The novel EhHSTF7 transcription factor displays an oligomer state and recognizes a heat shock element in the Entamoeba histolytica parasite

Heat stress regulation of human heat shock genes is mediated by human heat shock transcription factor hHSF1, which contains three 4-3 hydrophobic repeats (LZ1 to LZ3). In unstressed human cells (37 degrees C), hHSF1 appears to be in an inactive, monomeric state that may be maintained through intramolecular interactions stabilized by transient interaction with hsp70. Heat stress (39 to 42 degrees C) disrupts these interactions, and hHSF1 homotrimerizes and acquires heat shock element DNA-binding ability. hHSF1 expressed in Xenopus oocytes also assumes a monomeric, non-DNA-binding state and is converted to a trimeric, DNA-binding form upon exposure of the oocytes to heat shock (35 to 37 degrees C in this organism). Because endogenous HSF DNA-binding activity is low and anti-hHSF1 antibody does not recognize Xenopus HSF, we employed this system for mapping regions in hHSF1 that are required for the maintenance of the monomeric state. The results of mutagenesis analyses strongly suggest that the inactive hHSF1 monomer is stabilized by hydrophobic interactions involving all three leucine zippers which may form a triple-stranded coiled coil. Trimerization may enable the DNA-binding function of hHSF1 by facilitating cooperative binding of monomeric DNA-binding domains to the heat shock element motif. This view is supported by observations that several different LexA DNA-binding domain-hHSF1 chimeras bind to a LexA-binding site in a heat-regulated fashion, that single amino acid replacements disrupting the integrity of hydrophobic repeats render these chimeras constitutively trimeric and DNA binding, and that LexA itself binds stably to DNA only as a dimer but not as a monomer in our assays.

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Modular recognition of 5-base-pair DNA sequence motifs by human heat shock transcription factor

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3504-3514.1991 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3504-3514

Author(s):

N F Cunniff ◽

J Wagner ◽

W D Morgan

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Gene Promoter ◽

Heat Shock Transcription Factor ◽

Heat Shock Gene ◽

Gene Promoters ◽

Sequence Motifs ◽

Binding Modes ◽

Heat Shock Element

We investigated the recognition of the conserved 5-bp repeated motif NGAAN, which occurs in heat shock gene promoters of Drosophila melanogaster and other eukaryotic organisms, by human heat shock transcription factor (HSF). Extended heat shock element mutants of the human HSP70 gene promoter, containing additional NGAAN blocks flanking the original element, showed significantly higher affinity than the wild-type promoter element for human HSF in vitro. Protein-DNA contact positions were identified by hydroxyl radical protection, diethyl pyrocarbonate interference, and DNase I footprinting. New contacts in the mutant HSE constructs corresponded to the locations of additional NGAAN motifs. The pattern of binding indicated the occurrence of multiple DNA binding modes for HSF with the various constructs and was consistent with an oligomeric, possibly trimeric, structure of the protein. In contrast to the improved binding, the extended heat shock element mutant constructs did not exhibit dramatically increased heat-inducible transcription in transient expression assays with HeLa cells.

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Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple-stranded coiled-coil structure

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7557-7568.1994 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7557-7568

Author(s):

J Zuo ◽

R Baler ◽

G Dahl ◽

R Voellmy

Keyword(s):

Transcription Factor ◽

Heat Stress ◽

Heat Shock ◽

Dna Binding ◽

Hydrophobic Interactions ◽

Coiled Coil ◽

Heat Shock Transcription Factor ◽

Single Amino Acid ◽

Binding Ability ◽

Heat Shock Element

Heat stress regulation of human heat shock genes is mediated by human heat shock transcription factor hHSF1, which contains three 4-3 hydrophobic repeats (LZ1 to LZ3). In unstressed human cells (37 degrees C), hHSF1 appears to be in an inactive, monomeric state that may be maintained through intramolecular interactions stabilized by transient interaction with hsp70. Heat stress (39 to 42 degrees C) disrupts these interactions, and hHSF1 homotrimerizes and acquires heat shock element DNA-binding ability. hHSF1 expressed in Xenopus oocytes also assumes a monomeric, non-DNA-binding state and is converted to a trimeric, DNA-binding form upon exposure of the oocytes to heat shock (35 to 37 degrees C in this organism). Because endogenous HSF DNA-binding activity is low and anti-hHSF1 antibody does not recognize Xenopus HSF, we employed this system for mapping regions in hHSF1 that are required for the maintenance of the monomeric state. The results of mutagenesis analyses strongly suggest that the inactive hHSF1 monomer is stabilized by hydrophobic interactions involving all three leucine zippers which may form a triple-stranded coiled coil. Trimerization may enable the DNA-binding function of hHSF1 by facilitating cooperative binding of monomeric DNA-binding domains to the heat shock element motif. This view is supported by observations that several different LexA DNA-binding domain-hHSF1 chimeras bind to a LexA-binding site in a heat-regulated fashion, that single amino acid replacements disrupting the integrity of hydrophobic repeats render these chimeras constitutively trimeric and DNA binding, and that LexA itself binds stably to DNA only as a dimer but not as a monomer in our assays.

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Homoharringtonine Induces Heat Protection and Facilitates Dissociation of Heat Shock Transcription Factor and Heat Shock Element Complex

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1993.2579 ◽

1993 ◽

Vol 197 (2) ◽

pp. 1011-1018

Author(s):

Y.J. Lee ◽

Z.Z. Hou ◽

G. Erdos ◽

J.M. Cho ◽

P.M. Corry

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Heat Shock Transcription Factor ◽

Heat Shock Element ◽

Heat Protection

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Heat shock-induced interactions of heat shock transcription factor and the human hsp70 promoter examined by in vivo footprinting

Molecular and Cellular Biology ◽

10.1128/mcb.11.1.586-592.1991 ◽

1991 ◽

Vol 11 (1) ◽

pp. 586-592

Author(s):

K Abravaya ◽

B Phillips ◽

R I Morimoto

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Consensus Sequence ◽

Regulatory Elements ◽

Heat Shock Transcription Factor ◽

Hsp70 Promoter ◽

Heat Shock Element ◽

In Vivo Footprinting ◽

Human Hsp70

Genomic footprinting of the human hsp70 promoter reveals that heat shock induces a rapid binding of a factor, presumably heat shock transcription factor, to a region encompassing five contiguous NGAAN sequences, three perfect and two imperfect matches to the consensus sequence. Arrays of inverted NGAAN sequences have been defined as the heat shock element. No protein is bound to the heat shock element prior to or after recovery from heat shock. Heat shock does not perturb the binding of factors to other regulatory elements in the promoter which contribute to basal expression of the hsp70 gene.

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Heat Shock Element Architecture Is an Important Determinant in the Temperature and Transactivation Domain Requirements for Heat Shock Transcription Factor

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6340 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6340-6352 ◽

Cited By ~ 71

Author(s):

Nicholas Santoro ◽

Nina Johansson ◽

Dennis J. Thiele

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Transcriptional Activation ◽

Single Gene ◽

Limited Proteolysis ◽

Normal Growth ◽

Heat Shock Transcription Factor ◽

Transactivation Domain ◽

Heat Shock Element

ABSTRACT The baker’s yeast Saccharomyces cerevisiae possesses a single gene encoding heat shock transcription factor (HSF), which is required for the activation of genes that participate in stress protection as well as normal growth and viability. Yeast HSF (yHSF) contains two distinct transcriptional activation regions located at the amino and carboxyl termini. Activation of the yeast metallothionein gene, CUP1, depends on a nonconsensus heat shock element (HSE), occurs at higher temperatures than other heat shock-responsive genes, and is highly dependent on the carboxyl-terminal transactivation domain (CTA) of yHSF. The results described here show that the noncanonical (or gapped) spacing of GAA units in the CUP1HSE (HSE1) functions to limit the magnitude of CUP1transcriptional activation in response to heat and oxidative stress. The spacing in HSE1 modulates the dependence for transcriptional activation by both stresses on the yHSF CTA. Furthermore, a previously uncharacterized HSE in the CUP1 promoter, HSE2, modulates the magnitude of the transcriptional activation of CUP1, via HSE1, in response to stress. In vitro DNase I footprinting experiments suggest that the occupation of HSE2 by yHSF strongly influences the manner in which yHSF occupies HSE1. Limited proteolysis assays show that HSF adopts a distinct protease-sensitive conformation when bound to the CUP1HSE1, providing evidence that the HSE influences DNA-bound HSF conformation. Together, these results suggest that CUP1regulation is distinct from that of other classic heat shock genes through the interaction of yHSF with two nonconsensus HSEs. Consistent with this view, we have identified other gene targets of yHSF containing HSEs with sequence and spacing features similar to those ofCUP1 HSE1 and show a correlation between the spacing of the GAA units and the relative dependence on the yHSF CTA.

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Modular recognition of 5-base-pair DNA sequence motifs by human heat shock transcription factor.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3504 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3504-3514 ◽

Cited By ~ 14

Author(s):

N F Cunniff ◽

J Wagner ◽

W D Morgan

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Gene Promoter ◽

Heat Shock Transcription Factor ◽

Heat Shock Gene ◽

Gene Promoters ◽

Sequence Motifs ◽

Binding Modes ◽

Heat Shock Element

We investigated the recognition of the conserved 5-bp repeated motif NGAAN, which occurs in heat shock gene promoters of Drosophila melanogaster and other eukaryotic organisms, by human heat shock transcription factor (HSF). Extended heat shock element mutants of the human HSP70 gene promoter, containing additional NGAAN blocks flanking the original element, showed significantly higher affinity than the wild-type promoter element for human HSF in vitro. Protein-DNA contact positions were identified by hydroxyl radical protection, diethyl pyrocarbonate interference, and DNase I footprinting. New contacts in the mutant HSE constructs corresponded to the locations of additional NGAAN motifs. The pattern of binding indicated the occurrence of multiple DNA binding modes for HSF with the various constructs and was consistent with an oligomeric, possibly trimeric, structure of the protein. In contrast to the improved binding, the extended heat shock element mutant constructs did not exhibit dramatically increased heat-inducible transcription in transient expression assays with HeLa cells.

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Activation of heat-shock transcription factor 1 by hypertonic shock in 3T3 cells

Biochemical Journal ◽

10.1042/bj3190601 ◽

1996 ◽

Vol 319 (2) ◽

pp. 601-606 ◽

Cited By ~ 22

Author(s):

Roberta ALFIERI ◽

Pier Giorgio PETRONINI ◽

Simona URBANI ◽

Angelo F BORGHETTI

Keyword(s):

Transcription Factor ◽

Heat Shock ◽

Time Course ◽

Binding Activity ◽

Heat Shock Transcription Factor ◽

3T3 Cells ◽

Mobility Shift ◽

Heat Shock Element ◽

Competition Analysis ◽

Hypertonic Shock

The exposure of 3T3 cells to a medium made hypertonic by the addition of NaCl induced activation of a heat-shock transcription factor (HSF). This activation, as monitored by gel-mobility-shift assays, occurred within 10 min of hypertonic shock and was dose-dependent in relation to the osmotic strength of the medium up to 0.7 osM. Competition analysis indicated that the effect of hypertonic shock on HSF binding activity was specific. The magnitude of the heat-shock element (HSE)-HSF binding induced by incubating the cells in a 0.7 osM medium was comparable in intensity and time course with that induced by a 44 °C heat shock. Following removal of the stressors, the decrease in HSF-HSE binding was more rapid in hypertonicity-shocked than in heat-shocked cells. Treatment of the cells with cycloheximide did not inhibit HSF-HSE binding, indicating that the activation was independent of new protein synthesis. By using a specifically directed polyclonal serum, HSF1 was identified as the transcription factor involved in the hypertonicity-induced activation. HSF was also activated when a membrane-impermeable osmolyte such as sucrose was used to increase the osmolarity of the medium. However, no HSF-HSE binding was observed after addition of glycerol (a freely membrane-permeable osmolyte) in excess. There was a temporal relationship between the hypertonicity-induced volume decrease, the increase in the intracellular K+ concentration and the induction of HSF-HSE binding. In contrast, an increase in the intracellular Na+ concentration was not required to induce HSF-HSE binding. However, unlike the heat-shock response, the activation of HSF by hypertonic shock did not lead to elongation of the RNA transcript of heat-shock protein 70.

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