A Novel Non-conventional Heat Shock Element Regulates Expression of MDJ1 Encoding a DnaJ Homolog in Saccharomyces cerevisiae

Abstract

The heat shock factor (HSF) is a pivotal transcriptional factor that regulates the expression of genes encoding heat shock proteins (HSPs) via heat shock elements (HSEs). nGAAnnTTCnnGAAn functions as the minimum consensus HSE (cHSE) in vivo. Here we show that the expression of Saccharomyces cerevisiae MDJ1 encoding a mitochondrial DnaJ homolog is regulated by HSF via a novel non-consensus HSE (ncHSE MDJ1 ), which consists of three separated pentameric nGAAn motifs, nTTCn-(11 bp)-nGAAn-(5 bp)-nGAAn. This is the first evidence to show that the immediate contact of nGAAn motifs is dispensable for regulation by HSF in vivo. ncH-SE MDJ1 confers different heat shock responses versus cHSE and, unlike cHSE, definitively requires a carboxylterminal activation domain of HSF in the expression. ncHSE MDJ1 -like elements are found in promoter regions of some other DnaJ-related genes. The highly conserved HSF/HSE system suggests that similar ncHSEs may be used for the expression of HSP genes in other eukaryotes including humans.All organisms possess a highly conserved system that responds to elevated temperatures by transcriptionally inducing genes encoding heat shock proteins (HSPs) 1 to deal with heat stress. The induction requires heat shock transcription factor (HSF) and cis-heat shock elements (HSEs) (1). HSF has two conserved domains, a helix-turn-helix DNA binding domain and a coiled-coil hydrophobic repeat domain needed for trimer or higher order multimer formation (2, 3). HSFs of the budding yeasts (Saccharomyces cerevisiae and Kluyveromyces lactis) uniquely possess two activation domains in the amino terminus (AAD) and carboxyl terminus (CAD) (4, 5), whereas those of fission yeast (Shizosaccharomyces pombe), Drosophilla melanogaster, and vertebrates have CADs alone. HSF is moderately phosphorylated under non-stress conditions and is further activated by hyperphosphorylation upon heat shock to induce the expression of HSPs (6 -8). In fission yeast, D. melanogaster, and mammals, the binding activity of HSFs to HSEs is dramatically stimulated by heat shock as HSF monomers are converted to trimers (9 -11). In contrast, in the budding yeast, HSF constitutively binds to HSE, but the binding activity seems to increase after heat shock-induced hyperphosphorylation (7,(12)(13)(14)(15)(16).The HSE is composed of several contiguous inverted repeats of the 5-base pair sequence nGAAn (where n is any nucleotide) (1,17,18). The number of the pentameric units in HSE varies, but at least three units are thought to be the minimum required for heat regulation in vivo. Namely, nGAAnnTTCnnGAAn is the minimum consensus HSE (cHSE) (19,20). However, an HSE can tolerate and still function with a 5-bp insertion between two repeating units if the spacing and orientation of the pentameric elements are maintained, e.g. nGAAn-(5-bp)-nGAAn (21). The most characterized endogenous non-consensus HSE (ncHSE) is that of CUP1 in S. cerevisiae, nTTCnnGAAn-(5-bp)-nGAGn denoted ncHSE CUP1 (22)(23)(24). Similar elements regulate HSP82 and HSC82 (12,(25)(...

Identification of a Novel Class of Target Genes and a Novel Type of Binding Sequence of Heat Shock Transcription Factor in Saccharomyces cerevisiae

“…We have further analyzed the 5Ј-flanking regions of genes lacking the known types of HSE and identified a novel HSE consisting of three nTTCn direct repeats separated by 5-bp blocks. Some of the previously identified Hsf1 targets, such as HSP60, HSC82, UBI4, EDC2, and RPN4 (37,42,(47)(48)(49), were not recognized as Hsf1-regulated genes in the present work. The number of Hsf1-regulated genes identified was smaller than the number of Hsf1-bound genes, the latter being ϳ165 (20).…”

“…The notable enrichment of the sequence in the Hsf1-regulated genes (20%, 12 of 59 genes), relative to the entire genome, supports the functional role as the HSE. It was previously reported that MDJ1 and YDJ1 (containing the step-type HSE) have a non-conventional HSE comprising nTTCn(11 bp)nGAAn(5 bp)nGAAn (47). We suggest that interaction with the two sequences leads to cooperative binding of the Hsf1 trimers.…”

Interaction between Heat Shock Transcription Factors (HSFs) and Divergent Binding Sequences

“…In the PNC1 promoter, the heat shock response requires not only the directly repeated units but also several units that are located in varying positions. An exceptional HSE was also found in the MDJ1 promoter, in which the third nGAAn unit is far away from the two regularly arranged units (15). It was shown that a pair of direct repeats is not sufficient to function as the HSE (15,20).…”

“…An exceptional HSE was also found in the MDJ1 promoter, in which the third nGAAn unit is far away from the two regularly arranged units (15). It was shown that a pair of direct repeats is not sufficient to function as the HSE (15,20). Thus, although the presence of a regularly positioned third unit is needed for the binding of ScHsf1, functional HSEs tolerate slightly divergent configurations.…”

Genome-Wide Analysis of the Biology of Stress Responses through Heat Shock Transcription Factor

“…While the role of the highly conserved HSE in directing HSF binding to specific loci has been well established in yeasts and metazoans (33,51,52), a number of studies have demonstrated that there are distinct types of HSEs bound by S. cerevisiae HSF, as well as distinct HSEs that are preferentially bound by the HSF1 and HSF2 isoforms in mammals (1). Indeed, the cooperative binding of S. cerevisiae HSF to multiple nonconsensus HSEs may be enhanced by heat shock or other stresses (10,13,45). To attempt to understand the determinants that govern the heat shock-dependent binding distribution of HSF within the S. cerevisiae genome, we applied the pattern-finding program MDscan (29) to distinct subsets of the HSF target loci identified here by ChIP-DNA microarray experiments.…”