Antibody-mediated activation of Drosophila heat shock factor in vitro.

Eukaryotic cells respond to elevated temperatures by rapidly activating the expression of heat shock genes. Central to this activation is heat shock-inducible binding of the transcriptional activator, termed heat shock factor (HSF), to common regulatory elements, which are located upstream of all heat shock genes. The DNA binding activity of the inactive form of Drosophila HSF was induced in vitro by treatment with polyclonal antibodies to the purified, in vivo-activated factor. This finding, together with observations that high temperature and low pH activate HSF binding in vitro, suggests that the inactive form of HSF can directly recognize and transduce the heat shock signal without undergoing a covalent modification of protein structure.

Purification and properties of Drosophila heat shock activator protein.

Drosophila heat shock activator protein, a rare transacting factor which is induced upon heat shock to bind specifically to the heat shock regulatory sequence in vivo, has been purified from shocked cells to more than 95 percent homogeneity by sequence-specific duplex oligonucleotide affinity chromatography. The purified protein has a relative molecular mass of 110 kilodaltons, binds to the regulatory sequence with great affinity and specificity, and strongly stimulates transcription of the Drosophila hsp70 gene. Studies with this regulatory protein should lead to an understanding of the biochemical pathway underlying the heat shock phenomenon.

Intramolecular repression of mouse heat shock factor 1.

The pathway leading to transcriptional activation of heat shock genes involves a step of heat shock factor 1 (HSF1) trimerization required for high-affinity binding of this activator protein to heat shock elements (HSEs) in the promoters. Previous studies have shown that in vivo the trimerization is negatively regulated at physiological temperatures by a mechanism that requires multiple hydrophobic heptad repeats (HRs) which may form a coiled coil in the monomer. To investigate the minimal requirements for negative regulation, in this work we have examined mouse HSF1 translated in rabbit reticulocyte lysate or extracted from Escherichia coli after limited expression. We show that under these conditions HSF1 behaves as a monomer which can be induced by increases in temperature to form active HSE-binding trimers and that mutations of either HR region cause activation in both systems. Furthermore, temperature elevations and acidic buffers activate purified HSF1, and mild proteolysis excises fragments which form HSE-binding oligomers. These results suggest that oligomerization can be repressed in the monomer, as previously proposed, and that repression can be relieved in the apparent absence of regulatory proteins. An intramolecular mechanism may be central for the regulation of this transcription factor in mammalian cells, although not necessarily sufficient.

Complex modes of heat shock factor activation.

Eucaryotic organisms respond to elevated environmental temperatures by rapidly activating the expression of heat shock genes. The transcriptional activation of heat shock genes is mediated by a conserved upstream regulatory sequence, the heat shock element (HSE). Using an HSE-binding assay, we show that a cellular factor present in a range of vertebrate species binds specifically to the HSE. This factor is presumably the transcriptional activator of heat shock genes, heat shock factor (HSF). In vertebrates, the binding of HSF to the HSE was induced when cells were subjected to heat shock at high temperatures, even in the absence of protein synthesis. Under mild heat shock conditions, HSF binding was induced to a lesser extent, but this induction required protein synthesis, suggesting that synthesis of HSF itself, or an activating factor, is necessary for response to heat shock at intermediate temperatures. The inducibility of HSF binding in higher eucaryotes is contrasted with constitutive HSF binding activity in fungi. It appears that despite conservation of the HSE in evolution, the means by which HSF is activated to bind DNA in higher and lower eucaryotes may have diverged.

Analysis of heat shock transcription factor for suppression of polyglutamine toxicity.

Individually over-expressed chaperones can interfere with cytotoxicity and aggregation of polyglutamine proteins in disease models. As chaperones cooperate, the analysis of suppression or reversal of polyglutamine pathology may require ways to up-regulate multiple chaperone coding genes. This condition might be achieved by exogenous expression of de-repressed forms of heat shock transcription factor 1 (HSF1), which mediates induction of several genes coding cytosolic and nuclear chaperones. Here we present the rationale behind this possible approach and the caveats, and employ a non-neuronal cell system to test whether Ataxin-1 aggregation can be modulated by de-repressed HSF1 mutants through augmented expression of chaperone coding genes. In our experiments, HSF1 mutants have induced heat shock protein 70 and Human DnaJ (HDJ)-1 to intermediate levels. Cells expressing such mutants also showed partial reduction of Ataxin-1 [31Q] aggregation. A consolidated positive outcome of these tests in cellular models would encourage experiments in transgenic mice and prospects for pharmacological modulation of HSF1 activity or delivery.

Quinolinic acid induced neurodegeneration in the striatum: a combined in vivo and in vitro analysis of receptor changes and microglia activation.

PURPOSE: Huntington's disease (HD) is a progressive neurodegenerative disorder, which is characterised by prominent neuronal cell loss in the basal ganglia with motor and cognitive disturbances. One of the most well-studied pharmacological models of HD is produced by local injection in the rat brain striatum of the excitotoxin quinolinic acid (QA), which produces many of the distinctive features of this human neurodegenerative disorder. Here, we report a detailed analysis, obtained both in vivo and in vitro of this pharmacological model of HD. MATERIALS AND METHODS: By combining emission tomography (PET) with autoradiographic and immunocytochemical confocal laser techniques, we quantified in the QA-injected striatum the temporal behavior (from 1 to 60 days from the excitotoxic insult) of neuronal cell density and receptor availability (adenosine A(2A) and dopamine D(2) receptors) together with the degree of microglia activation. RESULTS: Both approaches showed a loss of adenosine A(2A) and dopamine D(2) receptors paralleled by an increase of microglial activation. CONCLUSION: This combined longitudinal analysis of the disease progression, which suggested an impairment of neurotransmission, neuronal integrity and a reversible activation of brain inflammatory processes, might represent a more quantitative approach to compare the differential effects of treatments in slowing down or reversing HD in rodent models with potential applications to human patients.

Induction of sequence-specific binding of Drosophila heat shock activator protein without protein synthesis.

Drosophila tissue culture cells stimulated by heat shock contain high levels of heat shock activator protein, which binds specifically to the heat-shock control DNA element. In contrast, nonshocked cells have low basal levels of binding activity. Here, we show that within 30 seconds of heat shock of intact cells the sequence-specific binding activity in whole cell extracts increases significantly, reaching a plateau by 5 min after the start of the shock; removal of the heat stimulus returns the activity to basal levels. Known chemical inducers of heat-shock genes elicit a similar pattern of specific binding activity. Moreover, this pattern is observed in the presence of protein synthesis inhibitors, even if the stimulus-withdrawal is repeated sequentially through five cycles. Our results are inconsistent with models which propose proteolysis as the chief means of mediating heat-shock transcriptional control. Rather, they suggest that heat shock activator pre-exists in normal cells in a nonbinding form, which is converted upon cell stimulus to a high affinity, sequence-specific binding form, most probably by a post-translational modification. This conversion may be crucial for the transcriptional activation of heat shock genes.

A cell type specific factor recognizes the rat thyroglobulin promoter.

We have fused a 900 base pair long DNA segment containing the transcriptional start site of the rat thyroglobulin (Tg) gene to the bacterial gene for chloramphenicol acetyltransferase (cat). The fusion gene has been introduced into three different cell lines derived from the rat thyroid gland and into a rat liver cell line. Expression of the fusion gene was detected only in the one thyroid cell line that is able to express the endogenous Tg gene. The minimum DNA sequence required for the cell type specific expression was determined by deletion analysis; it extends 170 nucleotides upstream of the transcription initiation site. The Tg promoter contains a readily detectable binding sites for a factor present in salt extracts of thyroid cell nuclei. This binding site is not recognized by the nuclear extracts of any other cell type that we have tested, suggesting that it may help mediate the cell type specific expression of the Tg gene.

Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability.

We have cloned two distinct mouse heat shock transcription factor genes, mHSF1 and mHSF2. The mHSF1 and mHSF2 open reading frames are similar in size, containing 503 and 517 amino acids, respectively. Although mHSF1 and mHSF2 are quite divergent overall (only 38% identity), they display extensive homology in the DNA-binding and oligomerization domains that are conserved in the heat shock factors of Saccharomyces cerevisiae, Kluyveromyces lactis, Drosophila, tomato, and human. The ability of these two mouse heat shock factors to bind to the heat shock element (HSE) is regulated by heat. mHSF1 is expressed in an in vitro translation system in an inactive form that is activated to DNA binding by incubation at temperatures greater than 41 degrees C, the same temperatures that activate heat shock factor DNA binding and the stress response in mouse cells in vivo. mHSF2, on the other hand, is expressed in a form that binds DNA constitutively but loses DNA binding by incubation at greater than 41 degrees C. Both mHSF1 and mHSF2 are encoded by single-copy genes, and neither is transcriptionally regulated by heat shock. However, there is a striking difference in the levels of mHSF1 mRNA in different tissues of the mouse.

Heat stress transcription factors from tomato can functionally replace HSF1 in the yeast Saccharomyces cerevisiae.

The fact that yeast HSF1 is essential for survival under nonstress conditions can be used to test heterologous Hsfs for the ability to substitute for the endogenous protein. Our results demonstrate that like Hsf of Drosophila, tomato Hsfs A1 and A2 can functionally replace the corresponding yeast protein, but Hsf B1 cannot. In addition to survival at 28 degrees C, we checked the transformed yeast strains for temperature sensitivity of growth, induced thermotolerance and activator function using two different lacZ reporter constructs. Tests with full-length Hsfs were supplemented by assays using mutant Hsfs lacking parts of their C-terminal activator region or oligomerization domain, or containing amino acid substitutions in the DNA-binding domain. Remarkably, results with the yeast system are basically similar to those obtained by the analysis of the same Hsfs as transcriptional activators in a tobacco protoplast assay. Most surprising is the failure of HsfB1 to substitute for the yeast Hsf. The defect can be overcome by addition to HsfB1 of a short C-terminal peptide motif from HsfA2 (34 amino acid residues), which represents a type of minimal activator necessary for interaction with the yeast transcription apparatus. Deletion of the oligomerization domain (HR-A/B) does not interfere with Hsf function for survival or growth at higher temperatures. But monomeric Hsf has a markedly reduced affinity for DNA, as shown by lacZ reporter and band-shift assays.

Complex expression of murine heat shock transcription factors.

A central step in the transcriptional activation of heat shock genes is the binding of the heat shock factor (HSF) to upstream heat shock elements (HSEs). In vertebrates, HSF1 mediates the ubiquitous response to stress stimuli, while the role of a second HSE-binding factor, HSF2, is still unclear. In this work we show that both factors are expressed in a wide range of murine tissues and each exists as two splicing isoforms. Although HSFs are virtually ubiquitous proteins, their abundance is predominant in testis and variable among other tissues, indicating specific regulations of their expression. A low level of DNA-binding activity of HSF1, detected in many tissues, is probably physiological and is not explained by an anomalous regulation of one of the two isoforms. Our observations suggest that these regulatory proteins may all have roles in fully developed tissues. This possibility is not mutually exclusive of a role of HSF2 during cellular differentiation and tissue development [L. Sistonen, K. D. Sarge and R. I. Morimoto (1994), Mol. Cell. Biol., 14, 2087-2099].

Changes in cellular gene expression in rat thyroid epithelial cells transformed by the Kirsten murine sarcoma virus.

We have exploited a recently characterized system of rat thyroid epithelial cells transformed by the wild-type (wt) and a temperature-sensitive (ts) mutant strain of the Kirsten murine sarcoma virus (Ki-MSV) in order to study the effects of the K-ras oncogene on the gene expression of differentiated thyroid epithelial cells. By using cDNAs isolated from normal thyroid glands as probes, we were able to identify three sets of cellular sequences whose expression is influenced by the v-K-ras oncogene. The first set of genes is irreversibly repressed by transformation with both the wt and the ts viruses. The second set of genes is repressed in the ts-Ki-MSV-transformed cells but not in the same cells grown at the nonpermissive temperature. A third set of genes is present at higher levels at the nonpermissive temperature than at the permissive temperature. This system has allowed us to isolate and characterize a number of cDNA clones belonging to each of these three sets of genes. These specific cDNAs are suitable probes to study phenotypical changes during transformation of epithelial cells.