Mutated Yeast Heat Shock Transcription Factor Activates Transcription Independently of Hyperphosphorylation

Abstract

The homotrimeric heat shock transcription factor (HSF) binds to the heat shock element of target genes and regulates transcription in response to various stresses. The Hsf1 protein of Saccharomyces cerevisiae is extensively phosphorylated upon heat shock; a modification that is under positive regulation by its C-terminal regulatory domain (CTM). Hyperphosphorylation has been implicated in gene-specific transcriptional activation. Here, we surveyed genes whose heat shock response is reduced by a CTM mutation. The CTM is indispensable for transcription via heat shock elements bound by a single Hsf1 trimer but is dispensable for transcription via heat shock elements bound by Hsf1 trimers in a cooperative manner. Intragenic mutations located within or near the wing region of the winged helix-turn-helix DNA-binding domain suppress the temperature-sensitive growth phenotype associated with the CTM mutation and enable Hsf1 to activate transcription independently of hyperphosphorylation. Deletion of the wing partially restores the transcriptional defects of the unphosphorylated Hsf1. These results demonstrate a functional link between hyperphosphorylation and the wing region and suggest that this modification is involved in a conformational change of a single Hsf1 trimer to an active form.The eukaryotic heat shock transcription factor HSF regulates the transcription of various genes under numerous stressful conditions. HSF proteins share common structural motifs, including a winged helix-turn-helix DNA-binding domain (DBD), 3 a hydrophobic repeat region essential for three-stranded coiled-coil formation, and a C-terminal transactivation domain (1-3). HSF binds to a conserved DNA sequence motif termed the heat shock element (HSE) by forming a homotrimer through the hydrophobic repeat regions, and the DBD of each monomer recognizes a 5-bp sequence, 5Ј-nGAAn-3Ј. The organization of the three nGAAn units varies among functional HSEs (4 -13). The perfect-type HSE consists of three or more contiguous inverted repeats of the unit (nTTCnnGAAnnTTCn), the gap-type HSE consists of two inverted units separated from a third unit by a 5-bp gap (nTTCnnGAAn(5 bp)nGAAn), and the step-type HSE consists of direct repeats of the nGAAn or nTTCn motif separated by 5 bp (nGAAn(5 bp)nGAAn(5 bp)nGAAn).In the yeast Saccharomyces cerevisiae, the HSF encoded by the HSF1 gene regulates transcription under normal physiological conditions as well as under stress conditions, and it is essential for cell viability. The genes targeted by Hsf1 encode proteins that function in a broad range of biological processes, including protein folding and degradation, detoxification, energy generation, carbohydrate metabolism, and cell wall organization (12, 13). Mammalian cells contain three HSF isoforms, HSF1, HSF2, and HSF4. Among these, HSF1 has roles in stress-induced transcription, extra-embryonic development, and postnatal growth (14, 15). Both S. cerevisiae Hsf1 and mammalian HSF1 are inducibly phosphorylated concomitant with activation (16 -22). Phosphoryla...

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…Temperatureresistant mutations near the DBD enable Hsf1 lacking its C-terminal AD to activate transcription (Hashikawa et al 2006). These suppressors implicate the DBD in mediating the response, as did single point mutations that constitutively enhanced transcription (Bulman et al 2001).…”

“…The N-and C-terminal ADs activate different sets of genes in the Hsf1 regulon during heat shock (Eastmond and Nelson 2006). Stress-induced activation is accompanied by Hsf1 hyperphosphorylation, but whether it is important for activation or for return to an inactive state is unclear (Sorger 1990;Jakobsen and Pelham 1991;Hoj and Jakobsen 1994;Hashikawa et al 2006).…”

“…Stress, such as increased temperature or oxidative damage, induces binding to new targets ) and activation of Hsf1 through two differentially acting ADs, one N-and one C-terminal to the central DBD (Nieto-Sotelo et al 1990;Sorger 1990). The essential C-terminal AD modulates the response to high temperature (Nieto-Sotelo et al 1990;Sorger 1990) and is indispensable for response to heat stress for genes containing a single heat shock element (Hashikawa et al 2006). The N-and C-terminal ADs activate different sets of genes in the Hsf1 regulon during heat shock (Eastmond and Nelson 2006).…”

In the Yeast Heat Shock Response, Hsf1-Directed Induction of Hsp90 Facilitates the Activation of the Slt2 (Mpk1) Mitogen-Activated Protein Kinase Required for Cell Integrity

“…Mammalian, fly, and plant HSFs have just a single trans-activation domain, whereas the HSFs of yeasts possess two distinct trans-activation domains: one close to the amino terminus and the other adjacent to the carboxy terminus (NTA and CTA, respectively). Loss of the C-terminal (CT) domain of Hsf1 (sequences 583 to 833, containing the CTA and modulator sequences) leads to a compromised induction of certain genes, but not others, in response to heat shock (8,16,18,35,43,45). It also causes loss of the ability to grow above about 35°C, revealing that gene expression directed by the Hsf1 CT domain is required for yeast to grow at high temperature.…”

A Novel Heat Shock Transcription Factor Family in Entamoeba histolytica

“…Although the molecular weight of the peptides recognized do not precisely correspond to the molecular weight of the predicted EhHSTF, in the literature has been reported that these proteins may occur postranslational modifications as phosphorylation provoking changes in their molecular weights [36]. Perhaps, have been reported that the HSTF requires being homotrimerized to binds to the HSE [37].…”