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. 1998 Nov;18(11):6436-46.
doi: 10.1128/MCB.18.11.6436.

Residues in the Swi5 zinc finger protein that mediate cooperative DNA binding with the Pho2 homeodomain protein

L T Bhoite  1 , D J Stillman
Affiliations

Residues in the Swi5 zinc finger protein that mediate cooperative DNA binding with the Pho2 homeodomain protein

L T Bhoite et al. Mol Cell Biol. 1998 Nov.

Abstract

The Swi5 zinc finger and the Pho2 homeodomain DNA-binding proteins bind cooperatively to the HO promoter. Pho2 (also known as Bas2 or Grf10) activates transcription of diverse genes, acting with multiple distinct DNA-binding proteins. We have performed a genetic screen to identify amino acid residues in Swi5 that are required for synergistic transcriptional activation of a reporter construct in vivo. Nine unique amino acid substitutions within a 24-amino-acid region of Swi5, upstream of the DNA-binding domain, reduce expression of promoters that require both Swi5 and Pho2 for activation. In vitro DNA binding experiments show that the mutant Swi5 proteins bind DNA normally, but some mutant Swi5 proteins (resulting from SWI5* mutations) show reduced cooperative DNA binding with Pho2. In vivo experiments show that these SWI5* mutations sharply reduce expression of promoters that require both SWI5 and PHO2, while expression of promoters that require SWI5 but are PHO2 independent is largely unaffected. This suggests that these SWI5* mutations do not affect the ability of Swi5 to bind DNA or activate transcription but specifically affect the region of Swi5 required for interaction with Pho2. Two-hybrid experiments show that amino acids 471 to 513 of Swi5 are necessary and sufficient for interaction with Pho2 and that the SWI5* point mutations cause a severe reduction in this two-hybrid interaction. Analysis of promoter activation by these mutants suggests that this small region of Swi5 has at least two distinct functions, conferring specificity for activation of the HO promoter and for interaction with Pho2.

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Figures

FIG. 1
FIG. 1
Activity of reporters in SWI5* mutants. Yeast strains were independently transformed with the HO(site B)-lacZ and the CTS1(46)-lacZ reporters, and transformants were grown in selective medium and assayed for β-galactosidase activity. Three independent transformants were assayed, and standard errors are shown. The normalized levels are also shown, as percentages of the wild-type level. All of the strains are ace2 mutants, and the SWI5* mutant alleles are present at the native SWI5 locus. The following strains were used: DY4854, DY1923, DY1143, DY1985, DY4684, DY4686, DY4852, DY4688, DY4690, DY4692, DY4695, DY4696, and DY4698.
FIG. 2
FIG. 2
SWI5* mutations specifically affect HO expression. S1 nuclease protection assays using probes specific for HO (A) and SIC1 (B) were performed with the CMD1 probe as the internal control by using yeast strains which contained the wild-type SWI5 gene or Pho2 interaction-defective SWI5* mutants at the SWI5 locus. RNAs from the following strains were prepared: DY150 (lane 1), DY161 (lane 2), DY1921 (lane 3), DY4854 (lane 4), DY1923 (lane 5), DY1936 (lane 6), DY1143 (lane 7), DY4684 (lane 8), DY4686 (lane 9), DY4852 (lane 10), DY4688 (lane 11), DY4690 (lane 12), DY4692 (lane 13), DY4695 (lane 14), DY4696 (lane 15), and DY4698 (lane 16). The numbers below lanes 1 to 7 indicate HO mRNA levels, normalized to that of wild type (lane 1, 100%). For lanes 4 to 16, the strains with the SWI5* mutations all contained an ace2 mutation, and the HO mRNA levels shown below these lanes are normalized to that of the ace2 strain (lane 4, 100%). The ACE2 and PHO2 genotypes are indicated at the top of the lanes. WT, wild type.
FIG. 3
FIG. 3
SWI5* mutations reduce expression from the HO(a1) promoter. Yeast strains which contained the wild-type SWI5 gene or Pho2 interaction-defective SWI5* mutants at the SWI5 locus along with the PHO2-dependent HO(a1) mutant promoter were constructed. S1 nuclease protection assays using probes specific for HO and the CMD1 probe (internal control) were performed on RNA extracted from the following strains: DY5031 (lane 1), DY4905 (lane 2), DY4907 (lane 3), DY4909 (lane 4), DY4911 (lane 5), DY4913 (lane 6), DY4915 (lane 7), DY4917 (lane 8), DY4919 (lane 9), DY4921 (lane 10), DY4843 (lane 11), and DY2406 (lane 12). The numbers below the lanes indicate the HO mRNA levels, normalized to that of a wild-type strain with the HO(a1) promoter (lane 1, 100%). The PHO2 genotype is given at the top of the lanes. WT, wild type.
FIG. 4
FIG. 4
DNA binding by mutant Swi5 proteins, without and with Pho2. (A to C) DNA binding to HO promoter DNA by Swi5 alone. (D to F) DNA binding to HO promoter DNA by Swi5 in the presence of 8.1 ng of Pho2. Each panel illustrates an independent gel retardation assay using mutant Swi5 proteins, with wild-type Swi5 included in each assay as a standard internal control. The experiments in panels A and B, C and D, and E and F were conducted in pairs, at the same time with the same probe and the same preparations of purified proteins. Thus, direct comparisons can be made within each set of paired panels. (A and B) The following amounts of Swi5 were added to each binding reaction mixture: 73, 145, 290, and 580 ng of wild-type Swi5 (lanes 2 to 5, respectively); 88, 175, 350, and 700 ng of Swi5(E482K) (lanes 6 to 9, respectively); 38, 75, 150, and 300 ng of Swi5(S483G) (lanes 10 to 13, respectively); 83, 165, 330, and 660 ng of Swi5(R484G) (lanes 14 to 17, respectively). (C and D) The following amounts of Swi5 were added to each binding reaction mixture: 20, 40, 80, and 160 ng of wild-type Swi5 (lanes 2 to 5, respectively); 17, 34, 75, and 150 ng of Swi5(R484S) (lanes 6 to 9, respectively); 22, 43, 85, and 170 ng of Swi5(F485S) (lanes 10 to 13, respectively); 32, 63, 125, and 250 ng of Swi5(V494A) (lanes 14 to 17, respectively). (E and F) The following amounts of Swi5 were added to each binding reaction mixture: 73, 145, 290, and 580 ng of wild-type Swi5 (lanes 2 to 5, respectively); 55, 110, 220, and 440 ng of Swi5(S497P) (lanes 6 to 9, respectively); 62, 123, 245, and 490 ng of Swi5(Q498R) (lanes 10 to 13, respectively); 42, 83, 165, and 330 ng of Swi5(S505P) (lanes 14 to 17, respectively). Lane 1 in all panels has no added protein. The reaction mixtures in lane 18 (panels A, C, D, and E) and lane 19 (panels B and F) contained 73 ng of Swi5 protein only. WT, wild type.
FIG. 5
FIG. 5
Swi5 residues important for interaction with Pho2. The diagram shows the Swi5 protein, with residues 545 to 632 comprising the three zinc fingers of Swi5 indicated. The region of amino acids 471 to 511 is expanded, and the 482-to-505 region important for Pho2 interaction is shaded. The primary amino acid sequence of Swi5 from residues 471 to 511 is shown, amino acid substitutions that reduce interaction with Pho2 are in boldface type, and those residues that affect expression of the native HO locus are indicated with a star. The putative phosphorylation sites for the Pho85 CDK are overlined, and putative phosphorylated residues (T490, S492, and S505) that were mutated to alanines have a box beneath them.
FIG. 6
FIG. 6
Two-hybrid analysis of Swi5-Pho2 interactions. Strain DY1641 was transformed with bait and prey plasmids (HIS3 and LEU2 markers, respectively), and transformants were grown in selective medium and assayed for β-galactosidase activity. Three independent transformants were assayed, and standard errors are shown. The normalized levels are also shown as a percentage of the wild-type level. The LexA-Swi5 plasmids contain amino acids 471 to 513 of Swi5 fused in frame to the LexA DNA-binding domain. The following plasmids were used: M1921, M3895, M3896, M3897, M3899, M3900, M3901, M3902, M3903, M3917, M3918, M3931, M3466, and M3913.
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