Lea Sistonen

Roles of the heat shock transcription factors in regulation of the heat shock response and beyond

The heat shock response, characterized by increased expression of heat shock proteins (Hsps) is induced by exposure of cells and tissues to extreme conditions that cause acute or chronic stress. Hsps function as molecular chaperones in regulating cellular homeostasis and promoting survival. If the stress is too severe, a signal that leads to programmed cell death, apoptosis, is activated, thereby providing a finely tuned balance between survival and death. In addition to extracellular stimuli, several nonstressful conditions induce Hsps during normal cellular growth and development. The enhanced heat shock gene expression in response to various stimuli is regulated by heat shock transcription factors (HSFs). After the discovery of the family of HSFs (i.e., murine and human HSF1, 2, and 4 and a unique avian HSF3), the functional relevance of distinct HSFs is now emerging. HSF1, an HSF prototype, and HSF3 are responsible for heat‐induced Hsp expression, whereas HSF2 is refractory to classical stressors. HSF4 is expressed in a tissue‐specific manner;similar to HSF1 and HSF2, alternatively spliced isoforms add further complexity to its regulation. Recently developed powerful genetic models have provided evidence for both cooperative and specific functions of HSFs that expand beyond the heat shock response. Certain specialized functions of HSFs may even include regulation of novel target genes in response to distinct stimuli.—Pirkkala, L., Nykanen, P, Sistonen, L. Roles of the heat shock transcription factors in regulation of the heat shock response and beyond.—Pirkkala, L., Nykäen, P, Sistonen, L. Roles of the heat shock transcription factors in regulation of the heat shock response and beyond. FASEB J. 15, 1118–1131 (2001)

Stress-Inducible Regulation of Heat Shock Factor 1 by the Deacetylase SIRT1

Heat shock factor 1 (HSF1) is essential for protecting cells from protein-damaging stress associated with misfolded proteins and regulates the insulin-signaling pathway and aging. Here, we show that human HSF1 is inducibly acetylated at a critical residue that negatively regulates DNA binding activity. Activation of the deacetylase and longevity factor SIRT1 prolonged HSF1 binding to the heat shock promoter Hsp70 by maintaining HSF1 in a deacetylated, DNA–binding competent state. Conversely, down-regulation of SIRT1 accelerated the attenuation of the heat shock response (HSR) and release of HSF1 from its cognate promoter elements. These results provide a mechanistic basis for the requirement of HSF1 in the regulation of life span and establish a role for SIRT1 in protein homeostasis and the HSR.

Multisite phosphorylation provides sophisticated regulation of transcription factors.

Reversible phosphorylation is a prevalent mechanism by which the activity of eukaryotic transcription factors is regulated rapidly in response to changes in the cellular environment. Accumulated evidence has expanded the concept of phosphorylation to a process that provides dynamic and precise tuning of the transactivating potential of a factor, rather than being a static on/off switch. In the case of transcription factors such as heat shock factor 1 (HSF1), p53 and nuclear factor of activated T cells (NFAT), multisite phosphorylation enables several effects to operate within a single factor, thereby functioning as a key to signal integration. Studies on these transcription factors illustrate recent progress in solving the dynamic nature of transcriptional regulation by multisite phosphorylation.

Phosphorylation of serine 230 promotes inducible transcriptional activity of heat shock factor 1

Heat shock factor 1 (HSF1) is a serine‐rich constitutively phosphorylated mediator of the stress response. Upon stress, HSF1 forms DNA‐binding trimers, relocalizes to nuclear granules, undergoes inducible phosphorylation and acquires the properties of a transactivator. HSF1 is phosphorylated on multiple sites, but the sites and their function have remained an enigma. Here, we have analyzed sites of endogenous phosphorylation on human HSF1 and developed a phosphopeptide antibody to identify Ser230 as a novel in vivo phosphorylation site. Ser230 is located in the regulatory domain of HSF1, and promotes the magnitude of the inducible transcriptional activity. Ser230 lies within a consensus site for calcium/calmodulin‐dependent protein kinase II (CaMKII), and CaMKII overexpression enhances both the level of in vivo Ser230 phosphorylation and transactivation of HSF1. The importance of Ser230 was further established by the S230A HSF1 mutant showing markedly reduced activity relative to wild‐type HSF1 when expressed in hsf1−/− cells. Our study provides the first evidence that phosphorylation is essential for the transcriptional activity of HSF1, and hence for induction of the heat shock response.

Heat Shock Factor 1 as a Coordinator of Stress and Developmental Pathways

The transition from normal growth conditions to stressful conditions is accompanied by a robust upregulation of heat shock proteins, which dampen the cytotoxicity caused by misfolded and denatured proteins. The most prominent part of this transition occurs on the transcriptional level. In mammals, protein-damaging stress leads to the activation of heat shock factor 1 (HSF1), which binds to upstream regulatory sequences in the promoters of heat shock genes. The activation of HSF1 proceeds through a multi-step pathway, involving a monomer-to-trimer transition, nuclear accumulation and extensive posttranslational modifications. In addition to its established role as the main regulator of heat shock genes, new data link HSF 1 to developmental pathways. In this chapter, we examine the established stress-related functions and prospect the intriguing role of HSF 1 as a developmental coordinator.

Heat Shock Factors at a Crossroad between Stress and Development

Abstract:u2002 Organisms must be able to sense and respond rapidly to changes in their environment in order to maintain homeostasis and survive. Induction of heat shock proteins (Hsps) is a common cellular defense mechanism for promoting survival in response to various stress stimuli. Heat shock factors (HSFs) are transcriptional regulators of Hsps, which function as molecular chaperones in protecting cells against proteotoxic damage. Mammals have three different HSFs that have been considered functionally distinct: HSF1 is essential for the heat shock response and is also required for developmental processes, whereas HSF2 and HSF4 are important for differentiation and development. Specifically, HSF2 is involved in corticogenesis and spermatogenesis, and HSF4 is needed for maintenance of sensory organs, such as the lens and the olfactory epithelium. Recent evidence, however, suggests a functional interplay between HSF1 and HSF2 in the regulation of Hsp expression under stress conditions. In lens formation, HSF1 and HSF4 have been shown to have opposite effects on gene expression. In this chapter, we present the different roles of the mammalian HSFs as regulators of cellular stress and developmental processes. We highlight the interaction between different HSFs and discuss the discoveries of novel target genes in addition to the classical Hsps.

Brain abnormalities, defective meiotic chromosome synapsis and female subfertility in HSF2 null mice.

Heat shock factor 2, one of the four vertebrate HSFs, transcriptional regulators of heat shock gene expression, is active during embryogenesis and spermatogenesis, with unknown functions and targets. By disrupting the Hsf2 gene, we show that, although the lack of HSF2 is not embryonic lethal, Hsf2−/− mice suffer from brain abnormalities, and meiotic and gameto genesis defects in both genders. The disturbances in brain are characterized by the enlargement of lateral and third ventricles and the reduction of hippocampus and striatum, in correlation with HSF2 expression in proliferative cells of the neuroepithelium and in some ependymal cells in adults. Many developing spermatocytes are eliminated via apoptosis in a stage‐specific manner in Hsf2−/− males, and pachytene spermatocytes also display structural defects in the synaptonemal complexes between homologous chromosomes. Hsf2−/− females suffer from multiple fertility defects: the production of abnormal eggs, the reduction in ovarian follicle number and the presence of hemorrhagic cystic follicles are consistent with meiotic defects. Hsf2−/− females also display hormone response defects, that can be rescued by superovulation treatment, and exhibit abnormal rates of luteinizing hormone receptor mRNAs.

The ubiquitin‐proteasome pathway

Regulating protein stability and turnover is a key task in the cell. Besides lysosomes, ubiquitin‐mediated proteasomal degradation comprises the major proteolytic pathway in eukaryotes. Proteins destined for degradation by the proteasome are conjugated by a ‘tag’, a ubiquitin chain to a lysine, through an extensively regulated enzymatic cascade. The ubiquitylated proteins are subsequently targeted for degradation by the 26S proteasome, the major proteolytic machinery for ubiquitylated proteins in the cell. Ubiquitylation can be considered as another covalent post‐translational modification and signal, comparable to acety‐lation, glycosylation, methylation, and phosphorylation. However, ubiquitylation has multiple roles in addition to targeting proteins for degradation. Depending on the number of ubiquitin moieties and the linkages made, ubiquitin also plays an important role in DNA repair, protein sorting and virus budding. Unregulated degradation of proteins, or abnormally stable proteins, interfere with several regulatory pathways, and the ubiquitin‐proteasome pathway is affected in a number of diseases, such as neurodegenerative diseases, cellular atrophies and malignancies. Therefore, dissecting the ubiquitin‐proteasome pathway and identifying proteins involved in conjunction with the signals required for specific degradation of certain substrates, would help in developing novel therapeutic approaches to treat diseases where the ubiquitin‐proteasome pathway is impaired.

Characterization of a hypoxia-inducible factor (HIF-1alpha ) from rainbow trout. Accumulation of protein occurs at normal venous oxygen tension.

The mammalian hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that controls the induction of several genes involved in glycolysis, erythropoiesis, and angiogenesis when cells are exposed to hypoxic conditions. Until now, the expression and function of HIF-1α have not been studied in fish, which experience wide fluctuations of oxygen tensions in their natural environment. Using electrophoretic mobility shift assay, we have ascertained that a hypoxia-inducible factor is present in rainbow trout cells. We have also cloned the full-length cDNA (3605 base pairs) of the HIF-1α from rainbow trout with a predicted protein sequence of 766 amino acids that showed a 61% similarity to human and mouse HIF-1α. Polyclonal antibodies against the N-terminal part (amino acids 12–363) and the C-terminal part (amino acids 330–730) of rainbow trout HIF-1α protein recognized rainbow trout and chinook salmon HIF-1α protein in Western blot analysis. Also, the human and mouse HIF-1α proteins were recognized by the N-terminal rainbow trout anti-HIF-1α antibody but not by the C-terminal HIF-1α antibody. The accumulation of HIF-1α was studied by incubating rainbow trout and chinook salmon cells at different oxygen concentrations from 20 to 0.2% O2 for 1 h. The greatest accumulation of HIF-1α protein occurred at 5% O2 (38 torr), a typical oxygen tension of venous blood in normoxic animals. The protein stability experiments in the absence or presence of a proteasome inhibitor, MG-132, demonstrated that the inhibitor is able to stabilize the protein, which normally is degraded via the proteasome pathway both in normoxia and hypoxia. Notably, the hypoxia response element of oxygen-dependent degradation domain is identical in mammalian, Xenopus, and rainbow trout HIF-1α proteins, suggesting a high degree of evolutionary conservation in degradation of HIF-1α protein.

HSF1 at a glance

ABSTRACT Heat shock factor 1 (HSF1) is an evolutionarily highly conserved transcription factor that coordinates stress-induced transcription and directs versatile physiological processes in eukaryotes. The central position of HSF1 in cellular homeostasis has been well demonstrated, mainly through its strong effect in transactivating genes that encode heat shock proteins (HSPs). However, recent genome-wide studies have revealed that HSF1 is capable of reprogramming transcription more extensively than previously assumed; it is also involved in a multitude of processes in stressed and non-stressed cells. Consequently, the importance of HSF1 in fundamental physiological events, including metabolism, gametogenesis and aging, has become apparent and its significance in pathologies, such as cancer progression, is now evident. In this Cell Science at a Glance article, we highlight recent advances in the HSF1 field, discuss the organismal control over HSF1, and present the processes that are mediated by HSF1 in the context of cell type, cell-cycle phase, physiological condition and received stimuli.