Transcription activator interactions with multiple SWI/SNF subunits

Abstract

We have previously shown that the yeast SWI/SNF complex stimulates in vitro transcription from chromatin templates in an ATP-dependent manner. SWI/SNF function in this regard requires the presence of an activator with which it can interact directly, linking activator recruitment of SWI/SNF to transcriptional stimulation. In this study, we determine the SWI/SNF subunits that mediate its interaction with activators. Using a photo-cross-linking label transfer strategy, we show that the Snf5, Swi1, and Swi2/Snf2 subunits are contacted by the yeast acidic activators, Gcn4 and Hap4, in the context of the intact native SWI/SNF complex. In addition, we show that the same three subunits can interact individually with acidic activation domains, indicating that each subunit contributes to binding activators. Furthermore, mutations that reduce the activation potential of these activators also diminish its interaction with each of these SWI/SNF subunits. Thus, three distinct subunits of the SWI/SNF complex contribute to its interactions with activation domains.

FIG. 1.

Affinity purification of the yeast SWI/SNF complex. (A) Diagram of the SWI/SNF purification scheme. Double affinity-purified yeast SWI/SNF complex was used in photo-cross-linking experiments. (B) The purified complex was separated on an SDS-4 to 15% gradient polyacrylamide gel and found to be largely free of contaminating proteins as visualized by silver staining. Known SWI/SNF subunits are indicated to the right of the panel, and unknown proteins are labeled according to their approximate molecular weight. E#1 and E#2 indicate two subsequent Flag peptide elutions from the Flag M2 agarose affinity gel.

FIG. 2.

Interaction of Snf5, Swi1, and Swi2/Snf2 in the context of intact SWI/SNF with Hap4. (A) Diagram of the photo-cross-linking reagent Sulfo-SBED. (B) Schematic of the photo-cross-linking experiments. Photo-cross-linking experiments were performed with purified SWI/SNF complex and an activator that was previously conjugated with Sulfo-SBED. Where indicated, the reaction mixtures were exposed to UV light (312 nm) at 6 cm for 8 min at room temperature. The activator was separated from interacting SWI/SNF subunits upon the addition of DTT, which reduces the disulfide bond. (C) Snf5, Swi1, and Swi2/Snf2 are cross-linked by GST-Hap4. The photo-cross-linking reaction mixtures were run on an SDS-4 to 15% gradient polyacrylamide gel. The proteins were transferred to PVDF membrane and immunoblotted with streptavidin-HRP or the indicated SWI/SNF-specific antibodies. The asterisks indicate photo-cross-linked SWI/SNF subunits. (D) SWI/SNF subunits are not cross-linked by GST alone. The photo-cross-linking experiment was performed as described for panels B and C.

FIG. 3.

Interaction of Snf5, Swi1, and Swi2/Snf2 in the context of intact SWI/SNF with Gcn4. (A) Photo-cross-linking reactions were carried out as in Fig. 2C, except that full-length Gcn4 (the GST tag was cleaved and removed) was conjugated with the cross-linking reagent and incubated with purified SWI/SNF. The asterisks indicate photo-cross-linked SWI/SNF subunits. (B) Reaction mixtures identical to those shown in panel A were separated on an SDS-5% polyacrylamide gel, instead of an SDS-4 to 15% gradient polyacrylamide gel. The asterisks indicate photo-cross-linked products.

FIG. 4.

Identification of biotinylated SWI/SNF subunits after photo-cross-linking. Reaction mixtures identical to those shown in Fig. 3 were heat denatured in the presence of SDS, followed by incubation with streptavidin-agarose beads. The precipitated material was separated on an SDS-4 to 15% gradient polyacrylamide gel, transferred to PVDF, and immunoblotted with SWI/SNF-specific antisera.

FIG. 5.

Interaction of Snf5, Swi1, and Swi2/Snf2 with acidic activators but not with activation domain mutants by GST pull-down analysis. (A) GST pull-down assays were performed with the GST fusion proteins indicated bound to glutathione-Sepharose beads and individually expressed ³⁵S-labeled SWI/SNF subunits. A total of 50% of each input and supernatant (S) and 100% of the proteins associated with the beads (B) were loaded on SDS-8, 10, or 12% polyacrylamide gels, depending on the size of the labeled subunit. The gels were fixed, enhanced, and dried, and radiolabeled proteins were visualized by autoradiography. (B) GST pull-down assays were performed as for panel A, with GST or GST-Hap4 (amino acids 330 through 554) and the ³⁵S-labeled SWI/SNF subunits indicated. (C) GST pull-down as-says were performed as for panels A and B, with the GST fusion proteins and ³⁵S-labeled SWI/SNF subunits indicated. Gcn4[3,5,6,7] contains mutations in hydrophobic residues within its activation domain that reduce its activation potential.

FIG. 6.

Interaction of Snf5, Swi1, and Swi2/Snf2 with acidic activators but not with activation domain mutants by far-Western analysis. Far-Western analysis was used to confirm the interactions of Snf5, Swi1, and Swi2/Snf2 with GST-VP16 and GST-Gcn4. GST fusion proteins were run on a SDS-10% polyacrylamide gel and transferred to PVDF membrane. After denaturation and renaturation steps, the blots were incubated with in vitro transcription and translation rabbit reticulocyte lysate reactions containing the ³⁵S-labeled SWI/SNF subunit indicated below each panel. A COOH-terminally truncated VP16 activation domain (GST-VP16Δ456) and full-length Gcn4 that contained hydrophobic cluster mutations in its activation domain (GST-Gcn4 [3,5,6,7]) were also tested for interaction with the ³⁵S-labeled subunits. The interaction of the radiolabeled subunits with the GST fusions on the blot was visualized by autoradiography.

FIG. 7.

Interaction of SWI/SNF subunits with the yeast activators Pho4 and Swi5. (A) Purified SWI/SNF interacts with the acidic activator Pho4. GST pull-down assays were done with the GST fusion proteins indicated and affinity-purified SWI/SNF complex. The presence of SWI/SNF in the supernatant or associated with the beads was detected by immunoblotting with antibodies against the SWI/SNF subunits Swi3 and Swp73. (B) Individual SWI/SNF subunit interactions with Pho4 and Swi5. GST pull-down assays were performed as for Fig. 5 with the GST fusion proteins and ³⁵S-labeled SWI/SNF subunits indicated. Radiolabeled SWI/SNF subunits were visualized by autoradiography.