scholarly journals Dual regulation of heat-shock transcription factor (HSF) activation and DNA-binding activity by H2O2: role of thioredoxin

1996 ◽  
Vol 318 (1) ◽  
pp. 187-193 ◽  
Author(s):  
Muriel R. JACQUIER-SARLIN ◽  
Barbara S POLLA
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Dna Binding ◽  
Cellular Response ◽  
Nuclear Translocation ◽  
Alkylating Agents ◽  
Iodoacetic Acid ◽  
Binding Activity ◽  
Dna Binding Activity

The heat-shock (HS) response is a ubiquitous cellular response to stress, involving the transcriptional activation of HS genes. Reactive oxygen species (ROS) have been shown to regulate the activity of a number of transcription factors. We investigated the redox regulation of the stress response and report here that in the human pre-monocytic line U937 cells, H2O2 induced a concentration-dependent transactivation and DNA-binding activity of heat-shock factor-1 (HSF-1). DNA-binding activity was, however, lower with H2O2 than with HS. We thus hypothesized a dual regulation of HSF by oxidants. We found that oxidizing agents, such as H2O2 and diamide, as well as alkylating agents, such as iodoacetic acid, abolished, in vitro, the HSF-DNA-binding activity induced by HS in vivo. The effects of H2O2in vitro were reversed by the sulphydryl reducing agent dithiothreitol and the endogenous reductor thioredoxin (TRX), while the effects of iodoacetic acid were irreversible. In addition, TRX also restored the DNA-binding activity of HSF oxidized in vivo, while it was found to be itself induced in vivo by both HS and H2O2. Thus, H2O2 exerts dual effects on the activation and the DNA-binding activity of HSF: on the one hand, H2O2 favours the nuclear translocation of HSF, while on the other, it alters HSF-DNA-binding activity, most likely by oxidizing critical cysteine residues within the DNA-binding domain. HSF thus belongs to the group of ROS-modulated transcription factors. We propose that the time required for TRX induction, which may restore the DNA-binding activity of oxidized HSF, provides an explanation for the delay in heat-shock protein synthesis upon exposure of cells to ROS.

scholarly journals Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells

1994 ◽  
Vol 14 (8) ◽  
pp. 5309-5317
Author(s):  
S P Murphy ◽  
J J Gorzowski ◽  
K D Sarge ◽  
B Phillips
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Embryonal Carcinoma ◽  
Embryonic Stem ◽  
Binding Activity ◽  
Hsp70 Promoter ◽  
Ec Cells

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

scholarly journals Specific DNA Binding and Regulation of Its Own Expression by the AidB Protein in Escherichia coli

2010 ◽  
Vol 192 (23) ◽  
pp. 6136-6142 ◽  
Author(s):  
Valentina Rippa ◽  
Angela Amoresano ◽  
Carla Esposito ◽  
Paolo Landini ◽  
Michael Volkert ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Alkylating Agents ◽  
Specific Interaction ◽  
Domain Architecture ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Preferential Binding

ABSTRACT Upon exposure to alkylating agents, Escherichia coli increases expression of aidB along with three genes (ada, alkA, and alkB) that encode DNA repair proteins. While the biological roles of the Ada, AlkA, and AlkB proteins have been defined, despite many efforts, the molecular functions of AidB remain largely unknown. In this study, we focused on the biological role of the AidB protein, and we demonstrated that AidB shows preferential binding to a DNA region that includes the upstream element of its own promoter, PaidB. The physiological significance of this specific interaction was investigated by in vivo gene expression assays, demonstrating that AidB can repress its own synthesis during normal cell growth. We also showed that the domain architecture of AidB is related to the different functions of the protein: the N-terminal region, comprising the first 439 amino acids (AidB “I-III”), possesses FAD-dependent dehydrogenase activity, while its C-terminal domain, corresponding to residues 440 to 541 (AidB “IV”), displays DNA binding activity and can negatively regulate the expression of its own gene in vivo. Our results define a novel role in gene regulation for the AidB protein and underline its multifunctional nature.

Abstract 121: GRK5-NT Regulates the Activity of Calcium-Calmodulin-Dependent Transcription Factors

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Daniela Sorriento ◽  
Anna Fusco ◽  
Gaetano Santulli ◽  
Bruno Trimarco ◽  
Guido Iaccarino
Keyword(s):  
Transcription Factors ◽  
Cardiac Hypertrophy ◽  
Nuclear Translocation ◽  
Binding Activity ◽  
Activated T Cells ◽  
Calmodulin Binding ◽  
Amino Terminal ◽  
Calcium Calmodulin Dependent ◽  
Dna Binding Activity

We have demonstrated that the amino terminal domain (GRK5-NT), which includes the RH domain and the region of binding to calmodulin, is able to regulate cardiac hypertrophy through inhibition of NFκB. Several studies indicate that mechanical stress and neuro-hormonal activation (Angiotensin, phenylephrine, endothelin) increase the levels of intracellular calcium, therefore activating calcineurin, a serine/threonine phosphatase that dephosphorylates nuclear factor of activated T cells (NFAT). Aim of this study is to evaluate the possibility that GRK5-NT regulates calcium-calmodulin dependent transcription factors. To this aim, we infected cardiomyoblasts in culture with an adenovirus encoding for GRK5-NT (AdGRK5-NT) or treated with a synthetic protein (TAT-RH), which reproduce the only RH domain of GRK5. Hypertrophic responses were induced by chronic stimulation of α 1 adrenergic receptors with phenylephrine (PE 10 -7 M,24h) and the levels of GATA4 and NFAT3 were assessed by western blot. PE induces activation and nuclear translocation of GATA4 and NFAT3 (NFAT:+38±3%;GATA4:+46±3% vs control), the treatment with AdGRK5-NT, but TAT-RH, reduces this effect (NFAT:-68±5%;GATA4:-56±2% vs PE). These data suggest that GRK5-NT using the NT domain regulates the activation of calcium-calmodulin dependent transcription factors through a mechanism of competition for binding to calmodulin. To confirm these data, we evaluated the effect of GRK5-NT in vivo in spontaneously hypertensive and hypertrophic rats (SHR). The overexpression of GRK5-NT in the heart was induced by intracardiac injection of 10 10 pfu/ml of AdGRK5-NT. The treatment inhibits nuclear translocation of NFAT3 and GATA4 (NFAT:-55±1%;GATA4:-34±3% vs PE) and is associated with a reduction in their DNA binding activity, as assessed by EMSA (NFAT:-47±1.5%;GATA4:-33±1% vs EP). In conclusion, our data indicate that GRK5-NT is able to regulate cardiac hypertrophy in two ways: inhibition of NFκB through binding and stabilization of IκB nuclear and inhibition activity of calcium- calmodulin dependent transcription factors, possibly by competing with calmodulin binding.

scholarly journals Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells.

1994 ◽  
Vol 14 (8) ◽  
pp. 5309-5317 ◽  
Author(s):  
S P Murphy ◽  
J J Gorzowski ◽  
K D Sarge ◽  
B Phillips
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Embryonal Carcinoma ◽  
Embryonic Stem ◽  
Binding Activity ◽  
Hsp70 Promoter ◽  
Ec Cells

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

scholarly journals Cysteine 64 of Ref-1 Is Not Essential for Redox Regulation of AP-1 DNA Binding

2003 ◽  
Vol 23 (12) ◽  
pp. 4257-4266 ◽  
Author(s):  
Jared M. Ordway ◽  
Derek Eberhart ◽  
Tom Curran
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Redox Regulation ◽  
Binding Activity ◽  
Regulation Of Transcription ◽  
Binding Domains ◽  
Dna Binding Activity ◽  
Reducing Activity

ABSTRACT Ref-1 participates in DNA repair as well as in redox regulation of transcription factor function. The redox function of Ref-1 involves reduction of oxidized cysteine residues within the DNA binding domains of several transcription factors, including Fos and Jun. Reduction of these residues is required for DNA binding, providing a redox-dependent mechanism for regulation of target gene expression. Previous in vitro studies implicated cysteine 65 of human Ref-1 (cysteine 64 of mouse Ref-1) as the redox catalytic site. We analyzed the in vivo role of cysteine 64 in redox regulation of AP-1 activity by introducing a cysteine-to-alanine point mutation into the endogenous mouse Ref-1 gene (ref-1 C64A). Unlike Ref-1 null mice, which die very early in embryonic development, homozygous ref-1 C64A mice are viable, they survive to normal life expectancy, and they display no overt abnormal phenotype. Although Ref-1 provides the major AP-1-reducing activity in murine cells, ref-1 C64A cells retain normal levels of endogenous AP-1 DNA binding activity in vivo as well as normal Fos- and Jun-reducing activity in vitro. These results demonstrate that Ref-1 cysteine 64/65 is not required for redox regulation of AP-1 DNA binding in vivo, and they challenge previous hypotheses regarding the mechanism by which Ref-1 regulates the redox-dependent activity of specific transcription factors.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

scholarly journals Direct inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furan-benzimidazole dication

2008 ◽  
Vol 36 (10) ◽  
pp. 3341-3353 ◽  
Author(s):  
Paul Peixoto ◽  
Yang Liu ◽  
Sabine Depauw ◽  
Marie-Paule Hildebrand ◽  
David W. Boykin ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Activity ◽  
Direct Inhibition ◽  
Selective Binding ◽  
Dna Binding Activity
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