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.

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...

Calcitonin gene-related peptide (CGRP) is a neuropeptide that has potent vasodilator properties and is involved in various behavioral disorders. The relationship between CGRP and depression-like behavior is unclear. In this study, we used chronically stressed mice to investigate whether CGRP is involved in depression-like behavior. Each mouse was exposed to restraint and water immersion stress for 15 days. After stress exposure, mice were assessed using behavioral tests: open field test, forced swim test and sucrose preference test. Serum corticosterone levels, hippocampal proliferation and mRNA expression of neurotrophins were measured. After stress exposure, mice exhibited depression-like behavior and decreased CGRP mRNA levels in the hippocampus. Although intracerebroventricular CGRP administration (0.5 nmol) did not alter depression-like behavior after 15-day stress exposure, a single CGRP administration into the brain, before the beginning of the 15-day stress exposure, normalized the behavioral dysfunctions and increased nerve growth factor (Ngf) mRNA levels in stressed mice. Furthermore, in the mouse E14 hippocampal cell line, CGRP treatment induced increased expression of Ngf mRNA. The NGF receptor inhibitor K252a inhibited CGRP's antidepressant-like effects in stressed mice. These results suggest that CGRP expression in the mouse hippocampus is associated with depression-like behavior and changes in Ngf mRNA levels.Depression is a major psychiatric disorder that is associated with high rates of suicide, and is considered one of the most important causes of human disability. Excessive exposure to stressful life events induces the onset of behavioral disorders, including depression and post-traumatic stress disorder. The mechanisms underlying the psychopathology of depression are multifaceted, however, it is known to be accompanied by a decrease or impairment of neurogenesis in the hippocampus 1,2 . Therefore, mechanistic investigations of depression that target neurogenesis in the hippocampus are crucial. Calcitonin gene-related peptide (CGRP), a potent vasodilator 3 and neurotransmitter in the central nervous system 4 , is a 37-residue amino acid. CGRP receptors are distributed in the hypothalamus, central gray matter, ventromedial nucleus of the thalamus, amygdala, hippocampus and dentate gyrus 5 . CGRP-containing neurons are found in the hypothalamus, preoptic area, amygdala, thalamus, hippocampus (CA3 pyramidal cells), and dentate gyrus granule cells 3,6 . CGRP is reported to be involved in various behaviors suggestive of anxiety. Intracerebroventricular (i.c.v.) CGRP infusions evoke fear-like freezing 7 and anxiety behavior 8 , and improve learning and memory processing 9 . However, it is less well understood whether CGRP is involved in depression-like behavior. Clinical research shows that CGRP levels may be altered in depressed patients 10 . With this in mind, the aim of the present study was to examine whether CGRP is involved in behavioral development using a 15-day stress exposure model in mice...

The heat shock transcription factor Hsf1 of the yeast Saccharomyces cerevisiae regulates the transcription of a set of genes that contain heat shock elements (HSEs) in their promoters and function in diverse cellular processes, including protein folding. Here, we show that Hsf1 activates the transcription of various target genes when cells are treated with oxidizing reagents, including the superoxide anion generators menadione and KO 2 and the thiol oxidants diamide and 1-chloro-2,4-dinitrobenzene (CDNB). Similar to heat shock, the oxidizing reagents are potent inducers of both efficient HSE binding and extensive phosphorylation of Hsf1. The inducible phosphorylation of Hsf1 is regulated by the intramolecular domain-domain interactions and affects HSE structure-specific transcription. Unlike the heat shock, diamide, or CDNB response, menadione or KO 2 activation of Hsf1 is inhibited by cyclic-AMP-dependent protein kinase (PKA) activity, which negatively regulates the activator functions of other transcriptional regulators implicated in the oxidative stress response. These results demonstrate that Hsf1 is a member of the oxidative stress-responsive activators and that PKA is a general negative regulator in the superoxide anion response.

The hydrophobic repeat is a conserved structural motif of eukaryotic heat shock transcription factor (HSF) that enables HSF to form a homotrimer. Homotrimeric HSF binds to heat shock elements (HSEs) consisting of three inverted repeats of the sequence nGAAn. Sequences consisting of four or more nGAAn units are bound cooperatively by two HSF trimers. We show that in Saccharomyces cerevisiae cells oligomerization-defective Hsf1 is not able to bind HSEs with three units and is not extensively phosphorylated in response to stress; it is therefore unable to activate genes containing this type of HSE. Several lines of evidence indicate that oligomerization is a prerequisite for stress-induced hyperphosphorylation of Hsf1. In contrast, oligomerization and hyperphosphorylation are not necessary for gene activation via HSEs with four units. Intragenic suppressor screening of oligomerization-defective hsf1 showed that an interface between adjacent DNA-binding domains is important for the binding of Hsf1 to the HSE. We suggest that Saccharomyces cerevisiae HSEs with different structures are regulated differently; HSEs with three units require Hsf1 to be both oligomerized and hyperphosphorylated, whereas HSEs with four or more units do not require either.The formation of homo-and hetero-oligomers of DNA binding transcription factors is important for their interactions with specific DNA sequences. The DNA-binding domain (DBD) 3 of a monomeric protein recognizes a relatively short sequence unit and does not bind stably. Oligomer formation increases the selectivity of protein-DNA interactions by causing the proteins to bind the more complex sequence consisting of the short units recognized by each DBD. The ability of these transcription factors to activate transcription is also regulated by protein-protein interactions among the monomers (1-3).The eukaryotic heat shock transcription factor (HSF) governs constitutive and stress-inducible mRNA synthesis of various genes, including heat shock protein (HSP) genes. A helix-turn-helix DBD and two hydrophobic repeat regions, HR-A and HR-B, are HSF motifs that are conserved from yeast to humans. The DBD recognizes a 5-bp sequence, 5Ј-nGAAn-3Ј, and HR-A/B mediates interactions between three monomers to form a trimer via a triple-stranded coiled-coil. Homotrimeric HSF binds to three inverted repeats of a unit, termed the heat shock element (HSE), located in the regulatory regions of target genes (4 -6).The Saccharomyces cerevisiae HSF encoded by the HSF1 gene regulates transcription of 1.5-3.0% of the total genes of yeast (7,8). The target genes of Hsf1 contain the "perfect type" HSE, which consists of three contiguous inverted repeats of the nGAAn unit (3P type, nTTCnnGAAnnTTCn), as well as an HSE containing one (gap type, nTTCnnGAAn(5 bp)nGAAn) or two (step type, nGAAn(5 bp)nGAAn(5 bp)nGAAn) gaps between the repeating units (8 -11). The three nGAAn units of these HSEs are bound by a single Hsf1 trimer, whereas HSEs consisting of four or five contiguous units (4P or 5P type) are boun...