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

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/.

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

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.

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

Heat 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.

Genomic Heat Shock Element Sequences Drive Cooperative Human Heat Shock Factor 1 DNA Binding and Selectivity

The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer.