Phosphorylation of the yeast heat shock transcription factor is implicated in gene-specific activation dependent on the architecture of the heat shock element

Abstract

Heat shock transcription factor (HSF) binds to the heat shock element (HSE) and regulates transcription, where the divergence of HSE architecture provides gene- and stress-specific responses. The phosphorylation state of HSF, regulated by stress, is involved in the activation and inactivation of the transcription activation function. A domain designated as CTM (C-terminal modulator) of the Saccharomyces cerevisiae HSF is required for the activation of genes containing atypical HSE but not typical HSE. Here, we demonstrate that CTM function is conserved among yeast HSFs and is necessary not only for HSE-specific activation but also for the hyperphosphorylation of HSF upon heat shock. Moreover, both transcription and phosphorylation defects due to CTM mutations were restored concomitantly by a set of intragenic suppressor mutations. Therefore, the hyperphosphorylation of HSF is correlated with the activation of genes with atypical HSE but is not involved in that of genes with typical HSE. The function of CTM was circumvented in an HSF derivative lacking CE2, a yeast-specific repression domain. Taken together, we suggest that CTM alleviates repression by CE2, which allows HSF to be heat-inducibly phosphorylated and presume that phosphorylation is a prerequisite for the activator function of HSF when it binds to an atypical HSE.

FIG.1.

Characterization of ScHsf1 derivatives. (A) Schematic representation of ScHsf1 protein and its derivatives. Each protein is illustrated as a rectangle, the designation of which is shown to the left. The structural motifs of ScHsf1 are indicated above ScHsf1; AR1 and AR2 refer to the N- and C-terminal activation domains, respectively; DBD, DNA-binding domain; Oligomer, oligomerization domain; CE2, conserved element 2; CTM, C-terminal regulatory domain. (26, 39, 49, 51, 57). The numbers represent amino acid positions. Bent lines connecting a pair of rectangles indicate AR1 deletion. The thick vertical line in CTM denotes the ba1 mutation (Arg826Glu and Arg830Glu). Thick-lined boxes represent the C-terminal regions of SpHSF and hHSF1, in which the amino acid numbers correspond to those in the original proteins. (B) Growth of cells expressing deletion and point mutants of ScHsf1. Cells expressing ScHsf1 (wild type [WT]), Hsf1-ΔAR1 (ΔAR1), Hsf1-ba1 (ba1), or Hsf1-ΔAR1/ba1 (ΔAR1/ba1) were streaked on a YPD plate and were incubated at 28 or 38°C for 2 days. (C) mRNA levels of CUP1 and SSA4 in cells expressing deletion and point mutants of ScHsf1. Cells expressing various Hsf1 derivatives were grown in YPD medium at 28°C, and then the temperature was shifted to 39°C. At the indicated times, aliquots of cells were removed and stored at −80°C before use. Total RNA prepared from each sample was subjected to RT-PCR analysis with sets of primers for CUP1, SSA4, and ACT1 (upper panel). The lower panel shows the kinetic profiles of the CUP1 or SSA4 mRNA, in which the levels are normalized to the value of ACT1 mRNA as 100%. (D) Carboxy-terminal sequences of various HSFs. The upper panel shows a schematic overview of the structures of ScHsf1, KlHSF, SpHSF, and hHSF1. Homologous regions are shaded equivalently. The lower panel shows their C-terminal amino acid sequences. The basic amino acids are indicated in bold letters. The numbers show amino acid positions. (E) Growth of cells expressing ScHsf1 chimeras. The growth of cells expressing ScHsf1 (WT), Hsf1-ΔAR1 (ΔAR1), Hsf1-Sp (Sp), Hsf1-Sp/ΔCTM, (Sp/ΔCTM), Hsf1-Hs (Hs), or Hsf1-Hs+CTM (Hs + CTM) was analyzed as described for panel B. (F) mRNA levels of CUP1 and SSA4 in cells expressing ScHsf1 chimeras. Total RNA samples prepared from cells shown in panel E were analyzed as described for panel C.

FIG. 2.

Phosphorylation of ScHsf1 derivatives. (A) Immunoblot analysis of ScHsf1. Cells expressing ScHsf1 (wild type [WT]) or Hsf1-ba1 (ba1) were grown in YPD medium at 28°C (lanes 2 and 7) or at 36, 39, or 42°C (lanes 3 to 5 and 8 to 10) for 15 min. For lanes 1 and 2, the cells were grown at 20°C. The prepared cell extracts were subjected to SDS-PAGE and immunoblot analysis with anti-ScHsf1 serum. (B) Phosphatase treatment of ScHsf1. Cells expressing ScHsf1 (WT) or Hsf1-ba1 (ba1) were grown at 28°C (heat shock −) or 39°C (heat shock +) for 15 min. The cell extracts were subjected to immunoprecipitation with anti-ScHsf1 serum. The precipitates were divided into two portions, incubated in the absence (CIP −) or presence (CIP +) of calf intestine alkaline phosphatase, and subjected to immunoblotting. (C) In vivo ³²P labeling of ScHsf1. Cells expressing ScHsf1 (WT) or Hsf1-ba1 (ba1) were grown at 28°C (heat shock −) or 39°C (heat shock +) for 15 min in the presence of [³²P]orthophosphate. The cell extracts were separated by SDS-PAGE and subjected to phosphorimaging (lanes 1 to 4) and immunoblot (lanes 5 to 8) analyses. (D) Immunoblot analysis of ScHsf1 chimeras. Cells expressing ScHsf1 (WT), Hsf1-ΔAR1 (ΔAR1), or various chimeras (Sp, Sp/ΔCTM, Hs, and Hs+CTM) were grown at 28°C (heat shock −) or 39°C (heat shock +) for 15 min. The cell extracts were subjected to immunoblot analysis as described for panel A. In lanes 9 and 10, Hsf1-Hs proteins migrated close to an unknown cross-reacting protein indicated by an asterisk on the right.

FIG. 3.

Characterization of intragenic suppressors of CTM mutation. (A) Location of suppressor mutations. The suppressor genes and their mutations are summarized. The structural motifs of Hsf1-ΔAR1/ba1 are shown in Fig. 1A. (B) Growth of hsf1-ΔAR1/ba1 cells containing suppressor mutations. Cells harboring HSF1 (WT), hsf1-ΔAR1/ba1 (ΔAR1/ba1), or various suppressor genes (IS-1, -3, -4, and -15) were streaked on a YPD plate and were incubated at 38°C for 2 days. (C) mRNA levels of CUP1 and SSA4 in hsf1-ΔAR1/ba1 cells containing suppressor mutations. Total RNA samples prepared from the cells shown in panel B were analyzed as described for Fig. 1C. For comparison, the kinetic profiles of the CUP1 or SSA4 mRNA in hsf1-ΔAR1/ba1 cells are also shown.

FIG. 4.

Effect of suppressor mutations on CTM function. (A) Growth of hsf1-ΔCTM cells containing suppressor mutations. The growth of cells expressing ScHsf1 (wild type [WT]), Hsf1-ΔCTM (ΔCTM), or Hsf1-ΔCTM containing the suppressor mutations (K491R, Y537C, and Q535R) was analyzed as described for Fig. 3B. (B) mRNA levels of CUP1 and SSA4 in hsf1-ΔCTM cells containing suppressor mutations. Total RNA samples prepared from the cells shown in panel A were analyzed as described for Fig. 1C, and a kinetic profile of the CUP1 mRNA is shown.

FIG. 5.

Effect of CE2 deletion on CTM function. (A) Amino acid sequences of the CE2 regions. The upper panel shows the structures of the ScHsf1 derivatives. The structural motifs are shown in Fig. 1A. The lower panel shows the amino acid sequence of the deleted region in Hsf1-ΔCE2 in the top row. The positions of two suppressor mutations are also indicated by solid triangles. In the second and third rows, the conserved sequences found in KlHSF and SpHSF, respectively, are shown. The CE2 element consists of a heptapeptide sequence and a serine stretch (26), which are indicated in capital letters. The amino acids that are identical in at least two orthologs of the HSFs are indicated in bold letters. The numbers show the amino acid positions. (B) Basal level of CUP1 mRNA in hsf1cells containing suppressor mutations. ace1 null mutant cells expressing ScHsf1 (WT), Hsf1-ΔCE2 (ΔCE2), Hsf1-K491R, Hsf1-Y537C, or Hsf1-Q535R were grown at 28°C. Total RNA prepared from each sample was subjected to RT-PCR analysis with sets of primers for CUP1 and ACT1. The relative levels of CUP1 mRNA, which are normalized to the value of ACT1 mRNA as 100%, represent the means ± standard errors of three independent experiments. (C) Growth of cells expressing Hsf1-ΔCE2 derivatives. The growth of cells expressing ScHsf1 (WT), Hsf1-ba1 (ba1), Hsf1-ΔCE2 (ΔCE2), or Hsf1-ΔCE2/ba1 (ΔCE2/ba1) was analyzed as described for Fig. 3B. (D) mRNA levels of CUP1 and SSA4 in cells expressing Hsf1-ΔCE2 derivatives. Total RNA samples prepared from the cells shown in panel C were analyzed as described for Fig. 1C. For comparison, the kinetic profiles of the CUP1 or SSA4 mRNA in HSF1 and hsf1-ba1 cells are also shown. (E) Immunoblot analysis of Hsf1-ba1 protein containing suppressors of the ba1 mutation. For lanes 1 to 6, the cells expressing Hsf1-ba1, which contains suppressor mutations (ba1/K491R, ba1/Y537C, and ba1/Q535R), were grown at 28°C (heat shock −) or 39°C (heat shock +) for 15 min. For lanes 7 to 10, the cells expressing Hsf1-ΔCE2 (ΔCE2) or Hsf1-ΔCE2/ba1 (ΔCE2/ba1) were used. The cell extracts were subjected to immunoblot analysis as described for Fig. 2A.

FIG. 6.

Physical interaction mediated by CTM and CE2. (A) Schematic representation of the polypeptides used for the binding assay. The structures of the N-terminal polypeptides (N583/WT, N583/ΔCE2, and N583 containing the K491R, Y537C, or Q535R mutation) and C-terminal polypeptides fused with GST (GST-C250/WT and GST-C250/ba1) are shown with motifs that are explained in the legend to Fig. 1A. (B) Coomassie brilliant blue staining of purified polypeptides. Purified polypeptides N583/WT (lane 1), N583/ΔCE2 (lane 2), N583/K491R (lane 3), N583/Y537C (lane 4), N583/Q535R (lane 5), GST (lane 6), GST-C250/WT (lane 7), and GST-C250/ba1 (lane 8) were subjected to SDS-PAGE and stained with Coomassie brilliant blue. Molecular mass markers are indicated to the left in kilodaltons. (C) Immunoblot analysis of bound proteins. Purified protein GST (lanes 2), GST-C250/WT (lanes 3), or GST-C250/ba1 (lane 4) was immobilized on glutathione Sepharose and incubated with N583/WT, N583/ΔCE2, N583/K491R, N583/Y537C, or N583/Q535R polypeptide. After extensive washing, bound polypeptides were subjected to immunoblot analysis. Lane 1 contains input N-terminal polypeptides (IN).