Eva Czarnecka-Verner

The Hsf world: classification and properties of plant heat stress transcription factors

Based on the partial or complete sequences of 14 plant heat stress transcription factors (Hsfs) from tomato, soybean, Arabidopsis and maize we propose a general nomenclature with two basic classes, i.e. classes A and B each containing two or more types of Hsfs (HsfA1, HsfA2 etc.). Despite some plant-specific peculiarities, essential functional domains and modules of these proteins are conserved among plants, yeast, Drosophila and vertebrates. A revised terminology of these parts follows recommendations agreed upon among the authors and representatives from other laboratories working in this field (see legend to Fig. 1). Similar to the situation with the small heat shock proteins (sHsps), the complexity of the hsf gene family in plants appears to be higher than in other eukaryotic organisms.

Plants contain a novel multi-member class of heat shock factors without transcriptional activator potential.

Based on phylogeny of DNA-binding domains and the organization of hydrophobic repeats, two families of heat shock transcription factors (HSFs) exist in plants. Class A HSFs are involved in the activation of the heat shock response, but the role of class B HSFs is not clear. When transcriptional activities of full-length HSFs were monitored in tobacco protoplasts, no class B HSFs from soybean or Arabidopsis showed activity under control or heat stress conditions. Additional assays confirmed the finding that the class B HSFs lacked the capacity to activate transcription. Fusion of a heterologous activation domain from human HSF1 (AD2) to the C-terminus of GmHSFB1-34 gave no evidence of synergistic enhancement of AD2 activity, which would be expected if weak activation domains were present. Furthermore, activity of AtHSFB1-4 (class B) was not rescued by coexpression with AtHSFA4-21 (class A) indicating that the class A HSF was not able to provide a missing function required for class B activity. The transcriptional activation potential of Arabidopsis AtHSFA4-21 was mapped primarily to a 39 amino acid fragment in the C-terminus enriched in bulky hydrophobic and acidic residues. Deletion mutagenesis of the C-terminal activator regions of tomato and Arabidopsis HSFs indicated that these plant HSFs lack heat-inducible regulatory regions analogous to those of mammalian HSF1. These findings suggest that heat shock regulation in plants may differ from metazoans by partitioning negative and positive functional domains onto separate HSF proteins. Class A HSFs are primarily responsible for stress-inducible activation of heat shock genes whereas some of the inert class B HSFs may be specialized for repression, or down-regulation, of the heat shock response.

Plant class B HSFs inhibit transcription and exhibit affinity for TFIIB and TBP

Plant heat shock transcription factors (HSFs) are capable of transcriptional activation (class A HSFs) or both, activation and repression (class B HSFs). However, the details of mechanism still remain unclear. It is likely, that the regulation occurs through interactions of HSFs with general transcription factors (GTFs), as has been described for numerous other transcription factors. Here, we show that class A HSFs may activate transcription through direct contacts with TATA-binding protein (TBP). Class A HSFs can also interact weakly with TFIIB. Conversely, class B HSFs inhibit promoter activity through an active mechanism of repression that involves the C-terminal regulatory region (CTR) of class B HSFs. Deletion analysis revealed two sites in the CTR of soybean GmHSFB1 potentially involved in protein–protein interactions with GTFs: one is the repressor domain (RD) located in the N-terminal half of the CTR, and the other is a TFIIB binding domain (BD) that shows affinity for TFIIB and is located C-terminally from the RD. A Gal4 DNA binding domain-RD fusion repressed activity of LexA-activators, while Gal4-BD proteins synergistically activated strong and weak transcriptional activators. In vitrobinding studies were consistent with this pattern of activity since the BD region alone interacted strongly with TFIIB, and the presence of RD had an inhibitory effect on TFIIB binding and transcriptional activation.

Isolation and characterization of six heat shock transcription factor cDNA clones from soybean

Thermal stress in soybean seedlings causes the activation of pre-existing heat shock transcription factor proteins (HSFs). Activation results in the induction of DNA binding activity which leads to the transcription of heat shock genes. From a soybean cDNA library we have isolated cDNA clones corresponding to six HSF genes. Two HSF genes are expressed constitutively at the transcriptional level, and the remaining four are heat-inducible. Two of the heat inducible genes are also responsive to cadmium stress. Comparative analysis of HSF sequences indicated higher conservation of the DNA binding domain among plant HSFs than those from yeast or other higher eukaryotes. The putative plant HSF oligomerization domain contains hydrophobic heptapeptide repeats characteristic of coiled coils and seems to exist in two structural variants. The carboxy-terminal domains are reduced in size and the C-terminal heptad repeat is degenerate.

Role of the TATA Binding Protein–Transcription Factor IIB Interaction in Supporting Basal and Activated Transcription in Plant Cells

The TATA binding protein (TBP) and transcription factor IIB (TFIIB) play crucial roles in transcription of class II genes. The requirement for TBP–TFIIB interactions was evaluated in maize cells by introducing mutations into the Arabidopsis TBP (AtTBP2) within the C-terminal stirrup. Protein binding experiments indicated that amino acid residues E-144 and E-146 of AtTBP2 are both essential for TFIIB binding in vitro. Activation domains derived from herpes simplex viral protein VP16, the Drosophila fushi tarazu glutamine-rich domain (ftzQ), and yeast Gal4 were tested in transient assays. TBP–TFIIB interactions were dispensable for basal transcription but were required for activated transcription. In general, activated transcription was more severely inhibited by TBP mutation E-146R than by mutation E-144R. However, these TBP mutations had little effect on activity of the full-length cauliflower mosaic virus 35S and maize ubiquitin promoters, thus demonstrating that strong TBP–TFIIB contacts are not always required for transcription driven by complex promoters.

Plant heat shock transcription factors: positive and negative aspects of regulation

Six heat shock transcription factors (HSFs) have been isolated and characterized from soybean and two from Arabidopsis (Czarnecka-Verner et al. 1995; Barros, Czarnecka-Verner and Gurley, unpublished). Based on a phylogeny analysis of the DNA binding domains and organization of oligomerization domains, they have been assigned to classes A2 and B of the plant HSF family (Nover et al. 1996). In vivo studies of full length HSFs were conducted in transient expression systems using a GUS reporter driven by a heat shock element (HSE) located upstream from the minimal 35S CaMV promoter. Neither soybean nor Arabidopsis HSF class B members were able to function as transcriptional activators under control or heat stress conditions. Conversely, class A HSFs from tomato and Arabidopsis have the capacity to activate transcription. Co-expression studies of activator HSFs from tomato and Arabidopsis, and inert HSFs from soybean and Arabidopsis demonstrated that the inert HSFs were able to trans-attenuate the transcriptional activity of activator HSFs. We suggest that heat shock regulation in plants may differ from metazoan systems by partitioning negative and positive functional domains onto separate HSF proteins. In plants two classes of HSFs exist: class A members, i.e. HSF activators, and a novel class B (inert HSFs) which is largely specialized for repression, or attenuation, of the heat shock response.

Yeast two-hybrid map of Arabidopsis TFIID

General transcription factor IID (TFIID) is a multisubunit protein complex involved in promoter recognition and is fundamental to the nucleation of the RNA polymerase II transcriptional preinitiation complex. TFIID is comprised of the TATA binding protein (TBP) and 12–15 TBP-associated factors (TAFs). While general transcription factors have been extensively studied in metazoans and yeast, little is known about the details of their structure and function in the plant kingdom. This work represents the first attempt to compare the structure of a plant TFIID complex with that determined for other organisms. While no TAF3 homolog has been observed in plants, at least one homolog has been identified for each of the remaining 14 TFIID subunits, including both TAF14 and TAF15 which have previously been shown to be unique to either yeast or humans. The presence of both TAFs 14 and 15 in plants suggests ancient roles for these proteins that were lost in metazoans and fungi, respectively. Yeast two-hybrid interaction assays resulted in a total of 65 binary interactions between putative subunits of Arabidopsis TFIID, including 26 contacts unique to plants. The interaction matrix of Arabidopsis TAFs is largely consistent with the three-lobed topological map for yeast TFIID, which suggests that the structure and composition of TFIID have been highly conserved among eukaryotes.

Isolation and characterization of class A4 heat shock transcription factor from alfalfa

Plant heat shock transcription factors (HSFs) regulate transcription of heat shock (HS) genes. In Arabidopsis thaliana, 21 HSFs have been classified into groups A-C. Members of class A act as typical transcriptional activators, whereas B HSFs function as coactivators or repressors depending on promoter context. The function of class C HSFs is still unclear. Here, we present the isolation and characterization of the first HSF from alfalfa (Medicago sativa L.) and designate it MsHSFA4 based on amino acid sequence analysis. The MsHSFA4 gene was determined to be single copy and was detected at two separate genetic loci in the tetraploid Medicago sativa. Overexpression of MsHSFA4 in tobacco mesophyll protoplasts resulted in weak transcriptional activity, similar to that exhibited by Arabidopsis AtHSFA4a. The MsHSFA4 proximal promoter contains three putative HSE elements, and the gene itself is activated both by heat and cold stress.

Expression of human heat shock transcription factors 1 and 2 in HeLa cells and yeast

We have examined reporter gene (beta-gal) expression directed by human heat shock transcription factors 1 and 2 (HSF1 and HSF2) in HeLa cells and in yeast (Saccharomyces cerevisiae). Transcriptional activation domains of both HSFs were mapped to the C-termini using chimeric proteins containing the GAL4 DNA binding domain (GAL4-DBD). Deletion analysis of HSF1 largely confirmed the mapping and expression pattern of activation domain 2 (AD2) previously reported by Green et al (1995) with the exception of the contribution of the oligomerization domain (hydrophobic region A) to basal repression in yeast, but not in HeLa cells. In addition, a C-terminal activation domain for HSF2 (amino acids 397 to 536) was identified by analysis in yeast. In contrast to HSF1, full length HSF2 and the isolated activation domain of HSF2 showed little activity in HeLa cells. Analysis of point mutations generated by low fidelity PCR within AD2 of HSF1 indicated that hydrophobic and charged amino acids in addition to proline, serine and threonine make critical contributions to transcriptional activity. Co-expression of GAL4-DBD fusions with AD2 of HSF1 and the C-terminal activation domain of HSF2 showed no evidence of synergism in the activation of transcription. Wild-type human HSF1 and HSF2 were both able to substitute for the endogenous yeast HSF under normal growth conditions.

Adaptation of the Agrobacterium tumefaciens VirG response regulator to activate transcription in plants

The Agrobacterium tumefaciens VirG response regulator of the VirA/VirG two-component system was adapted to function in tobacco protoplasts. The subcellular localization of VirG and VirA proteins transiently expressed in onion cells was determined using GFP fusions. Preliminary studies using Gal4DBD-VP16 fusions with VirG and Escherichia coli UhpA, and NarL response regulators indicated compatibility of these bacterial proteins with the eukaryotic transcriptional apparatus. A strong transcriptional activator based on tandem activation domains from the Drosophila fushi tarazu and Herpes simplex VP16 was created. Selected configurations of the two-site Gal4-vir box GUS reporters were activated by chimeric effectors dependent on either the yeast Gal4 DNA-binding domain or that of VirG. Transcriptional induction of the GUS reporter was highest for the VirE19-element promoter with both constitutive and wild-type VirG-tandem activation domain effectors. Multiple VirE19 elements increased the reporter activity proportionately, indicating that the VirG DNA binding domain was functional in plants. The VirG constitutive-Q-VP16 effector was more active than the VirG wild-type. In both the constitutive and wild-type forms of VirG, Q-VP16 activated transcription of the GUS reporter best when located at the C-terminus, i.e. juxtaposed to the VirG DNA binding domain. These results demonstrate the possibility of using DNA binding domains from bacterial response regulators and their cognate binding elements in the engineering of plant gene expression.Graphical Abstract