scholarly journals The DNA-binding properties of two heat shock factors, HSF1 and HSF3, are induced in the avian erythroblast cell line HD6.

1995 ◽  
Vol 15 (10) ◽  
pp. 5268-5278 ◽  
Author(s):  
A Nakai ◽  
Y Kawazoe ◽  
M Tanabe ◽  
K Nagata ◽  
R I Morimoto
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Cell Line ◽  
Biological Properties ◽  
Binding Activity ◽  
Heat Shock Factors ◽  
Reporter Construct ◽  
Heat Shock Element ◽  
Dna Binding Activity ◽  
Dna Binding Properties

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. HSF 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.

Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells

1992 ◽  
Vol 12 (9) ◽  
pp. 4104-4111
Author(s):  
L Sistonen ◽  
K D Sarge ◽  
B Phillips ◽  
K Abravaya ◽  
R I Morimoto
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Binding Activity ◽  
Erythroid Cell ◽  
Hsp70 Gene ◽  
Mobility Shift ◽  
Heat Shock Element ◽  
Dna Binding Activity ◽  
Erythroleukemia Cells

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

scholarly journals Inhibition of DNA Binding by Differential Sumoylation of Heat Shock Factors

2006 ◽  
Vol 26 (3) ◽  
pp. 955-964 ◽  
Author(s):  
Julius Anckar ◽  
Ville Hietakangas ◽  
Konstantin Denessiouk ◽  
Dennis J. Thiele ◽  
Mark S. Johnson ◽  
...  
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Regulation Of Gene Expression ◽  
Binding Activity ◽  
Covalent Modification ◽  
Heat Shock Factors ◽  
Substrate Selection ◽  
Binding Domains ◽  
Dna Binding Activity ◽  
Specific Regulation

ABSTRACT Covalent modification of proteins by the small ubiquitin-related modifier SUMO regulates diverse biological functions. Sumoylation usually requires a consensus tetrapeptide, through which the binding of the SUMO-conjugating enzyme Ubc9 to the target protein is directed. However, additional specificity determinants are in many cases required. To gain insights into SUMO substrate selection, we have utilized the differential sumoylation of highly similar loop structures within the DNA-binding domains of heat shock transcription factor 1 (HSF1) and HSF2. Site-specific mutagenesis in combination with molecular modeling revealed that the sumoylation specificity is determined by several amino acids near the consensus site, which are likely to present the SUMO consensus motif to Ubc9. Importantly, we also demonstrate that sumoylation of the HSF2 loop impedes HSF2 DNA-binding activity, without affecting its oligomerization. Hence, SUMO modification of the HSF2 loop contributes to HSF-specific regulation of DNA binding and broadens the concept of sumoylation in the negative regulation of gene expression.

scholarly journals Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells.

1992 ◽  
Vol 12 (9) ◽  
pp. 4104-4111 ◽  
Author(s):  
L Sistonen ◽  
K D Sarge ◽  
B Phillips ◽  
K Abravaya ◽  
R I Morimoto
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Binding Activity ◽  
Erythroid Cell ◽  
Hsp70 Gene ◽  
Mobility Shift ◽  
Heat Shock Element ◽  
Dna Binding Activity ◽  
Erythroleukemia Cells

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

Interaction between heat shock factor and hsp70 is insufficient to suppress induction of DNA-binding activity in vivo

1994 ◽  
Vol 14 (10) ◽  
pp. 6552-6560
Author(s):  
S K Rabindran ◽  
J Wisniewski ◽  
L Li ◽  
G C Li ◽  
C Wu
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Heat Shock Proteins ◽  
Expression System ◽  
Stress Protein ◽  
Binding Activity ◽  
Protein Family ◽  
Oligomeric State ◽  
Dna Binding Activity ◽  
Shock Proteins

The intracellular level of free heat shock proteins, in particular the 70-kDa stress protein family, has been suggested to be the basis of an autoregulatory mechanism by which the cell measures the level of thermal stress and regulates the synthesis of heat shock proteins. It has been proposed that the DNA-binding and oligomeric state of the heat shock transcription factor (HSF) is a principal step in the induction pathway that is responsive to the level of 70-kDa stress protein. To test this hypothesis, we investigated the association between HSF and 70-kDa stress protein by means of a coimmunoprecipitation assay. We found that 70-kDa stress proteins associate to similar extents with both latent and active forms of HSF, although unlike other 70-kDa stress protein substrates, the association with HSF was not significantly disrupted in the presence of ATP. Gel mobility shift assays indicated that active HSF trimers purified from a bacterial expression system could not be substantially deactivated in vitro with purified 70-kDa stress protein and ATP. In addition, elevated concentrations of hsp70 alone could not significantly inhibit induction of the DNA-binding activity of endogenous HSF in cultured rat cells, and the induction was also not inhibited in cultured rat cells or Drosophila cells containing elevated levels of all members of the heat shock protein family. However, the deactivation of HSF to the non-DNA-binding state after prolonged heat stress or during recovery could be accelerated by increased levels of heat shock proteins. Hence, the level of heat shock proteins may affect the rate of disassembly of HSF trimers, but another mechanism, as yet undefined, appears to control the onset of the oligomeric transitions.

scholarly journals The specific DNA binding activity of the dioxin receptor is modulated by the 90 kd heat shock protein.

1990 ◽  
Vol 9 (1) ◽  
pp. 69-76 ◽  
Author(s):  
A. Wilhelmsson ◽  
S. Cuthill ◽  
M. Denis ◽  
A.C. Wikström ◽  
J.A. Gustafsson ◽  
...  
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Heat Shock Protein ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Dioxin Receptor

Over-Expression of the P65 Subunit of NF-kB and Increased DNA Binding Activity of NF-kB Is a Common Event Both in Philadelphia Positive and Negative Chronic Myeloproliferative Disorders.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4759-4759
Author(s):  
Alessandro Morotti ◽  
Veronica Ullmannova ◽  
Daniela Cilloni ◽  
Francesca Messa ◽  
Manuela Messa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Cell Growth ◽  
Dna Binding ◽  
Western Blot ◽  
Cell Line ◽  
Myeloproliferative Disorders ◽  
Binding Activity ◽  
K562 Cell Line ◽  
Dna Binding Activity

Abstract The chronic myeloproliferative diseases (CMPD) are clonal disorders characterized by increased proliferation of cells from one or more myeloid lineages. The most common CMPD is Chronic Myeloid Leukemia which is characterized by the Philadelphia t(9;22) chromosomal translocation. The pathogenesis of Philadelphia negative CMPD is poorly understood, although the activation of tyrosine kinases appears to be an essential feature. For example, a constitutively activated PDGF receptor tyrosine kinase (FIP1L1-PDGFRA) is involved in some cases of the hypereosinophilic syndrome (HES), which is a form of CMPD characterized by increased proliferation of eosinophils. Different reports have demonstrated that the transcription factor NF-kB is essential for Bcr-Abl mediated transformation. NF-kB is a transcription factor which is composed of two subunits (generally p65 and p50). NF-kB dimers are retained into the cytoplasm by the inhibitory protein IkB. Different stimuli trigger the Serine phosphorylation of IkB and its proteolitc degradation. Free NF-kB translocates into the nucleus where it mediates the transcription of different genes involved in cellular proliferation, transformation and in apoptosis resistance. The aim of this work is to evaluate whether NF-kB is active both in Ph positive and in Ph negative CMPD. Bone marrow samples of 8 myeloproliferative disorders (3 Philadelphia positive CML, 3 Ph negative CML-like, 1 HES, 1 Idiopathic Myelofibrosis) have been collected at the diagnosis. The t(9;22) positive K562 cell line, derived form a CML blast crysis, has been used as a positive cellular control of the following experiments. Each samples have been lysed to obtain cytosolic and nuclear extracts. Western blot have been performed to evaluate the expression of the p65 subunit of NF-kB, the regulatory protein IkB and the antiapoptotic protein Bcl-2, whose expression may be regulated by NF-kB. Subsequently the DNA binding activity of NF-kB have been measured with an ELISA method. Our data shows that in all samples p65 is over-expressed both in the cytosol and in the nucleus respect to normal peripheral blood and normal bone marrow samples. The antiapoptotic Bcl-2 is also detectable by western blot in all pathological samples. In normal samples IkB is detected only in the cytosol and not in the nucleus while in CMPD samples it is expressed predominately in the nucleus. Basal DNA binding activity of NF-kB is increased in all the nuclear samples but not in normal samples. To assess whether NF-kB is directly involved in the control of cell growth and apoptosis, we have developed a stable K562 cell line expressing the super repressor IkB. The SR-IkB is a mutated for of IkB which can not be degraded causing an cytosolic sequestration of NF-kB. This stable cell line has a marked reduction of cell growth and is more sensible to the apoptotic stimuli. The data described above suggest that NF-kB activation may be a common mechanism of transformation in both Ph positive and negative CMPD and that its inhibition may be a powerful targeted molecular therapy.

Unusual levels of heat shock element-binding activity in embryonal carcinoma cells

1989 ◽  
Vol 9 (9) ◽  
pp. 3888-3896
Author(s):  
V Mezger ◽  
O Bensaude ◽  
M Morange
Keyword(s):  
Heat Shock ◽  
Cell Line ◽  
Gene Transcription ◽  
Embryonal Carcinoma ◽  
Binding Activity ◽  
In Vitro Differentiation ◽  
Heat Shock Element ◽  
Heat Induction ◽  
Ec Cells

In contrast to differentiated somatic cells, mouse embryonal carcinoma (EC) cell lines spontaneously express high levels of major members of the heat shock protein (HSP) family. In addition, some EC cell lines (noninducible) are not able to induce HSP gene transcription and HSP synthesis after a stress. However, after in vitro differentiation, constitutive HSP expression decreases and the differentiated derivatives become able to induce HSP gene transcription after a stress. These cells were tested by gel shift assays for the presence of an activity able to bind the heat shock element (HSE) before and after a stress. Control fibroblasts grown at 37 degrees C did not contain significant levels of HSE-binding activity, but heat shock dramatically increased the level of HSE-binding activity. In contrast to control fibroblasts, all EC cells contained significant levels of HSE-binding activity at 37 degrees C. In the inducible EC cell line F9, as in fibroblasts, heat shock strongly increased the level of HSE-binding activity. In the noninducible EC cells, however, HSE-binding activity markedly decreased upon heat shock. During in vitro differentiation of the noninducible cell line PCC7-S-1009, the constitutive HSE-binding activity found at 37 degrees C disappeared and heat induction of the HSE-binding activity appeared. Therefore, a good correlation exists between the high spontaneous expression of some members of the HSP family and the constitutive level of HSE-binding activity in EC cells at 37 degrees C. Heat induction of HSP gene transcription correlates with a strong increase in HSE-binding activity, whereas a deficiency in heat induction of HSP gene transcription is associated with a loss of HSE-binding activity upon heat shock.

DNA-binding properties of the E1A-associated 300-kilodalton protein

1992 ◽  
Vol 12 (6) ◽  
pp. 2826-2836
Author(s):  
Y Rikitake ◽  
E Moran
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Biochemical Properties ◽  
Binding Activity ◽  
Physical Interaction ◽  
Nf Kappa B ◽  
Biochemical Basis ◽  
Dna Binding Activity ◽  
P300 Binding ◽  
Dna Binding Properties

One of the major E1A-associated cellular proteins is a 300-kDa product (p300) that binds to the N-terminal region of the E1A products. The p300 binding site is distinct from sequences involved in binding the retinoblastoma product and other E1A-associated cellular products such as p60-cyclin A and p107. p300 binding to E1A is linked genetically to the enhancer repression function of E1A and the other E1A-mediated gene-regulating functions as well as to the transforming functions of E1A. However, the biochemical properties of p300 have not yet been characterized. We report here that p300 has an intrinsic DNA-binding activity and shows a preferential affinity for specific DNA sequences. The sequences selectively bound by p300 are related to those of a series of enhancer elements that are recognized by NF-kappa B. The direct physical interaction of p300 with enhancer elements provides a biochemical basis for the genetic evidence linking the E1A-mediated enhancer repression function with the p300-binding activity of E1A.

Down-regulation of nuclear aryl hydrocarbon receptor DNA-binding and transactivation functions: requirement for a labile or inducible factor

1994 ◽  
Vol 14 (9) ◽  
pp. 5653-5660
Author(s):  
M Reick ◽  
R W Robertson ◽  
D S Pasco ◽  
J B Fagan
Keyword(s):  
Dna Binding ◽  
Cell Line ◽  
Binding Activity ◽  
Ah Receptor ◽  
Down Regulation ◽  
Response Elements ◽  
Dna Binding Activity ◽  
Nuclear Extracts ◽  
Xenobiotic Response

Aryl hydrocarbons (AHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene activate the sequence-specific DNA-binding activity of the AH receptor. In the rat hepatocyte-derived cell line LCS7, DNA-binding activity peaked after 30 min and was then down-regulated, reaching negligible levels by 2 h. Down-regulation could be blocked, and DNA-binding activity maintained at maximum for many hours by inhibiting protein or RNA synthesis, implying that down-regulation is a mediated process requiring a labile or inducible protein. CYP1A1 transcription and in vivo DNA-protein interactions at xenobiotic response elements were down-regulated in parallel with DNA-binding activity in nuclear extracts, and these changes could also be blocked by inhibitors of protein synthesis. The correlation between AH receptor DNA-binding activity, intensity of in vivo footprints at xenobiotic response elements, and CYP1A1 transcription rate implies that down-regulation of AH receptor DNA-binding activity is important in regulating CYP1A1 transcription and that receptor is required continuously to maintain transcription. This correlation extends to the murine hepatoma cell line Hepa-1c1c7, in which slower kinetics of activation and down-regulation of CYP1A1 transcription paralleled slower activation and down-regulation of AH receptor DNA-binding activity. The difference in kinetics between cell lines also implies that AH receptor DNA-binding activity is modulated by a mechanism that may be influenced by cell-specific regulatory pathways. The above observations in conjunction with mixing experiments and comparisons of cytoplasmic and nuclear extracts indicate that down-regulation of AH receptor DNA-binding activity is probably due either to degradation or to conversion of the receptor to form that is inactive in both DNA binding and transactivation.

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