Functional interplay between chromatin remodeling complexes RSC, SWI/SNF and ISWI in regulation of yeast heat shock genes.

Chromatin remodeling is an essential part of transcription initiation. We show that at heat shock gene promoters functional interactions between individual ATP-dependent chromatin remodeling complexes play critical role in both nucleosome displacement and Pol II recruitment. Using HSP12, HSP82 and SSA4 gene promoters as reporters, we demonstrated that while inactivation of SNF2, a critical ATPase of the SWI/SNF complex, primarily affects the HSP12 promoter, depletion of STH1- a SNF2 homolog from the RSC complex reduces histone displacement and abolishes the Pol II recruitment at all three promoters. From these results, we conclude that redundancy between SWI/SNF and RSC complexes is only partial and likely is affecting different chromatin remodeling steps. While inactivation of other individual ATP-dependent chromatin remodeling complexes negligibly affects reporter promoters, combinatorial inactivation of SNF2 and ISW1 has a synergistic effect by diminishing histone loss during heat induction and eliminating Pol II recruitment. Importantly, it also eliminates preloading of HSF on HSP82 and SSA4 promoters before heat shock and diminishes HSF binding during heat shock. These observations suggest that prior action of chromatin remodeling complexes is necessary for the activator binding.

Displacement of histones at promoters of Saccharomyces cerevisiae heat shock genes is differentially associated with histone H3 acetylation.

Chromatin remodeling at promoters of activated genes spans from mild histone modifications to outright displacement of nucleosomes in trans. Factors affecting these events are not always clear. Our results indicate that histone H3 acetylation associated with histone displacement differs drastically even between promoters of such closely related heat shock genes as HSP12, SSA4, and HSP82. The HSP12 promoter, with the highest level of histone displacement, showed the highest level of H3 acetylation, while the SSA4 promoter, with a lower histone displacement, showed only modest H3 acetylation. Moreover, for the HSP12 promoter, the level of acetylated H3 is temporarily increased prior to nucleosome departure. Individual promoters in strains expressing truncated versions of heat shock factor (HSF) showed that deletion of either one of two activating regions in HSF led to the diminished histone displacement and correspondingly lower H3 acetylation. The deletion of both regions simultaneously severely decreased histone displacement for all promoters tested, showing the dependence of these processes on HSF. The level of histone H3 acetylation at individual promoters in strains expressing truncated HSF also correlated with the extent of histone displacement. The beginning of chromatin remodeling coincides with the polymerase II loading on heat shock gene promoters and is regulated either by HSF binding or activation of preloaded HSF.

Cell cycle-dependent binding of yeast heat shock factor to nucleosomes.

In the nucleus, transcription factors must contend with the presence of chromatin in order to gain access to their cognate regulatory sequences. As most nuclear DNA is assembled into nucleosomes, activators must either invade a stable, preassembled nucleosome or preempt the formation of nucleosomes on newly replicated DNA, which is transiently free of histones. We have investigated the mechanism by which heat shock factor (HSF) binds to target nucleosomal heat shock elements (HSEs), using as our model a dinucleosomal heat shock promoter (hsp82-DeltaHSE1). We find that activated HSF cannot bind a stable, sequence-positioned nucleosome in G(1)-arrested cells. It can do so readily, however, following release from G(1) arrest or after the imposition of either an early S- or late G(2)-phase arrest. Surprisingly, despite the S-phase requirement, HSF nucleosomal binding activity is restored in the absence of hsp82 replication. These results contrast with the prevailing paradigm for activator-nucleosome interactions and implicate a nonreplicative, S-phase-specific event as a prerequisite for HSF binding to nucleosomal sites in vivo.

The Skn7 response regulator of Saccharomyces cerevisiae interacts with Hsf1 in vivo and is required for the induction of heat shock genes by oxidative stress.

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein-protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

Cooperative binding of heat shock factor to the yeast HSP82 promoter in vivo and in vitro.

Previous work has shown that heat shock factor (HSF) plays a central role in remodeling the chromatin structure of the yeast HSP82 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 approximately 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 HSP82 promoter 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.

In vivo analyses of upstream promoter sequence elements in the 5 S rRNA gene from Saccharomyces cerevisiae.

Upstream promoter elements of the Saccharomyces cerevisiae 5 S rRNA gene have been characterized by genomic DNase I "footprinting" and by in vivo mutational analyses using base substitutions and deletions. A high copy shuttle-vector was used to efficiently express the mutant 5 S rRNA genes in vivo and a structural mutation in the 5 S rRNA, which was previously shown to be functionally neutral but easily detected by gel electrophoresis, allowed for an accurate measure of gene expression. The results provide direct evidence for upstream regulatory elements which confirms a start site element (sse) from -1 to -8 and identifies a new independent upstream promoter element (upe) centered from about -17 to -20. In contrast to previous reports with reconstituted systems, both elements dramatically affect the efficiency of gene expression and suggest that the saturated conditions which are used in reconstituted studies mask sequence dependence; a dependency that could be physiologically significant and play a role in the regulation of 5 S rRNA expression. The footprint analyses support an extended region of protein interaction as recently observed in reconstituted systems but again provide evidence of significant structural rearrangements when the upstream sequence is changed.

Heat shock factor gains access to the yeast HSC82 promoter independently of other sequence-specific factors and antagonizes nucleosomal repression of basal and induced transcription.

Transcription in eukaryotic cells occurs in the context of chromatin. Binding of sequence-specific regulatory factors must contend with the presence of nucleosomes for establishment of a committed preinitiation complex. Here we demonstrate that the high-affinity binding site for heat shock transcription factor (HSF) is occupied independently of other cis-regulatory elements and is critically required for preventing nucleosomal assembly over the yeast HSC82 core promoter under both noninducing (basal) and inducing conditions. Chromosomal mutation of this sequence, termed HSE1, erases the HSF footprint and abolishes both transcription and in vivo occupancy of the TATA box. Moreover, it dramatically reduces promoter chromatin accessibility to DNase I and TaqI, as the nuclease-hypersensitive region is replaced by a localized nucleosome. By comparison, in situ mutagenesis of two other promoter elements engaged in stable protein-DNA interactions in vivo, the GRF2/REB1 site and the TATA box, despite reducing transcription three- to fivefold, does not compromise the nucleosome-free state of the promoter. The GRF2-binding factor appears to facilitate the binding of proteins to both HSE1 and TATA, as these sequences, while still occupied, are less protected from in vivo dimethyl sulfate methylation in a deltaGRF2 strain. Finally, deletion of a consensus upstream repressor sequence (URS1), positioned immediately upstream of the GRF2-HSE1 region and only weakly occupied in chromatin, has no expression phenotype, even under meiotic conditions. However, deletion of URS1, like mutation of GRF2, shifts the translational setting of an upstream nucleosomal array flanking the promoter region. Taken together, our results argue that HSF, independent of and dominant among sequence-specific factors binding to the HSC82 upstream region, antagonizes nucleosomal repression and creates an accessible chromatin structure conducive to preinitiation complex assembly and transcriptional activation.

Multiple protein-DNA interactions over the yeast HSC82 heat shock gene promoter.

We have utilized DNase I and micrococcal nuclease (MNase) to map the chromatin structure of the HSC82 heat shock gene of Saccharomyces cerevisiae. The gene is expressed at a high basal level which is enhanced 2-3-fold by thermal stress. A single, heat-shock invariant DNase I hypersensitive domain is found within the HSC82 chromosomal locus; it maps to the gene's 5' end and spans 250 bp of promoter sequence. DNase I genomic footprinting reveals that within this hypersensitive region are four constitutive protein-DNA interactions. These map to the transcription initiation site, the TATA box, the promoter-distal heat shock element (HSE1) and a consensus GRF2 (REB1/Factor Y) sequence. However, two other potential regulatory sites, the promoter-proximal heat shock element (HSE0) and a consensus upstream repressor sequence (URS1), are not detectably occupied under either transcriptional state. In contrast to its sensitivity to DNAase I, the nucleosome-free promoter region is relatively protected from MNase; the enzyme excises a stable nucleoprotein fragment of approximately 210 bp. As detected by MNase, there are at least two sequence-positioned nucleosomes arrayed 5' of the promoter; regularly spaced nucleosomes exhibiting an average repeat length of 160-170 bp span several kilobases of both upstream and downstream regions. Similarly, the body of the gene, which exhibits heightened sensitivity to DNase I, displays a nucleosomal organization under both basal and induced states, but these nucleosomes are not detectably positioned with respect to the underlying DNA sequence and may be irregularly spaced and/or structurally altered. We present a model of the chromatin structure of HSC82 and compare it to one previously derived for the closely related, but differentially regulated, HSP82 heat shock gene.

The upstream sequences of the HSP82 and HSC82 genes of Saccharomyces cerevisiae: regulatory elements and nucleosome positioning motifs.

We present the upstream sequences of HSP82 and HSC82, two closely related, but differentially regulated, heat-shock genes of Saccharomyces cerevisiae. Several dozen potential regulatory elements are identified within each upstream region; interestingly, only a few are conserved between the two genes. These include a consensus heat-shock element, an upstream repressor element, and a consensus TATA element. A search for motifs known actively to position nucleosomes in vitro revealed that such sequences are three- to seven-fold enriched within each promoter; a comparable enrichment is seen near the 3' end of each transcription unit. Located approximately 1100 bp upstream of HSC82 is an open reading frame (ORF) of 255 amino acids; approximately 800 bp upstream of HSP82 is an ORF of 132 amino acids. The latter ORF contains several conserved ankyrin motifs and appears to be expressed under normal growth conditions. Finally, we show by clamped homogeneous electric field gel electrophoresis that the two genetic loci map to different chromosomes: HSP82 to chromosome XVI and HSC82 to chromosome XIII. The sequences have been deposited in the GenBank database under Accession Numbers U20323 and U20349.

In vivo analyses of the internal control region in the 5S rRNA gene from Saccharomyces cerevisiae.

The internal control region of the Saccharomyces cerevisiae 5S rRNA gene has been characterized in vivo by genomic DNase I footprinting and by mutational analyses using base substitutions, deletions or insertions. A high copy shuttle vector was used to efficiently express mutant 5S rRNA genes in vivo and isotope labelling kinetics were used to distinguish impeded gene expression from nascent RNA degradation. In contrast to mutational studies in reconstituted systems, the analyses describe promoter elements which closely resemble the three distinct sequence elements that have been observed in Xenopus laevis 5S rRNA. The results indicate a more highly conserved structure than previously reported with reconstituted systems and suggest that the saturated conditions which are used in reconstitution studies mask sequence dependence which may be physiologically significant. Footprint analyses support the extended region of protein interaction which has recently been observed in some reconstituted systems, but mutational analyses indicate that these interactions are not sequence specific. Periodicity in the footprint provides further detail regarding the in vivo topology of the interacting protein.