Recruitment of the SWI/SNF chromatin remodeling complex by transcriptional activators

Abstract

SWI/SNF is a chromatin remodeling complex that facilitates expression of a number of yeast genes. Here we demonstrate that SWI/SNF can be recruited from yeast nuclear extracts by a transcriptional activator. Recruitment is dependent on an activation domain but not on promoter sequences, TBP, or RNA polymerase II holoenzyme. We also show that acidic activation domains can target SWI/SNF remodeling activity. These results demonstrate that SWI/SNF activity can be targeted by gene-specific activators and that this recruitment can occur independently of Pol II holoenzyme.

Figure 1

SWI/SNF is recruited to promoters in the immobilized template assay independently of holoenzyme and TBP. (A) Immobilized templates used in this study. (Top) The wild-type template contains the HIS4 core promoter and transcription start sites; (bottom) the core promoter, including the TATA box, was deleted and replaced by downstream nonpromoter sequences to create the ΔPromoter template. (B) Recruitment of Swi3p and Snf5p in wild-type and mutant NEs. PIC assembly was performed with the nuclear extracts indicated at top and analyzed by Western blotting using antibodies against the components indicated at right. All reactions included the activator Gal4–AH. rSrb2 (200 ng) and rTBP (400 ng) were added where indicated.

Figure 1

SWI/SNF is recruited to promoters in the immobilized template assay independently of holoenzyme and TBP. (A) Immobilized templates used in this study. (Top) The wild-type template contains the HIS4 core promoter and transcription start sites; (bottom) the core promoter, including the TATA box, was deleted and replaced by downstream nonpromoter sequences to create the ΔPromoter template. (B) Recruitment of Swi3p and Snf5p in wild-type and mutant NEs. PIC assembly was performed with the nuclear extracts indicated at top and analyzed by Western blotting using antibodies against the components indicated at right. All reactions included the activator Gal4–AH. rSrb2 (200 ng) and rTBP (400 ng) were added where indicated.

Figure 2

SWI/SNF is recruited to DNA by activators independently of promoter sequences. PICs were assembled on both wild-type (W) and ΔPromoter (Δ) templates as stated in Fig. 1, with the nuclear extracts (NE) indicated. Reactions were performed either with no activator (−), the GAL4 (1–94) DNA-binding domain (G), or Gal4–AH (A) and Gal4–VP16 (V), as indicated. rTBP (400 ng) was added where indicated. PICs were analyzed by Western blotting with antibodies against the components indicated at right. (Lane 1) Dynabeads without templates were used in PIC assembly as a control for nonspecific binding to beads.

Figure 3

SWI/SNF remodeling activity can be targeted by a transcriptional activator. (A) Schematic of nucleosomal array templates. (B) The ³²P-labeled arrays (0.2 nm final) were mixed with unlabeled arrays (3 nm final) in the four possible combinations and then either incubated with HincII alone (○) or exposed to 0.8 nm SWI/SNF and HincII in the absence (crossed circles), or presence of 10 nm GAL4–VP16 (●). The digestion of the labeled array with time was quantified and graphed at left.

Figure 3

SWI/SNF remodeling activity can be targeted by a transcriptional activator. (A) Schematic of nucleosomal array templates. (B) The ³²P-labeled arrays (0.2 nm final) were mixed with unlabeled arrays (3 nm final) in the four possible combinations and then either incubated with HincII alone (○) or exposed to 0.8 nm SWI/SNF and HincII in the absence (crossed circles), or presence of 10 nm GAL4–VP16 (●). The digestion of the labeled array with time was quantified and graphed at left.

Figure 4

SWI/SNF activity is recruited by acidic activation domains. HincII cleavage kinetics of reactions containing 0.2 nm labeled 208-11S–GAL4 array, 3 nm unlabeled 208-11S competitor array in the presence or absence of GAL4 derivatives, and in the absence (A) or presence (B) of 0.8 nm SWI/SNF. Note that the GAL4 derivatives did not affect HincII cleavage kinetics in the absence of SWI/SNF. (□) No Gal4/no SWI/SNF; (█) +SWI/SNF; (▴) Gal4 (1–94); (●) Gal4–AH; (▾) Gal4–Pro; (♦) Gal4–VP16.

Figure 4

SWI/SNF activity is recruited by acidic activation domains. HincII cleavage kinetics of reactions containing 0.2 nm labeled 208-11S–GAL4 array, 3 nm unlabeled 208-11S competitor array in the presence or absence of GAL4 derivatives, and in the absence (A) or presence (B) of 0.8 nm SWI/SNF. Note that the GAL4 derivatives did not affect HincII cleavage kinetics in the absence of SWI/SNF. (□) No Gal4/no SWI/SNF; (█) +SWI/SNF; (▴) Gal4 (1–94); (●) Gal4–AH; (▾) Gal4–Pro; (♦) Gal4–VP16.