Maximal stress-induced transcription from the human HSP70 promoter requires interactions with the basal promoter elements independent of rotational alignment.

Transcription of the human HSP70 gene is regulated by a complex array of cis-acting promoter elements that respond to conditions that include normal conditions of cell growth and induction following physiological stress. We have examined the requirements of the basal and inducible promoter elements by using promoter mutations and a transient transfection assay. Multiple forms of stress-induced transcription, including heat shock and incubation with heavy metals or amino acid analogs, are mediated by a single heat shock element (HSE) between -105 and -91 consisting of three contiguous 5-base-pair units, NGAAN, that are inverted relative to adjacent units. Maximal inducible expression requires a fully functional basal promoter. Spacing mutations which alter the relative helical orientation of adjacent genetic elements have only minimal effects on basal and stress-inducible expression and show no effects of periodicity. In addition, placement of the HSE adjacent to the basal promoter removes the requirements for a fully functional basal promoter for maximal stress-inducible expression. These results suggest that factors bound at the HSE and the basal promoter can function through multiple interactions.

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Identification of cis and trans components of a novel heat shock stress regulatory pathway in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.248-256.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 248-256

Author(s):

N Kobayashi ◽

K McEntee

Keyword(s):

Saccharomyces Cerevisiae ◽

Stress Response ◽

Heat Shock ◽

Reporter Gene ◽

Heat Shock Element ◽

Cis Acting ◽

Heat Shock Stress ◽

Specific Complex ◽

Cis And Trans ◽

Shock Stress

The stress-responsive DDR2 gene (previously called DDRA2) of Saccharomyces cerevisiae is transcribed at elevated levels following stress caused by heat shock or DNA damage. Previously, we identified a 51-bp promoter fragment, oligo31/32, which conferred heat shock inducibility on the heterologous CYC1-lacZ reporter gene in S. cerevisiae (N. Kobayashi and K. McEntee, Proc. Natl. Acad. Sci. USA 87:6550-6554, 1990). Using a series of synthetic oligonucleotides, we have identified a pentanucleotide, CCCCT (C4T), as an essential component of this stress response sequence. This element is not a binding site for the well-characterized heat shock transcription factor which recognizes a distinct cis-acting heat shock element in the promoters of many heat shock genes. Here we demonstrate the ability of oligonucleotides containing the C4T sequence to confer heat shock inducibility on the reporter gene and show that the presence of two such elements produces more than additive effects on induction. Gel retardation experiments have been used to demonstrate specific complex formation between C4T-containing fragments and one or more yeast proteins. Formation of these complexes was not competed by fragments containing mutations in the C4T sequence nor by heat shock element-containing competitor DNAs. Fragments containing the C4T element bound to a single 140-kDa polypeptide, distinct from heat shock transcription factors in yeast crude extracts. These experiments identify key cis- and trans-acting components of a novel heat shock stress response pathway in S. cerevisiae.

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

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1906-1916.1987 ◽

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.

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A bipartite operator interacts with a heat shock element to mediate early meiotic induction of Saccharomyces cerevisiae HSP82.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6754 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6754-6769 ◽

Cited By ~ 23

Author(s):

C Szent-Gyorgyi

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Point Mutations ◽

Early Step ◽

Heat Shock Element ◽

Cis Acting ◽

Major Mode ◽

Dna Elements ◽

Insight Into ◽

Activation Sequence

Although key genetic regulators of early meiotic transcription in Saccharomyces cerevisiae have been well characterized, the activation of meiotic genes is still poorly understood in terms of cis-acting DNA elements and their associated factors. I report here that induction of HSP82 is regulated by the early meiotic IME1-IME2 transcriptional cascade. Vegetative repression and meiotic induction depend on interactions of the promoter-proximal heat shock element (HSE) with a nearby bipartite repression element, composed of the ubiquitous early meiotic motif, URS1 (upstream repression sequence 1), and a novel ancillary repression element. The ancillary repression element is required for efficient vegetative repression, is spatially separable from URS1, and continues to facilitate repression during sporulation. In contrast, URS1 also functions as a vegetative repression element but is converted early in meiosis into an HSE-dependent activation element. An early step in this transformation may be the antagonism of URS1-mediated repression by IME1. The HSE also nonspecifically supports a second major mode of meiotic activation that does not require URS1 but does require expression of IME2 and concurrent starvation. Interestingly, increased rather than decreased URS1-mediated vegetative transcription can be artificially achieved by introducing rare point mutations into URS1 or by deleting the UME6 gene. These lesions offer insight into mechanisms of URS-dependent repression and activation. Experiments suggest that URS1-bound factors functionally modulate heat shock factor during vegetative transcription and early meiotic induction but not during heat shock. The loss of repression and activation observed when the IME2 activation element, T4C, is substituted for the HSE suggests specific requirements for URS1-upstream activation sequence interactions.

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

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1906 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1906-1916 ◽

Cited By ~ 67

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.

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B-Myb and cyclin D1 mediate heat shock element dependent activation of the human HSP70 promoter

Oncogene ◽

10.1038/sj.onc.1200945 ◽

1997 ◽

Vol 14 (10) ◽

pp. 1223-1229 ◽

Cited By ~ 25

Author(s):

Hiroshi Kamano ◽

Karl-Heinz Klempnauer

Keyword(s):

Heat Shock ◽

Cyclin D1 ◽

Hsp70 Promoter ◽

Heat Shock Element ◽

Human Hsp70

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HSEAT: A Tool for Plant Heat Shock Element Analysis, Motif Identification and Analysis

Current Bioinformatics ◽

10.2174/1574893614666190102151956 ◽

2020 ◽

Vol 15 (3) ◽

pp. 196-203 ◽

Cited By ~ 1

Author(s):

Sarah Rizwan Qazi ◽

Noor ul Haq ◽

Shakeel Ahmad ◽

Samina N. Shakeel

Keyword(s):

Heat Shock ◽

Differential Expression ◽

Promoter Region ◽

Analysis Tool ◽

Promoter Regions ◽

Heat Shock Element ◽

Element Analysis ◽

Cis Acting ◽

Plant Genes ◽

Hsp Genes

Background: Previous methods used to discover cis-regulatory motifs in promoter region of plant genes possess very limited performance, especially for analysis of novel and rare motifs. Different plant genes have differential expression under different environmental or experimental conditions and modular regulation of cis-regulatory sequences in promoter regions of the same or different genes. It has previously been revealed that Heat Shock Proteins (HSPs) creation is correlated with plant tolerance under heat and other stress conditions. Regulation of these HSP genes is controlled by interactions between heat shock factors (HSFs) with cis-acting motifs present in the promoter region of the genes. Differential expression of these HSP genes is because of their unique promoter architecture, cis-acting sequences and their interaction with HSFs. Objective: A versatile promoter analysis tool was proposed for identification and analysis of promoters of HSPs. Methods: Heat Shock Element Analysis Tool (HSEAT) has been implemented in java programming language using pattern recognition approach. This tool has build-in MS access database for storing different motifs. Results: HSEAT has been designed to detect different types of Heat Shock Elements (HSEs) in promoter regions of plant HSPs with integration of complete analysis of plant promoters to the tool. HSEAT is user-friendly, interactive application to discover various types of HSEs e.g. TTC Rich Types, Gap Types and Prefect HSE as well as STRE in HSPs. Here we examined and evaluated some known HSP promoters from different plants using this tool with already available tools. Conclusion: HSEAT has extensive potential to explore conserved or semi-conserved motifs or potential binding sites of different transcription factors for other stress regulating genes. This tool can be found at https://sourceforge.net/projects/heast/.

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The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.877-890.1993 ◽

1993 ◽

Vol 13 (2) ◽

pp. 877-890 ◽

Cited By ~ 1

Author(s):

K Kohno ◽

K Normington ◽

J Sambrook ◽

M J Gething ◽

K Mori

Keyword(s):

Endoplasmic Reticulum ◽

Heat Shock ◽

Mammalian Cells ◽

Yeast Cells ◽

Regulatory Sequences ◽

Upstream Sequence ◽

Unfolded Proteins ◽

Heat Shock Element ◽

Yeast Saccharomyces Cerevisiae ◽

Cis Acting

The endoplasmic reticulum (ER) of eukaryotic cells contains an abundant 78,000-Da protein (BiP) that is involved in the translocation, folding, and assembly of secretory and transmembrane proteins. In the yeast Saccharomyces cerevisiae, as in mammalian cells, BiP mRNA is synthesized at a high basal rate and is further induced by the presence of increased amounts of unfolded proteins in the ER. However, unlike mammalian BiP, yeast BiP is also induced severalfold by heat shock, albeit in a transient fashion. To identify the regulatory sequences that respond to these stimuli in the yeast KAR2 gene that encodes BiP, we have cloned a 1.3-kb segment of DNA from the region upstream of the sequences coding for BiP and fused it to a reporter gene, the Escherichia coli beta-galactosidase gene. Analysis of a series of progressive 5' truncations as well as internal deletions of the upstream sequence showed that the information required for accurate transcriptional regulation of the KAR2 gene in S. cerevisiae is contained within a approximately 230-bp XhoI-DraI fragment (nucleotides -245 to -9) and that this fragment contains at least two cis-acting elements, one (heat shock element [HSE]) responding to heat shock and the other (unfolded protein response element [UPR]) responding to the presence of unfolded proteins in the ER. The HSE and UPR elements are functionally independent of each other but work additively for maximum induction of the yeast KAR2 gene. Lying between these two elements is a GC-rich region that is similar in sequence to the consensus element for binding of the mammalian transcription factor Sp1 and that is involved in the basal expression of the KAR2 gene. Finally, we provide evidence suggesting that yeast cells monitor the concentration of free BiP in the ER and adjust the level of transcription of the KAR2 gene accordingly; this effect is mediated via the UPR element in the KAR2 promoter.

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TATA-dependent and TATA-independent function of the basal and heat shock elements of a human hsp70 promoter

Molecular and Cellular Biology ◽

10.1128/mcb.10.4.1319-1328.1990 ◽

1990 ◽

Vol 10 (4) ◽

pp. 1319-1328

Author(s):

J M Greene ◽

R E Kingston

Keyword(s):

Heat Shock ◽

Consensus Sequence ◽

Simian Virus 40 ◽

Simian Virus ◽

Hsp70 Promoter ◽

Heat Shock Element ◽

Tata Element ◽

Sequence Elements ◽

Human Heat Shock Protein ◽

Human Hsp70

We have characterized the interactions between the TATA element and other sequence elements of a human heat shock protein 70 (hsp70) promoter by a mutational approach. Expression of a distal element of this promoter requires an intact TATA element in human cell lines. The hsp70 TATA element can be functionally replaced for this interaction by TATA elements from the simian virus 40 early and adenovirus EIIa promoters. The TATA element in this promoter therefore both determines the appropriate start site and determines strength by allowing function of the distal element. In contrast, three proximal upstream elements necessary for basal and heat-regulated transcription have no requirement either for a TATA element or for any other proximal element. The behavior of promoters multiply mutant in these proximal elements implies that these elements function independently. We examined the interaction between the heat shock element (HSE) and the TATA element as the distance between the two factor-binding sites was increased. It was necessary to create a mutant HSE with an extended consensus sequence in order for the HSE to function at a distance. Moving this extended HSE 500 bases upstream did not increase its dependence on the TATA element, suggesting that the TATA independence of this element is intrinsic to its function and is not determined by distance from the promoter.

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Genetic and epigenetic determinants establish a continuum of Hsf1 occupancy and activity across the yeast genome

Molecular Biology of the Cell ◽

10.1091/mbc.e18-06-0353 ◽

2018 ◽

Vol 29 (26) ◽

pp. 3168-3182 ◽

Cited By ~ 17

Author(s):

David Pincus ◽

Jayamani Anandhakumar ◽

Prathapan Thiru ◽

Michael J. Guertin ◽

Alexander M. Erkine ◽

...

Keyword(s):

Heat Shock ◽

Chromatin Immunoprecipitation ◽

Fold Increase ◽

Yeast Genome ◽

Heat Shock Factor 1 ◽

Rna Seq ◽

Heat Shock Element ◽

Cis Acting ◽

Chromatin Immunoprecipitation Sequencing ◽

Pioneer Factors

Heat shock factor 1 is the master transcriptional regulator of molecular chaperones and binds to the same cis-acting heat shock element (HSE) across the eukaryotic lineage. In budding yeast, Hsf1 drives the transcription of ∼20 genes essential to maintain proteostasis under basal conditions, yet its specific targets and extent of inducible binding during heat shock remain unclear. Here we combine Hsf1 chromatin immunoprecipitation sequencing (seq), nascent RNA-seq, and Hsf1 nuclear depletion to quantify Hsf1 binding and transcription across the yeast genome. We find that Hsf1 binds 74 loci during acute heat shock, and these are linked to 46 genes with strong Hsf1-dependent expression. Notably, Hsf1’s induced DNA binding leads to a disproportionate (∼7.5-fold) increase in nascent transcription. Promoters with high basal Hsf1 occupancy have nucleosome-depleted regions due to the presence of “pioneer factors.” These accessible sites are likely critical for Hsf1 occupancy as the activator is incapable of binding HSEs within a stably positioned, reconstituted nucleosome. In response to heat shock, however, Hsf1 accesses nucleosomal sites and promotes chromatin disassembly in concert with the Remodels Structure of Chromatin (RSC) complex. Our data suggest that the interplay between nucleosome positioning, HSE strength, and active Hsf1 levels allows cells to precisely tune expression of the proteostasis network.

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