Temperature-dependent regulation of a heterologous transcriptional activation domain fused to yeast heat shock transcription factor.

Abstract

The heat shock transcription factor (HSF) of the yeast Saccharomyces cerevisiae is posttranslationally modified. At low growth temperatures, it activates transcription of heat shock genes only poorly; after shift to high temperatures, it activates transcription readily. In an effort to elucidate the mechanism of this regulation, we constructed a series of HSF-VP16 fusions that join the HSF DNA-binding domain to the strong transcriptional activation domain from the VP16 gene of herpes simplex virus. Replacement of the endogenous C-terminal transcriptional activation domain with that of VP16 generates an HSF derivative that exhibits behavior reminiscent of HSF itself: low transcriptional activation activity at normal growth temperature and high activity after heat shock. HSF can thus restrain the activity of the heterologous VP16 transcriptional activation domain. To determine what is required for repression of activity at low temperature, we deleted portions of HSF from this HSF-VP16 fusion to map the regulatory domain. We also isolated point mutations that convert the HSF-VP16 fusion into a constitutive transcriptional activator. We conclude that the central, evolutionarily conserved domain of HSF, encompassing the DNA-binding and multimerization domains, contains a major determinant of temperature-dependent regulation.Stress proteins, or heat shock proteins, are highly regulated, exhibiting low-level expression at low growth temperatures, intermediate levels at intermediate temperatures, and high levels at high temperatures. For Escherichia coli and the yeast Saccharomyces cerevisiae, the response to a temperature upshift is similar: a rapid increase in expression well above the original basal level, followed by adaptation to a new basal level that is only fewfold above the original.In eukaryotes, the major regulatory protein involved in heat shock protein expression is the transcriptional activator heat shock transcription factor (HSF). Indeed, the addition of a synthetic oligonucleotide bearing the HSF-binding site (a heat shock element) suffices to bring a heterologous gene under the control of the heat shock system (19, 29; this report). Therefore, to understand the regulation of heat shock protein expression, it is necessary to know how the activity of HSF is modulated such that it stimulates different levels of transcription at different temperatures.In Drosophila (30,39) example, reference 6) or of transcription factor activity (18,26,33,36) by phosphorylation. In most of these cases, it is either known or speculated that the effect of phosphorylation is to stabilize a conformation of the protein that exhibits higher (or lower) activity. It is relatively easy to imagine that HSF, whether from humans, flies, or yeasts, undergoes a conformational change upon phosphorylation and that the new conformation exhibits higher activity in DNA binding, transcriptional activation, or both (17,38).Existing data suggest that yeast HSF almost certainly undergoes a conformational change upon temperature upshift. First,...

Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis

“…However, while the C-terminal AD from yeast Hsf is sufficient to strongly drive gene expression when fused to a heterologous DNA binding domain, this activator is repressed at low temperatures in the native context of Hsf in spite of constitutive DNA binding (Sorger, 1990). Further, even when the Hsf C-terminal AD is replaced with a heterologous AD, this Hsf∆C-AD fusion retains near wild type activity under basal and heat shock conditions, though intergenic mutants can bypass basal repression of this fusion (Bonner et al, 1992). These observations suggest that interdomain interactions restrain Hsf activity in the absence of stress.…”

“…shown that expression of chimera containing the Hsf1 DNA binding domain fused to a viral transcriptional activator results in constitutive expression of Hsf1 targets (Bonner et al, 1992). Thus, we replaced the Gal4 DNA binding domain in our GEM construct with …”

A Functional Module of Yeast Mediator That Governs the Dynamic Range of Heat-Shock Gene Expression

“…Several ''masking'' domains have been identified. These include an N-terminal repressor domain (Sorger 1990), a region that overlaps the DNA-binding and trimerization domains (NietoSotelo et al 1990;Bonner et al 1992;Chen and Parker 2002), and a heptapeptide sequence termed CE2 located adjacent to the C-terminal activation domain ( Jakobsen and Pelham 1991). While these have been traditionally thought to physically interact with one or both activation domains, the possibility of alternative or additional mechanisms of repression, including the recruitment of corepressors, has not been ruled out.…”

“…Experimental rationale: The ability of yeast HSF to activate transcription in response to thermal stress, and the role of its activation and regulatory domains in this process, have been extensively investigated (e.g., NietoSotelo et al 1990;Sorger 1990;Bonner et al 1992;Bulman et al 2001;Chen and Parker 2002;Erkine and Gross 2003;Hashikawa and Sakurai 2004). However, a poorly understood function of HSF is its ability to stimulate basal transcription.…”

Identification of the C-terminal activator domain in yeast heat shock factor: independent control of transient and sustained transcriptional activity.

“…Finally, a mutation in the DNA-binding domain causes constitutive activity, albeit in a fusion protein where the CTA had been exchanged with a heterologous activator (Bonner et al, 1992).…”