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

2018 ◽  
Vol 29 (26) ◽  
pp. 3168-3182 ◽  
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.

scholarly journals Genetic and Epigenetic Determinants Establish a Continuum of Hsf1 Occupancy and Activity Across the Yeast Genome

2018 ◽  
Author(s):  
David Pincus ◽  
Jayamani Anandhakumar ◽  
Prathapan Thiru ◽  
Michael J. Guertin ◽  
Alexander M. Erkine ◽  
...  
Keyword(s):  
Heat Shock ◽  
Nucleosome Occupancy ◽  
Fold Increase ◽  
Yeast Genome ◽  
Heat Shock Factor 1 ◽  
Rna Seq ◽  
Heat Shock Element ◽  
Pioneer Factors ◽  
Accessible Chromatin

AbstractHeat Shock Factor 1 (Hsf1) is the master transcriptional regulator of molecular chaperones and binds to the same cis-acting element - Heat Shock Element (HSE) - across the eukaryotic lineage. In budding yeast, Hsf1 drives 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 ChIP-seq, nascent RNA-seq and Hsf1 nuclear depletion to quantify Hsf1 binding and transcription across the yeast genome. Hsf1 binds 74 loci during acute heat shock, 46 of which are linked to genes with strong Hsf1-dependent transcription. Most of these targets show detectable Hsf1 binding under basal conditions, but basal occupancy and heat shock-inducible binding both vary over two orders of magnitude. Notably, Hsf1’s induced DNA binding leads to a disproportionate (up to 50-fold) increase in nascent transcription. While variation in basal Hsf1 occupancy poorly correlates with the strength of the HSE, promoters with high basal Hsf1 occupancy have nucleosome-depleted regions due to the presence of ‘pioneer’ factors. Such accessible chromatin may be critical for Hsf1 occupancy of its genomic sites as the activator is incapable of binding HSEs embedded within a stable nucleosome in vitro. In response to heat shock, however, Hsf1 is able to gain access to nucleosomal sites and promotes chromatin remodeling with the RSC complex playing a key role. We propose that the interplay between nucleosome occupancy, HSE strength and active Hsf1 levels allows cells to precisely tune expression of the proteostasis network.

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

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.

scholarly journals Expression of a constitutively active mutant of heat shock factor 1 under the control of testis-specific hst70 gene promoter in transgenic mice induces degeneration of seminiferous epithelium.

2003 ◽  
Vol 50 (2) ◽  
pp. 535-541 ◽  
Author(s):  
Wiesława Widłak ◽  
Konrad Benedyk ◽  
Natallia Vydra ◽  
Magdalena Głowala ◽  
Dorota Scieglińska ◽  
...  
Keyword(s):  
Heat Shock ◽  
Transgenic Mice ◽  
Target Genes ◽  
Gene Promoter ◽  
Basic Mechanism ◽  
Seminiferous Epithelium ◽  
High Rate ◽  
Heat Shock Factor 1 ◽  
Heat Shock Element ◽  
Constitutively Active

Heat shock activates in somatic cells a set of genes encoding heat shock proteins which function as molecular chaperones. The basic mechanism by which these genes are activated is the interaction of the specific transcription factor HSF1 with a regulatory DNA sequence called heat shock element (HSE). In higher eukaryotes HSF1 is present in unstressed cells as inactive monomers which, in response to cellular stress, aggregate into transcriptionally competent homotrimers. In the present paper we showed that the expression of a transgene encoding mutated constitutively active HSF1 placed under the control of a spermatocyte-specific promoter derived from the hst70 gene severely affects spermatogenesis. We found the testes of transgenic mice to be significantly smaller than those of wild-type males and histological analysis showed massive degeneration of the seminiferous epithelium. The lumen of tubules was devoid of spermatids and spermatozoa and using the TUNEL method we demonstrated a high rate of spermatocyte apoptosis. The molecular mechanism by which constitutively active HSF1 arrests spermatogenesis is not known so far. One can assume that HSF1 can either induce or repress so far unknown target genes involved in germ cell apoptosis.

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

1995 ◽  
Vol 15 (12) ◽  
pp. 6754-6769 ◽  
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.

scholarly journals ChIP-seq and RNA-seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy

2018 ◽  
Author(s):  
Noémie Vimont ◽  
Fu Xiang Quah ◽  
David Guillaume-Schöpfer ◽  
François Roudier ◽  
Elisabeth Dirlewanger ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Immunoprecipitation ◽  
Sweet Cherry ◽  
Prunus Persica ◽  
Prunus Avium ◽  
Rna Extraction ◽  
Biological Processes ◽  
Mads Box ◽  
Rna Seq ◽  
Chromatin Immunoprecipitation Sequencing

ABSTRACTChromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors, and DNA. This technique in combination with RNA-sequencing (RNA-seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-seq protocol for tree buds (Prunus avium L., Prunus persica L Batch, Malus x domestica Borkh.) that has also been successfully tested on Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. Furthermore, one of the advantages of this protocol is that samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-seq on the same starting material. Focusing on dormant buds in sweet cherry, we explored the link between expression level and H3K4me3 enrichment for all genes, including a strong correlation between H3K4me3 enrichment at the DORMANCY-ASSOCIATED MADS-box 5 (PavDAM5) loci and its expression pattern. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.

scholarly journals ChIP-seq and RNA-seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Noémie Vimont ◽  
Fu Xiang Quah ◽  
David Guillaume Schöepfer ◽  
François Roudier ◽  
Elisabeth Dirlewanger ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Immunoprecipitation ◽  
Sweet Cherry ◽  
Prunus Persica ◽  
Prunus Avium ◽  
Rna Extraction ◽  
Biological Processes ◽  
Mads Box ◽  
Rna Seq ◽  
Chromatin Immunoprecipitation Sequencing

AbstractChromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors and DNA. This technique in combination with RNA-sequencing (RNA-seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-seq protocol for tree buds (Prunus avium L., Prunus persica L Batch, Malus x domestica Borkh.) that has also been successfully tested on Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. Furthermore, one of the advantages of this protocol is that samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-seq on the same starting material. Focusing on dormant buds in sweet cherry, we explored the link between expression level and H3K4me3 enrichment for all genes, including a strong correlation between H3K4me3 enrichment at the DORMANCY-ASSOCIATED MADS-BOX 5 (PavDAM5) loci and its expression pattern. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.

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

2020 ◽  
Vol 15 (3) ◽  
pp. 196-203 ◽  
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/.

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

1993 ◽  
Vol 13 (2) ◽  
pp. 877-890 ◽  
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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