the GAL1 and GAL7 UASG elements. (A) ChIP analysis of Swi1-Myc binding to the GAL1 and GAL7 UASG in WT (KLY022) and gcn5 (KLY024) strains upon galactose induction. (B) Quantification of the experiment illustrated in (A). (C, D) Same experiment as in (A, B) with the exception that Swi1-Myc binding was analyzed in WT (KLY020) and spt20 (KLY028) cells. (E) Ability of Swi1-Myc- and Snf5-Myc-expressing yeast cells to induce the GAL1 gene. Primer extension assays were carried out with RNA purified from WT (KLY020, KLY021, KLY022, and KLY023), gcn5 (KLY024 and KLY025), and spt20 cells (KLY028 and KLY029) induced with galactose for 3 h.
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 | Figure 4 Swi/Snf and SAGA are recruited to the GAL1 UASG by artificial recruitment of the Mediator. (A) Artificial recruitment of the Mediator bypasses the requirement for a transcriptional activator. A strain deleted for GAL4 and bearing a Myc-tagged version of Snf5 was transformed by a plasmid expressing the Gal4 DBD alone (KLY019) or fused to the C-terminal end of Gal11 (KLY018). Primer extension analyses were carried out with purified RNA from both strains. Yeast cells were grown in the presence of raffinose (-) and then galactose (+) was added in order to induce GAL1 gene expression. (B) ChIP analysis of the binding of Swi/Snf to the GAL1 UASG in yeast cells expressing Gal4-Gal11. Binding of Snf5-Myc in the strains used in (A). (C) Quantification of experiments as illustrated in (B) is shown. (D, E) Same experiment as in (B, D) with the exception that the binding of Gcn5-Myc (KLY044 and KLY043) was assessed. (F) ChIP analysis of the binding of Srb4-Myc and Rpb1 to the GAL1 UASG in yeast cells expressing a Gal4-Gal11 fusion (KLY091 and KLY092). (G) Quantification of experiments as illustrated in (F).
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Figure 5 The Mediator is required for Swi/Snf recruitment to the GAL1 UASG. (A) ChIP analysis of the binding of Swi1-Myc to the GAL1 UASG in the absence of the Mediator. Temperature-sensitive srb4-138 mutant (KLY042) containing Swi1-Myc was grown at 28°C and either shifted to 37°C for 45 min to inactivate srb4 (37°C) or kept at the permissive temperature (28°C). Galactose was added to media as in previous experiments GAL gene expression. (B) Quantification of experiments as shown in (A). (C, D) Similar experiment as in (A, B) except that binding of Gal11-Myc was analyzed (KLY090). PCRs contain chromosome II telomere primers used here as an internal control to normalize signals for each lane. (E) Persistence of the Swi/Snf complex in the absence of the Mediator. KLY042 cells were grown at the permissive temperature and galactose was added to the media for 30 min to induced GAL gene expression. Cells were then shifted to inducing media at permissive (28°C) or nonpermissive temperature (37°C). Aliquots were taken 30, 60, 120, and 180 min after a shift to the nonpermissive temperature. PCRs contain chromosome II telomere primers as an internal control. (F) Quantification of experiments as illustrated in (E).
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 | Figure 6 RNA polII and TAFIIs are required for Swi/Snf recruitment to the GAL1 UASG. (A) ChIP analysis of the binding of Swi2-Myc (YJR598) to the GAL1 UASG in an rpb1-1 yeast strain. The experiment was performed as described in Figure 5A. (B) Quantification of experiments as illustrated in (A). (C, D) Similar experiments as in (A, B) except that binding of Swi2-Myc was assessed in a taf1-2 mutant (YJR595). (E, F) Analysis of the binding of Swi2-Myc in a taf12-9 mutant (YJR592).
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