Heat shock factor can activate transcription while bound to nucleosomal DNA in Saccharomyces cerevisiae

1994 ◽  
Vol 14 (1) ◽  
pp. 189-199
Author(s):  
D S Pederson ◽  
T Fidrych
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Transcription Initiation ◽  
Heat Shock Factor ◽  
Micrococcal Nuclease ◽  
Nucleosome Assembly ◽  
Heat Shock Element ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nucleosomal Dna

After each round of replication, new transcription initiation complexes must assemble on promoter DNA. This process may compete with packaging of the same promoter sequences into nucleosomes. To elucidate interactions between regulatory transcription factors and nucleosomes on newly replicated DNA, we asked whether heat shock factor (HSF) could be made to bind to nucleosomal DNA in vivo. A heat shock element (HSE) was embedded at either of two different sites within a DNA segment that directs the formation of a stable, positioned nucleosome. The resulting DNA segments were coupled to a reporter gene and transfected into the yeast Saccharomyces cerevisiae. Transcription from these two plasmid constructions after induction by heat shock was similar in amount to that from a control plasmid in which HSF binds to nucleosome-free DNA. High-resolution genomic footprint mapping of DNase I and micrococcal nuclease cleavage sites indicated that the HSE in these two plasmids was, nevertheless, packaged in a nucleosome. The inclusion of HSE sequences within (but relatively close to the edge of) the nucleosome did not alter the position of the nucleosome which formed with the parental DNA fragment. Genomic footprint analyses also suggested that the HSE-containing nucleosome was unchanged by the induction of transcription. Quantitative comparisons with control plasmids ruled out the possibility that HSF was bound only to a small fraction of molecules that might have escaped nucleosome assembly. Analysis of the helical orientation of HSE DNA in the nucleosome indicated that HSF contacted DNA residues that faced outward from the histone octamer. We discuss the significance of these results with regard to the role of nucleosomes in inhibiting transcription and the normal occurrence of nucleosome-free regions in promoters.

scholarly journals Heat shock factor can activate transcription while bound to nucleosomal DNA in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (1) ◽  
pp. 189-199 ◽  
Author(s):  
D S Pederson ◽  
T Fidrych
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Transcription Initiation ◽  
Heat Shock Factor ◽  
Micrococcal Nuclease ◽  
Nucleosome Assembly ◽  
Heat Shock Element ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nucleosomal Dna

After each round of replication, new transcription initiation complexes must assemble on promoter DNA. This process may compete with packaging of the same promoter sequences into nucleosomes. To elucidate interactions between regulatory transcription factors and nucleosomes on newly replicated DNA, we asked whether heat shock factor (HSF) could be made to bind to nucleosomal DNA in vivo. A heat shock element (HSE) was embedded at either of two different sites within a DNA segment that directs the formation of a stable, positioned nucleosome. The resulting DNA segments were coupled to a reporter gene and transfected into the yeast Saccharomyces cerevisiae. Transcription from these two plasmid constructions after induction by heat shock was similar in amount to that from a control plasmid in which HSF binds to nucleosome-free DNA. High-resolution genomic footprint mapping of DNase I and micrococcal nuclease cleavage sites indicated that the HSE in these two plasmids was, nevertheless, packaged in a nucleosome. The inclusion of HSE sequences within (but relatively close to the edge of) the nucleosome did not alter the position of the nucleosome which formed with the parental DNA fragment. Genomic footprint analyses also suggested that the HSE-containing nucleosome was unchanged by the induction of transcription. Quantitative comparisons with control plasmids ruled out the possibility that HSF was bound only to a small fraction of molecules that might have escaped nucleosome assembly. Analysis of the helical orientation of HSE DNA in the nucleosome indicated that HSF contacted DNA residues that faced outward from the histone octamer. We discuss the significance of these results with regard to the role of nucleosomes in inhibiting transcription and the normal occurrence of nucleosome-free regions in promoters.

Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae

1987 ◽  
Vol 7 (5) ◽  
pp. 1906-1916
Author(s):  
M R Slater ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Promoter Region ◽  
Inducible Expression ◽  
Proximal Promoter ◽  
Heat Shock Gene ◽  
Heat Shock Element ◽  
Yeast Saccharomyces Cerevisiae ◽  
Hybrid Promoter ◽  
Shock Gene

The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100. The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.

scholarly journals Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.

1992 ◽  
Vol 12 (8) ◽  
pp. 3490-3498 ◽  
Author(s):  
N Hosokawa ◽  
K Hirayoshi ◽  
H Kudo ◽  
H Takechi ◽  
A Aoike ◽  
...  
Keyword(s):  
Heat Shock ◽  
Transcriptional Activation ◽  
Heat Shock Factor ◽  
Mobility Shift ◽  
Human Colon Cancer ◽  
Heat Shock Element ◽  
Mrna Accumulation ◽  
Cell Extracts

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.

scholarly journals Regulation of heat shock factor in Schizosaccharomyces pombe more closely resembles regulation in mammals than in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (1) ◽  
pp. 281-288 ◽  
Author(s):  
G J Gallo ◽  
T J Schuetz ◽  
R E Kingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Dna Binding ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Heat Shock Response ◽  
Heat Shock Factor ◽  
Regulatory Sequence ◽  
Heat Shock Element ◽  
Shock Response

The heat shock response appears to be universal. All eucaryotes studied encode a protein, heat shock factor (HSF), that is believed to regulate transcription of heat shock genes. This protein binds to a regulatory sequence, the heat shock element, that is absolutely conserved among eucaryotes. We report here the identification of HSF in the fission yeast Schizosaccharomyces pombe. HSF binding was not observed in extracts from normally growing S. pombe (28 degrees C) but was detected in increasing amounts as the temperature of heat shock increased between 39 and 45 degrees C. This regulation is in contrast to that observed in Saccharomyces cerevisiae, in which HSF binding is detectable at both normal and heat shock temperatures. The S. pombe factor bound specifically to the heat shock element, as judged by methylation interference and DNase I protection analysis. The induction of S. pombe HSF was not inhibited by cycloheximide, suggesting that induction occurs posttranslationally, and the induced factor was shown to be phosphorylated. S. pombe HSF was purified to near homogeneity and was shown to have an apparent mobility of approximately 108 kDa. Since heat-induced DNA binding by HSF had previously been demonstrated only in metazoans, the conservation of heat-induced DNA binding by HSF among S. pombe and metazoans suggests that this mode of regulation is evolutionarily ancient.

scholarly journals Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (5) ◽  
pp. 1906-1916 ◽  
Author(s):  
M R Slater ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Promoter Region ◽  
Inducible Expression ◽  
Proximal Promoter ◽  
Heat Shock Gene ◽  
Heat Shock Element ◽  
Yeast Saccharomyces Cerevisiae ◽  
Hybrid Promoter ◽  
Shock Gene

The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100. The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.

scholarly journals Cooperative Binding of Heat Shock Factor to the Yeast HSP82 Promoter In Vivo and In Vitro

1999 ◽  
Vol 19 (3) ◽  
pp. 1627-1639 ◽  
Author(s):  
Alexander M. Erkine ◽  
Serena F. Magrogan ◽  
Edward A. Sekinger ◽  
David S. Gross
Keyword(s):  
Heat Shock ◽  
Single Point ◽  
Heat Shock Factor ◽  
Single Point Mutation ◽  
Heat Shock Element ◽  
Cooperative Interactions ◽  
Before And After ◽  
Order Of Magnitude

ABSTRACT Previous work has shown that heat shock factor (HSF) plays a central role in remodeling the chromatin structure of the yeastHSP82 promoter via constitutive interactions with its high-affinity binding site, heat shock element 1 (HSE1). The HSF-HSE1 interaction is also critical for stimulating both basal (noninduced) and induced transcription. By contrast, the function of the adjacent, inducibly occupied HSE2 and -3 is unknown. In this study, we examined the consequences of mutations in HSE1, HSE2, and HSE3 on HSF binding and transactivation. We provide evidence that in vivo, HSF binds to these three sites cooperatively. This cooperativity is seen both before and after heat shock, is required for full inducibility, and can be recapitulated in vitro on both linear and supercoiled templates. Quantitative in vitro footprinting reveals that occupancy of HSE2 and -3 by Saccharomyces cerevisiae HSF (ScHSF) is enhanced ∼100-fold through cooperative interactions with the HSF-HSE1 complex. HSE1 point mutants, whose basal transcription is virtually abolished, are functionally compensated by cooperative interactions with HSE2 and -3 following heat shock, resulting in robust inducibility. Using a competition binding assay, we show that the affinity of recombinant HSF for the full-length HSP82promoter is reduced nearly an order of magnitude by a single-point mutation within HSE1, paralleling the effect of these mutations on noninduced transcript levels. We propose that the remodeled chromatin phenotype previously shown for HSE1 point mutants (and lost in HSE1 deletion mutants) stems from the retention of productive, cooperative interactions between HSF and its target binding sites.

Basal-level expression of the yeast HSP82 gene requires a heat shock regulatory element

1989 ◽  
Vol 9 (11) ◽  
pp. 4789-4798
Author(s):  
D McDaniel ◽  
A J Caplan ◽  
M S Lee ◽  
C C Adams ◽  
B R Fishel ◽  
...  
Keyword(s):  
Heat Shock ◽  
Regulatory Element ◽  
Heat Shock Factor ◽  
Tata Box ◽  
Hypersensitive Site ◽  
Heat Shock Element ◽  
Hsp82 Promoter ◽  
Basal Level Expression

Previous studies have shown that heat shock factor is constitutively bound to heat shock elements in Saccharomyces cerevisiae. We demonstrate that mutation of the heat shock element closest to the TATA box of the yeast HSP82 promoter abolishes basal-level transcription without markedly affecting inducibility. The mutated heat shock element no longer bound putative heat shock factor, either in vitro or in vivo, but still resided within a nuclease-hypersensitive site in the chromatin. Thus, constitutive binding of heat shock factor to heat shock elements in S. cerevisiae appears to functionally direct basal-level transcription.

scholarly journals A novel 66-kilodalton protein complexes with Rrn6, Rrn7, and TATA-binding protein to promote polymerase I transcription initiation in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (11) ◽  
pp. 6436-6443 ◽  
Author(s):  
C W Lin ◽  
B Moorefield ◽  
J Payne ◽  
P Aprikian ◽  
K Mitomo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Protein Complexes ◽  
Binding Protein ◽  
Strong Association ◽  
Tata Binding Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pol I ◽  
Polymerase I

We report the cloning of RRN11, a gene coding for a 66-kDa protein essential for transcription initiation by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. Rrn11 specifically complexes with two previously identified transcription factors, Rrn6 and Rrn7 (D. A. Keys, J. S. Steffan, J. A. Dodd, R. T. Yamamoto, Y. Nogi, and M. Nomura, Genes Dev. 8:2349-2362, 1994). The Rrn11-Rrn6-Rrn7 complex also binds the TATA-binding protein and is required for transcription by the core domain of the Pol I promoter. Therefore, we have designated the Rrn11-Rrn6-Rrn7-TATA-binding protein complex the yeast Pol I core factor. A two-hybrid assay was used to demonstrate involvement of short leucine heptad repeats on both Rrn11 and Rrn6 in the in vivo association of these two proteins. This assay also verified the previously described strong association between Rrn6 and Rrn7, independent of the Rrn6 leucine repeat.

scholarly journals HSP90 Interacts with and Regulates the Activity of Heat Shock Factor 1 in Xenopus Oocytes

1998 ◽  
Vol 18 (9) ◽  
pp. 4949-4960 ◽  
Author(s):  
Adnan Ali ◽  
Steven Bharadwaj ◽  
Ruth O’Carroll ◽  
Nick Ovsenek
Keyword(s):  
Heat Shock ◽  
Transcriptional Activation ◽  
Cellular Signaling ◽  
Heat Shock Factor ◽  
Heat Shock Factor 1 ◽  
Heat Shock Element ◽  
Nuclear Extracts ◽  
Multistep Process

ABSTRACT Transcriptional activation of heat shock genes is a reversible and multistep process involving conversion of inactive heat shock factor 1 (HSF1) monomers into heat shock element (HSE)-binding homotrimers, hyperphosphorylation, and further modifications that induce full transcriptional competence. HSF1 is controlled by multiple regulatory mechanisms, including suppression by additional cellular factors, physical interactions with HSP70, and integration into different cellular signaling cascades. However, the signaling mechanisms by which cells respond to stress and control the HSF1 activation-deactivation pathway are not known. Here we demonstrate that HSP90, a cellular chaperone known to regulate several signal transduction molecules and transcription factors, functions in the regulation of HSF1. The existence of HSF1-HSP90 heterocomplexes was shown by coimmunoprecipitation of HSP90 with HSF1 from unshocked and heat-shocked nuclear extracts, recognition of HSF1-HSE complexes in vitro by using HSP90 antibodies (Abs), and recognition of HSF1 in vivo by HSP90 Abs microinjected directly into oocyte nuclei. The functional impact of HSP90-HSF1 interactions was analyzed by using two strategies: direct nuclear injection of HSP90 Abs and treatment of cells with geldanamycin (GA), an agent that specifically blocks the chaperoning activity of HSP90. Both HSP90 Abs and GA delayed the disassembly of HSF1 trimers during recovery from heat shock and specifically inhibited heat-induced transcription from a chloramphenicol acetyltransferase reporter construct under control of the hsp70 promoter. HSP90 Abs activated HSE binding in the absence of heat shock, an effect that could be reversed by subsequent injection of purified HSP90. GA did not activate HSE binding under nonshock conditions but increased the quantity of HSE binding induced by heat shock. On the basis of these findings and the known properties of HSP90, we propose a new regulatory model in which HSP90 participates in modulating HSF1 at different points along the activation-deactivation pathway, influencing the interconversion between monomeric and trimeric conformations as well as transcriptional activation. We also put forth the hypothesis that HSP90 links HSF1 to cellular signaling molecules coordinating the stress response.

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