The Snf1 protein kinase and Sit4 protein phosphatase have opposing functions in regulating TATA-binding protein association with the Saccharomyces cerevisiae INO1 promoter

Abstract

To identify the mechanisms by which multiple signaling pathways coordinately affect gene expression, we investigated regulation of the S. cerevisiae INO1 gene. Full activation of INO1 transcription occurs in the absence of inositol and requires the Snf1 protein kinase in addition to other signaling molecules and transcription factors. Here, we present evidence that the Sit4 protein phosphatase negatively regulates INO1 transcription. A mutation in SIT4 was uncovered as a suppressor of the inositol auxotrophy of snf1Delta strains. We found that sit4 mutant strains exhibit an Spt(-) phenotype, suggesting a more general role for Sit4 in transcription. In fact, like the gene-specific regulators of INO1 transcription, Opi1, Ino2, and Ino4, both Snf1 and Sit4 regulate binding of TBP to the INO1 promoter, as determined by chromatin immunoprecipitation analysis. Experiments involving double-mutant strains indicate that the negative effect of Sit4 on INO1 transcription is unlikely to occur through dephosphorylation of histone H3 or Opi1. Sit4 is a known component of the target of rapamycin (TOR) signaling pathway, and treatment of cells with rapamycin reduces INO1 activation. However, analysis of rapamycin-treated cells suggests that Sit4 represses INO1 transcription through multiple mechanisms, only one of which may involve inhibition of TOR signaling.

Figure 1.—

Mutations in SIT4 suppress the inositol auxotrophy of snf1Δ strains. (A) Equal numbers of cells in 10-fold serial dilutions were spotted onto synthetic media lacking or containing 200 μm inositol and allowed to grow for 5 days at 30°. Note that strains containing sit4 mutations are less healthy. The following strains were examined: PY165, PY133, PY731, and PY515. (B) Northern blot analysis of INO1 mRNA levels. Samples were grown to early log phase in 200 μm inositol (+) or washed into medium lacking inositol and allowed to grow an additional 4 hr (−). The following strains were examined: PY165, PY133, PY523, PY859, PY499, and PY515. (C) β-Galactosidase assays of INO1-lacZ expression. Cells were grown to early log phase in medium containing 200 μm inositol (0 hr), washed in medium lacking inositol, and harvested at the indicated times. Results presented are the mean average from three colonies assayed at two extract concentrations. Error bars represent one standard deviation of the mean. The following strains were used: PY177, PY539, PY520, PY518, PY498, and PY536.

Figure 2.—

Characterization of the sit4-731 allele. (A, left) Representative tetratype tetrads derived from a cross between a snf1Δ strain (PY130) and a sit4-731 strain (PY859) grown on YPD for 3 days. (A, right) Representative tetratype tetrads derived from sporulation of diploid PY504 grown on YPD for 6 days. Open circles indicate SIT4 SNF1 progeny, circles with a vertical line indicate snf1Δ SIT4 progeny, circles with a horizontal line indicate SNF1 sit4 progeny, and circles with crossed lines indicate snf1 sit4 double-mutant progeny. (B) Diagram of the Sit4-731 and Sit4-TT proteins. sit4-731 contains a G → T mutation that introduces a stop codon at amino acid 128; the box labeled “??” represents the potential readthrough product. The sit4-TT mutation was created by removing the DNA sequences that would encode amino acids 128–311 (see materials and methods). Solid lines indicate the position of residues that are important for metal chelation, and dashed lines indicate the position of additional residues that are important for the active site in the crystal structure of a phosphatase with a similar catalytic domain, rabbit PP-1. (C) Western blot analysis of extracts of PY502 expressing untagged or HA₃-tagged forms of Sit4, Sit4-731, or Sit4-TT from CEN/ARS (C/A) or 2μ plasmids (see materials and methods). Arrows indicate the predicted positions of the 344- or 160-amino-acid HA₃-tagged proteins. (D) Equal numbers of cells in 10-fold serial dilutions were spotted onto YPD media lacking or containing 200 ng/ml rapamycin and allowed to grow for 3 days at 30°. The following strains were examined: PY165, PY133, PY523, PY859, PY538, and PY502.

Figure 3.—

Mutations in SIT4 cause an Spt⁻ phenotype. Equal numbers of cells in 10-fold serial dilutions were spotted onto minimal media containing or lacking histidine and allowed to grow for 5 days at 30°. (Bottom) Diagram of the his4-912δ locus, which contains a Ty δ-element (square with a solid triangle), and the predicted position of the RNA transcripts in Spt⁺ or Spt⁻ strains. The following strains were examined: FY653, PY667, and PY688.

Figure 4.—

ChIP analysis of TBP binding to the INO1 promoter. (A and C) Cells were grown to early log phase in 200 μm inositol (+Ino) or washed into medium lacking inositol and allowed to grow an additional 4 hr (−Ino). An anti-TBP antibody was used to immunoprecipitate crosslinked chromatin. Two dilutions of input DNA (I) and two amounts of precipitated DNA (P) were amplified with primers surrounding the INO1 TATA sequence and multiplexed with primers from a subtelomeric sequence of chromosome VI as a control (TELVI). Representative experiments are shown. The following strains were used: PY165, PY528, PY635, PY133, and PY523. (B and D) Quantitation of ChIP analysis. The amount of radiolabeled INO1 DNA amplified by quantitative PCR from input and precipitated samples was normalized to the TELVI levels in each lane. The relative TBP occupancy was determined by dividing the average of the two normalized precipitated samples by the average of the two normalized input samples. The mean relative occupancy from three independent ChIP experiments is presented. Error bars represent one standard deviation from the mean.

Figure 5.—

Phosphorylation of histone H3 at serine 10 is not required for suppression of snf1Δ by sit4-731. SNF1 SIT4 (FY1716) or snf1Δ sit4-731 (PY636) strains deleted for both copies of the histone H3 and H4 genes, but containing a URA3-marked plasmid with HHT1-HHF1, were transformed with plasmids containing no histone genes (vector; pRS314), wild-type histone H4 and H3 (H3-WT; pWZ414-F13), or wild-type histone H4 and histone H3 with an alanine substitution at serine 10 (H3-S10A; pRS414-59). Transformants selected on SC-Trp medium were purified on medium containing 5-FOA to select against the URA3-marked plasmid. (A) Strains grown on 5-FOA medium were replica plated to synthetic medium lacking inositol or containing 200 μm inositol and allowed to grow for 1 day (SNF1 SIT4) or 2 days (snf1Δ sit4-731) at 30°. Tryptophan was omitted to maintain selection for the histone plasmids. (B) S1 nuclease protection analysis of the strains described in A. Cells were grown to early log phase in medium containing 100 μm inositol (+Ino) or washed into medium lacking inositol and allowed to grow an additional 4 hr (−Ino). INO1 mRNA levels were normalized to DED1 mRNA levels, and the results from a representative experiment are shown.

Figure 6.—

Opi1 is unlikely to be the only target of Sit4. β-Galactosidase assays of INO1-lacZ expression. Cells were processed and β-galactosidase activity was quantitated as in Figure 1C. The following strains were used: PY177, PY533, PY498, and PY526.

Figure 7.—

The TOR pathway inhibits INO1 induction. Northern blot analysis of INO1 transcription. Cell cultures were grown to early log phase in medium containing 100 μm inositol, and samples (−30 min) were harvested from strains prior to rapamycin treatment. The cultures were divided and incubated with (0 min) or without (data not shown) 200 ng/ml rapamycin for an additional 30 min. Samples were washed into medium lacking inositol, in the presence or absence of rapamycin, and incubated for the indicated times (30, 60, or 90 min). The following strains were examined: PY165, PY859, and PY502.