scholarly journals Repression of the heat shock factor 1 transcriptional activation domain is modulated by constitutive phosphorylation.

1997 ◽  
Vol 17 (4) ◽  
pp. 2107-2115 ◽  
Author(s):  
M P Kline ◽  
R I Morimoto
Keyword(s):  
Heat Shock ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Physiological Stress ◽  
Heat Shock Factor 1 ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
High Level ◽  
Constitutive Phosphorylation

Heat shock transcription factor 1 (HSF1) is constitutively expressed in mammalian cells and negatively regulated for DNA binding and transcriptional activity. Upon exposure to heat shock and other forms of chemical and physiological stress, these activities of HSF1 are rapidly induced. In this report, we demonstrate that constitutive phosphorylation of HSF1 at serine residues distal to the transcriptional activation domain functions to repress transactivation. Tryptic phosphopeptide analysis of a collection of chimeric GAL4-HSF1 deletion and point mutants identified a region of constitutive phosphorylation encompassing serine residues 303 and 307. The significance of phosphorylation at serines 303 and 307 in the regulation of HSF1 transcriptional activity was demonstrated by transient transfection and assay of a chloramphenicol acetyltransferase reporter construct. Whereas the transfected wild-type GAL4-HSF1 chimera is repressed for transcriptional activity and derepressed by heat shock, mutation of serines 303 and 307 to alanine results in derepression to a high level of constitutive activity. Similar results were obtained with mutation of these serine residues in the context of full-length HSF1. These data reveal that constitutive phosphorylation of serines 303 and 307 has an important role in the negative regulation of HSF1 transcriptional activity at control temperatures.

scholarly journals The carboxyl-terminal transactivation domain of heat shock factor 1 is negatively regulated and stress responsive.

1995 ◽  
Vol 15 (8) ◽  
pp. 4309-4318 ◽  
Author(s):  
Y Shi ◽  
P E Kroeger ◽  
R I Morimoto
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Heat Shock Factor ◽  
Binding Domain ◽  
Heat Shock Factor 1 ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Carboxyl Terminal

We have characterized a stress-responsive transcriptional activation domain of mouse heat shock factor 1 (HSF1) by using chimeric GAL4-HSF1 fusion proteins. Fusion of the GAL4 DNA-binding domain to residues 124 to 503 of HSF1 results in a chimeric factor that binds DNA yet lacks any transcriptional activity. Transactivation is acquired upon exposure to heat shock or by deletion of a negative regulatory domain including part of the DNA-binding-domain-proximal leucine zippers. Analysis of a collection of GAL4-HSF1 deletion mutants revealed the minimal region for the constitutive transcriptional activator to map within the extreme carboxyl-terminal 108 amino acids, corresponding to a region rich in acidic and hydrophobic residues. Loss of residues 395 to 425 or 451 to 503, which are located at either end of this activation domain, severely diminished activity, indicating that the entire domain is required for transactivation. The minimal activation domain of HSF1 also confers enhanced transcriptional response to heat shock or cadmium treatment. These results demonstrate that the transcriptional activation domain of HSF1 is negatively regulated and that the signal for stress induction is mediated by interactions between the amino-terminal negative regulator and the carboxyl-terminal transcriptional activation domain.

scholarly journals Fos and jun cooperate in transcriptional regulation via heterologous activation domains.

1990 ◽  
Vol 10 (10) ◽  
pp. 5532-5535 ◽  
Author(s):  
C Abate ◽  
D Luk ◽  
E Gagne ◽  
R G Roeder ◽  
T Curran
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Regulation Of Gene Expression ◽  
Negative Influence ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Transcriptional Stimulation

The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site. Here we have examined the contribution of Fos and Jun toward transcriptional activity by using Fos and Jun polypeptides purified from Escherichia coli. Fos contained a transcriptional activation domain as well as a region which exerted a negative influence on transcriptional activity in vitro. Moreover, distinct activation domains in both Fos and Jun functioned cooperatively in transcriptional stimulation. Thus, regulation of gene expression by Fos and Jun results from an integration of several functional domains in a bimolecular complex.

scholarly journals Transcriptional activation upon pheromone stimulation mediated by a small domain of Saccharomyces cerevisiae Ste12p.

1997 ◽  
Vol 17 (11) ◽  
pp. 6410-6418 ◽  
Author(s):  
H Pi ◽  
C T Chien ◽  
S Fields
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Map Kinase ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Activation Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Activation Domains ◽  
Hybrid Proteins ◽  
High Level ◽  
Transcriptional Induction

In the yeast Saccharomyces cerevisiae, Ste12p induces transcription of pheromone-responsive genes by binding to a DNA sequence designated the pheromone response element. We generated a series of hybrid proteins of Ste12p with the DNA-binding and activation domains of the transcriptional activator Gal4p to define a pheromone induction domain of Ste12p sufficient to mediate pheromone-induced transcription by these hybrid proteins. A minimal pheromone induction domain, delineated as residues 301 to 335 of Ste12p, is dependent on the pheromone mitogen-activated protein (MAP) kinase pathway for induction activity. Mutation of the three serine and threonine residues within the minimal pheromone induction domain did not affect transcriptional induction, indicating that the activity of this domain is not directly regulated by MAP kinase phosphorylation. By contrast, mutation of the two tyrosines or their preceding acidic residues led to a high level of transcriptional activity in the absence of pheromone and consequently to the loss of pheromone induction. This constitutively high activity was not affected by mutations in the MAP kinase cascade, suggesting that the function of the pheromone induction domain is normally repressed in the absence of pheromone. By two-hybrid analysis, this minimal domain interacts with two negative regulators, Dig1p and Dig2p (also designated Rst1p and Rst2p), and the interaction is abolished by mutation of the tyrosines. The pheromone induction domain itself has weak and inducible transcriptional activity, and its ability to potentiate transcription depends on the activity of an adjacent activation domain. These results suggest that the pheromone induction domain of Ste12p mediates transcriptional induction via a two-step process: the relief of repression and synergistic transcriptional activation with another activation domain.

scholarly journals Intramolecular Repression of Mouse Heat Shock Factor 1

1998 ◽  
Vol 18 (2) ◽  
pp. 906-918 ◽  
Author(s):  
Thomas Farkas ◽  
Yulia A. Kutskova ◽  
Vincenzo Zimarino
Keyword(s):  
Heat Shock ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Heat Shock Factor ◽  
Heat Shock Factor 1 ◽  
Reticulocyte Lysate ◽  
Heptad Repeats ◽  
Intramolecular Mechanism

ABSTRACT 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.

Fos and jun cooperate in transcriptional regulation via heterologous activation domains

1990 ◽  
Vol 10 (10) ◽  
pp. 5532-5535
Author(s):  
C Abate ◽  
D Luk ◽  
E Gagne ◽  
R G Roeder ◽  
T Curran
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Regulation Of Gene Expression ◽  
Negative Influence ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Transcriptional Stimulation

The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site. Here we have examined the contribution of Fos and Jun toward transcriptional activity by using Fos and Jun polypeptides purified from Escherichia coli. Fos contained a transcriptional activation domain as well as a region which exerted a negative influence on transcriptional activity in vitro. Moreover, distinct activation domains in both Fos and Jun functioned cooperatively in transcriptional stimulation. Thus, regulation of gene expression by Fos and Jun results from an integration of several functional domains in a bimolecular complex.

scholarly journals Regulation of the Amino-Terminal Transcription Activation Domain of Progesterone Receptor by a Cofactor-Induced Protein Folding Mechanism

2005 ◽  
Vol 25 (20) ◽  
pp. 8792-8808 ◽  
Author(s):  
Suzanne E. Wardell ◽  
Stanley C. Kwok ◽  
Lori Sherman ◽  
Robert S. Hodges ◽  
Dean P. Edwards
Keyword(s):  
Progesterone Receptor ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Leucine Zipper ◽  
Activation Domain ◽  
Basic Leucine Zipper ◽  
Transcriptional Activation Domain ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Helical Content

ABSTRACT We previously identified a small basic leucine zipper (bZIP) protein, Jun dimerization protein 2 (JDP-2), that acts as a coregulator of the N-terminal transcriptional activation domain of progesterone receptor (PR). We show here that JDP-2, through interaction with the DNA binding domain (DBD), induces or stabilizes structure in the N-terminal domain in a manner that correlates with JDP-2 stimulation of transcriptional activity. Circular dichroism spectroscopy experiments showed that JDP-2 interaction caused a significant increase in overall helical content of a two-domain PR polypeptide containing the N-terminal domain and DBD and that the change in structure resides primarily in the N-terminal domain. Thermal melt curves showed that the JDP-2/PR complex is significantly more stable than either protein alone, and partial proteolysis confirmed that JDP-2 interaction alters conformation of the N-terminal domain of PR. Functional analysis of N-terminal domain mutants and receptor chimeras provides evidence that the stimulatory effect of JDP-2 on transcriptional activity of PR is mediated through an interdomain communication between the DBD and the N-terminal domain and that transcriptional activity and functional response to JDP-2 are mediated by multiple elements of the N-terminal domain as opposed to a discrete region.

scholarly journals Temperature-dependent regulation of a heterologous transcriptional activation domain fused to yeast heat shock transcription factor.

1992 ◽  
Vol 12 (3) ◽  
pp. 1021-1030 ◽  
Author(s):  
J J Bonner ◽  
S Heyward ◽  
D L Fackenthal
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Heat Shock Transcription Factor ◽  
Temperature Dependent ◽  
Activation Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transcriptional Activation Domain ◽  
Vp16 Fusion

The heat shock transcription factor (HSF) of the yeast Saccharomyces cerevisiae is posttranslationally modified. At low growth temperatures, it activates transcription of heat shock genes only poorly; after shift to high temperatures, it activates transcription readily. In an effort to elucidate the mechanism of this regulation, we constructed a series of HSF-VP16 fusions that join the HSF DNA-binding domain to the strong transcriptional activation domain from the VP16 gene of herpes simplex virus. Replacement of the endogenous C-terminal transcriptional activation domain with that of VP16 generates an HSF derivative that exhibits behavior reminiscent of HSF itself: low transcriptional activation activity at normal growth temperature and high activity after heat shock. HSF can thus restrain the activity of the heterologous VP16 transcriptional activation domain. To determine what is required for repression of activity at low temperature, we deleted portions of HSF from this HSF-VP16 fusion to map the regulatory domain. We also isolated point mutations that convert the HSF-VP16 fusion into a constitutive transcriptional activator. We conclude that the central, evolutionarily conserved domain of HSF, encompassing the DNA-binding and multimerization domains, contains a major determinant of temperature-dependent regulation.

B-myc inhibits neoplastic transformation and transcriptional activation by c-myc

1993 ◽  
Vol 13 (2) ◽  
pp. 1130-1136
Author(s):  
L M Resar ◽  
C Dolde ◽  
J F Barrett ◽  
C V Dang
Keyword(s):  
Dna Binding ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Neoplastic Transformation ◽  
Binding Motif ◽  
Activation Domain ◽  
Transcriptional Activation Domain ◽  
Amino Terminal

B-myc is a recently described myc gene whose product has not been functionally characterized. The predicted product of B-myc is a 168-amino-acid protein with extensive homology to the c-Myc amino-terminal region, previously shown to contain a transcriptional activation domain. We hypothesized that B-Myc might also function in transcriptional regulation, although its role in regulating gene expression is predicted to be unique, because B-Myc lacks the specific DNA-binding motif found in other Myc proteins. To determine whether B-Myc could interact with the transcriptional machinery, we studied the transcriptional activation properties of a chimeric protein containing B-Myc sequences fused to the DNA-binding domain of the yeast transcriptional activator GAL4 (GAL4-B-Myc). We found that GAL4-B-Myc strongly activated expression of a GAL4-regulated reporter gene in mammalian cells. In addition, full-length B-Myc was able to inhibit or squelch reporter gene activation by a GAL4 chimeric protein containing the c-Myc transcriptional activation domain. We also observed that B-Myc dramatically inhibited the neoplastic cotransforming activity of c-Myc and activated Ras in rat embryo cells. Because B-Myc inhibits both neoplastic transformation and transcriptional activation by c-Myc, we suggest that the transforming activity of c-Myc is related to its ability to regulate transcription. Whether B-Myc functions biologically to squelch transcription and/or to regulate transcription through a specific DNA-binding protein remains unestablished.

Temperature-dependent regulation of a heterologous transcriptional activation domain fused to yeast heat shock transcription factor

1992 ◽  
Vol 12 (3) ◽  
pp. 1021-1030
Author(s):  
J J Bonner ◽  
S Heyward ◽  
D L Fackenthal
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Heat Shock Transcription Factor ◽  
Temperature Dependent ◽  
Activation Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transcriptional Activation Domain ◽  
Vp16 Fusion

The heat shock transcription factor (HSF) of the yeast Saccharomyces cerevisiae is posttranslationally modified. At low growth temperatures, it activates transcription of heat shock genes only poorly; after shift to high temperatures, it activates transcription readily. In an effort to elucidate the mechanism of this regulation, we constructed a series of HSF-VP16 fusions that join the HSF DNA-binding domain to the strong transcriptional activation domain from the VP16 gene of herpes simplex virus. Replacement of the endogenous C-terminal transcriptional activation domain with that of VP16 generates an HSF derivative that exhibits behavior reminiscent of HSF itself: low transcriptional activation activity at normal growth temperature and high activity after heat shock. HSF can thus restrain the activity of the heterologous VP16 transcriptional activation domain. To determine what is required for repression of activity at low temperature, we deleted portions of HSF from this HSF-VP16 fusion to map the regulatory domain. We also isolated point mutations that convert the HSF-VP16 fusion into a constitutive transcriptional activator. We conclude that the central, evolutionarily conserved domain of HSF, encompassing the DNA-binding and multimerization domains, contains a major determinant of temperature-dependent regulation.

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