Heat shock factor 1 is predominantly a nuclear protein before and after heat stress

Department of Zoology, University of Toronto, Mississauga, Ontario, Canada L5L 1C6.
Journal of Cell Science (Impact Factor: 5.43). 09/1999; 112 ( Pt 16)(16):2765-74.
Source: PubMed


The induction of the heat shock genes in eukaryotes by heat and other forms of stress is mediated by a transcription factor known as heat shock factor 1 (HSF1). HSF1 is present in unstressed metazoan cells as a monomer with low affinity for DNA, and upon exposure to stress it is converted to an 'active' homotrimer that binds the promoters of heat shock genes with high affinity and induces their transcription. The conversion of HSF1 to its active form is hypothesized to be a multistep process involving physical changes in the HSF1 molecule and the possible translocation of HSF1 from the cytoplasm to the nucleus. While all studies to date have found active HSF1 to be a nuclear protein, there have been conflicting reports on whether the inactive form of HSF is predominantly a cytoplasmic or nuclear protein. In this study, we have made antibodies against human HSF1 and have reexamined its localization in unstressed and heat-shocked human HeLa and A549 cells, and in green monkey Vero cells. Biochemical fractionation of heat-shocked HeLa cells followed by western blot analysis showed that HSF1 was mostly found in the nuclear fraction. In extracts made from unshocked cells, HSF1 was predominantly found in the cytoplasmic fraction using one fractionation procedure, but was distributed approximately equally between the cytoplasmic and nuclear fractions when a different procedure was used. Immunofluorescence microscopy revealed that HSF1 was predominantly a nuclear protein in both heat shocked and unstressed cells. Quantification of HSF1 staining showed that approximately 80% of HSF1 was present in the nucleus both before and after heat stress. These results suggest that HSF1 is predominantly a nuclear protein prior to being exposed to stress, but has low affinity for the nucleus and is easily extracted using most biochemical fractionation procedures. These results also imply that HSF1 translocation is probably not part of the multistep process in HSF1 activation for many cell types.

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    • "Inactive HSF1 monomers are predominantly located in the nucleus due to a potent bipartite nuclear localization signal, but HSF1 shuttling between the nucleus and cytosol has been reported. Thermal stress inhibits HSF1 export, leading to further nuclear accumulation (Anckar and Sistonen, 2011; Mercier et al., 1999). Monomeric HSF1 is stabilized by binding to chaperones, including Hsp70, Hsp90, and their cofactors. "
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    ABSTRACT: When exposed to proteotoxic environmental conditions, mammalian cells activate the cytosolic stress response in order to restore protein homeostasis. A key feature of this response is the heat shock transcription factor 1 (HSF1)-dependent expression of molecular chaperones. Here, we describe the results of an RNA interference screen in HeLa cells to identify modulators of stress response induction and attenuation. The modulator proteins are localized in multiple cellular compartments, with chromatin modifiers and nuclear protein quality control playing a central regulatory role. We find that the acetyltransferase, EP300, controls the cellular level of activatable HSF1. This involves acetylation of HSF1 at multiple lysines not required for function and results in stabilization of HSF1 against proteasomal turnover. Acetylation of functionally critical lysines during stress serves to fine-tune HSF1 activation. Finally, the nuclear proteasome system functions in attenuating the stress response by degrading activated HSF1 in a manner linked with the clearance of misfolded proteins.
    Full-text · Article · Feb 2014 · Cell
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    • "Likewise, human HSF1 has been reported both as a nuclear protein under all conditions (Martinez-Balbas et al., 1995; Mercier et al., 1999) and as a predominantly cytoplasmic protein that undergoes stressinduced nuclear translocation (Baler et al., 1993; Sarge et al., 1993). The reasons for these inconsistencies are unclear but may be due to artifacts of overexpression or biochemical preparations that artificially place inactive HSF1 in the cytoplasm (Mercier et al., 1999). Recently, Chiang et al. (2012) reported that an HSF-1::GFP protein in C. elegans exhibited diffuse nucleo-cytoplasmic fluorescence under control conditions , converting to weak nuclear localization after heat shock. "
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    ABSTRACT: The heat shock transcription factor (HSF) is a conserved regulator of heat shock-inducible gene expression. Organismal roles for HSF in physiological processes such as development, aging, and immunity have been defined largely through studies of the single C. elegans HSF homolog, hsf-1. However, the molecular and cell biological properties of hsf-1 in C. elegans are incompletely understood. We generated animals expressing physiological levels of an HSF-1::GFP fusion protein and examined its function, localization, and regulation in vivo. HSF-1::GFP was functional as measured by its ability to rescue phenotypes associated with two hsf-1 mutant alleles. Rescue of hsf-1 stress, aging, and development phenotypes was abolished in a DNA-binding-deficient mutant, demonstrating that the transcriptional targets of hsf-1 are critical to its function even in the absence of stress. Under non-stress conditions, HSF-1::GFP was found primarily in the nucleus. Following heat shock, HSF-1::GFP rapidly and reversibly redistributed into dynamic, sub-nuclear structures that share many properties with human nuclear stress granules, including colocalization with markers of active transcription. Rapid formation of HSF-1 stress granules required HSF-1 DNA binding activity and the threshold for stress granule formation was altered by growth temperature. HSF-1 stress granule formation was not induced by inhibition of IGF signaling, a pathway previously suggested to function upstream of hsf-1. Our findings suggest that development, stress, and aging pathways may regulate HSF-1 function in distinct ways, and that HSF-1 nuclear stress granule formation is an evolutionarily conserved aspect of HSF-1 regulation in vivo. © 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.
    Preview · Article · Oct 2012 · Aging cell
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    • "While the formation of active oligomers to acquire DNA binding activity is conserved across species, there has been some controversy regarding the subcellular localization of HSF1 under unstressed conditions in different metazoan systems. Some studies have suggested that HSF1 is predominantly cytoplasmic prior to heat-shock and nuclear after stress (Sarge et al., 1993; Sistonen et al., 1994), whereas others have suggested that HSF1 is always nuclear (Mercier et al., 1999). Using a transgenic line that expresses gfp-tagged hsf-1 under the control of its own endogenous promoter, HSF-1-GFP was observed in intestinal cells, body wall muscle cells, hypodermal cells as well as many neurons in the head and tail. "
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    ABSTRACT: Extended longevity is often correlated with increased resistance against various stressors. Insulin/IGF-1-like signaling (IIS) is known to have a conserved role in aging and cellular mechanisms against stress. In C. elegans, genetic studies suggest that heat-shock transcription factor HSF-1 is required for IIS to modulate longevity. Here, we report that the activity of HSF-1 is regulated by IIS. This regulation occurs at an early step of HSF-1 activation via two HSF-1 regulators, DDL-1 and DDL-2. Inhibition of DDL-1/2 increases longevity and thermotolerance in an hsf-1-dependent manner. Furthermore, biochemical analyses suggest that DDL-1/2 negatively regulate HSF-1 activity by forming a protein complex with HSF-1. The formation of this complex (DHIC) is affected by the phosphorylation status of DDL-1. Both the formation of DHIC and the phosphorylation of DDL-1 are controlled by IIS. Our findings point to DDL-1/2 as a link between IIS and the HSF-1 pathway.
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