Induction temperature of human heat shock factor is reprogrammed in a Drosophila cell environment

The pathway leading to transcriptional activation of heat shock genes involves a step of heat shock factor 1 (HSF1) trimerization required for high-affinity binding of this activator protein to heat shock elements (HSEs) in the promoters. Previous studies have shown that in vivo the trimerization is negatively regulated at physiological temperatures by a mechanism that requires multiple hydrophobic heptad repeats (HRs) which may form a coiled coil in the monomer. To investigate the minimal requirements for negative regulation, in this work we have examined mouse HSF1 translated in rabbit reticulocyte lysate or extracted from Escherichia coli after limited expression. We show that under these conditions HSF1 behaves as a monomer which can be induced by increases in temperature to form active HSE-binding trimers and that mutations of either HR region cause activation in both systems. Furthermore, temperature elevations and acidic buffers activate purified HSF1, and mild proteolysis excises fragments which form HSE-binding oligomers. These results suggest that oligomerization can be repressed in the monomer, as previously proposed, and that repression can be relieved in the apparent absence of regulatory proteins. An intramolecular mechanism may be central for the regulation of this transcription factor in mammalian cells, although not necessarily sufficient.The increased synthesis of heat shock proteins (HSPs) is a response of cells of many, if not all, organisms to temperatures above normal and to diverse physiological and experimental stress stimuli (14,25). In eukaryotes, the induction of HSPencoding genes is regulated at the transcriptional level by heat shock factor (HSF), which binds multiple copies of an upstream sequence, the heat shock element (HSE), consisting of contiguous 5-bp modules (nGAAn) in alternating orientations (12,26).HSFs from a broad range of species are characterized by a conserved DNA-binding motif in the amino terminus and adjacent hydrophobic heptad repeats (HR-A and HR-B [HR-A,B]) which mediate subunit trimerization via an ␣-helical coiled-coil structure (17,34,41,43,46). A carboxy-terminal hydrophobic heptad repeat (HR-C) is also found in many members of this transcription factor family (26,36,49).In vertebrates, which contain multiple HSFs encoded by distinct genes (30,31,49), the transcriptional response to heat stress is mediated by HSF1. This protein is constitutively synthesized and mostly held in the nucleus in the apparent form of a monomer modified by phosphorylation (10,21,30,31,49). The heat shock stimulus rapidly activates the DNA-binding function of HSF1 by a reversible step of subunit trimerization (3,5,20,33,36,43,(47)(48)(49)(50), while a distinct step enables the function of a constitutively active transcriptional activator domain in the carboxy terminus (16,19,21,32,42,54).Previous studies have shown that the carboxy-terminal hydrophobic repeat (HR-C) is required to repress trimerization of HSF1 in human cells at physiological temperatures, and a similar requirement w...

Section: Discussionmentioning

confidence: 99%

Intramolecular Repression of Mouse Heat Shock Factor 1

Farkas

Kutskova

Zimarino

1998

“…The copper-inducible Gal4 expression vector pMT-G4 was constructed by inserting a PCR-amplified DNA fragment encoding the Gal4 DNAbinding domain (G4DBD) (amino acids 1 to 93) into the EcoRI-BamHI sites of pRmHa-3 (8). pMT-G4 was used in the construction of expression vectors encoding PCR-generated activator protein fragments or full-length Mediator proteins; these genes or gene fragments were inserted into the BamHI-SalI sites of pMT-G4.…”

Section: Methodsmentioning

confidence: 99%

Signal-Induced Transcriptional Activation by Dif Requires the dTRAP80 Mediator Module

Park

Kim

et al. 2003

The Mediator complex is the major multiprotein transcriptional coactivator complex in Drosophila melanogaster. Mediator components interact with diverse sets of transcriptional activator proteins to elicit the sophisticated regulation of gene expression. The distinct phenotypes associated with certain mutations in some of the Mediator genes and the specific in vitro interactions of Mediator gene products with transcriptional activator proteins suggest the presence of activator-specific binding subunits within the Mediator complex. However, the physiological relevance of these selective in vitro interactions has not been addressed. Therefore, we analyzed dTRAP80, one of the putative activator-binding subunits of the Mediator, for specificity of binding to a number of natural transcriptional activators from Drosophila. Among the group of activator proteins that requires the Mediator complex for transcriptional activation, only a subset of these proteins interacted with dTRAP80 in vitro and only these dTRAP80-interacting activators were defective for activation under dTRAP80-deficient in vivo conditions. In particular, activation of Drosophila antimicrobial peptide drosomycin gene expression by the NF-B-like transcription factor Dif during induction of the Toll signaling pathway was dependent on the dTRAP80 module. These results, and the indirect support from the dTRAP80 artificial recruitment assay, indicate that dTRAP80 serves as a genuine activator-binding target responsible for a distinct group of activators.

“…First, alteration of the temperature at which cells are growing modulates the response to a given temperature (2). Second, human HSF1 transfected into Drosophila cells or into tobacco protoplasts is activated at a lower temperature than is required in human cells (11,54). One candidate as a component of the stress-sensing apparatus is the molecular chaperone hsp70 (13).…”

mentioning

confidence: 99%

The DNA-Binding Properties of Two Heat Shock Factors, HSF1 and HSF3, Are Induced in the Avian Erythroblast Cell Line HD6

Nakai

Kawazoe

Tanabe

et al. 1995

108

166

Avian cells express three heat shock transcription factor (HSF) genes corresponding to a novel factor, HSF3, and homologs of mouse and human HSF1 and HSF2. Analysis of the biochemical and cell biological properties of these HSFs reveals that HSF3 has properties in common with both HSF1 and HSF2 and yet has features which are distinct from both. HSF3 is constitutively expressed in the erythroblast cell line HD6, the lymphoblast cell line MSB, and embryo fibroblasts, and yet its DNA-binding activity is induced only upon exposure of HD6 cells to heat shock. Acquisition of HSF3 DNA-binding activity in HD6 cells is accompanied by oligomerization from a non-DNA-binding dimer to a DNA-binding trimer, whereas the effect of heat shock on HSF1 is oligomerization of an inert monomer to a DNA-binding trimer. Induction of HSF3 DNA-binding activity is delayed compared with that of HSF1. As occurs for HSF1, heat shock leads to the translocation of HSF3 to the nucleus. HSF3 exhibits the properties of a transcriptional activator, as judged from the stimulatory activity of transiently overexpressed HSF3 measured by using a heat shock element-containing reporter construct and as independently assayed by the activity of a chimeric GAL4-HSF3 protein on a GAL4 reporter construct. These results reveal that HSF3 is negatively regulated in avian cells and acquires DNA-binding activity in certain cells upon heat shock.Heat shock genes are transcriptionally induced upon exposure of cells to physiological conditions, including stresses such as heat shock, heavy metals, oxidative stress, and amino acid analogs, and during nonstressful conditions such as cell growth, differentiation, and viral infections (27). The effects of heat shock and other stresses on heat shock gene transcription are mediated through interactions of the heat shock element (HSE) that is composed of at least three pentanucleotide modules (nGAAn) arranged as contiguous inverted repeats (33) with heat shock factor (HSF), the inducible transcriptional activator that regulates the transcription of these stress-inducible genes (25). In the budding yeasts Saccharomyces cerevisiae and Kluyveromyces lactis, HSF binds to HSE constitutively as a trimer and stimulates transcription upon heat shock (22,49,50). During deactivation of the heat shock response, the Cterminal activator domain is unmasked and becomes phosphorylated (9, 21, 32). In contrast, HSF does not bind to the HSE under normal growth conditions in the fission yeast Schizosaccharomyces pombe, Drosophila melanogaster, and higher eukaryotes but acquires HSE-binding activity by oligomerization to a trimer upon exposure to stress conditions (17,29,50,57,58,61,62). HSF trimers exhibit cooperative interactions between adjacent and distantly spaced HSEs as measured by DNA binding studies and transcriptional activation (3, 63).Recently, HSF genes from a number of eukaryotes have been cloned, thus allowing a comparative analysis to identify regulatory domains. The HSF family includes HSF1, HSF2, and HSF3 in higher eukaryotes ...