Human Heat Shock Factor Research Articles

Protein Misfolding Releases Human HSF1 from HSP70 Latency Control

Heat shock factor 1 (HSF1) responds to stress to mount the heat shock response (HSR), a conserved transcriptional program that allows cells to maintain proteostasis by upregulating heat shock proteins (HSPs). The homeostatic stress regulation of HSF1 plays a key role in human physiology and health but its mechanism has remained difficult to pinpoint. Recent work in the budding yeast model has implicated stress-inducible chaperones of the HSP70 family as direct negative regulators of HSF1 activity. Here, we have investigated the latency control and activation of human HSF1 by HSP70 and misfolded proteins. Purified oligomeric HSF1-HSP70 (HSPA1A) complexes exhibited basal DNA binding activity that was inhibited by increasing the levels of HSP70 and, importantly, misfolded proteins reverted the inhibitory effect. Using site-specific UV photo-crosslinking, we monitored HSP70-HSF1 complexes in HEK293T cells. While HSF1 was bound by the substrate binding domain of HSP70 in unstressed cells, activation of HSF1 by heat shock as well as by inducing the misfolding of newly synthesized proteins resulted in release of HSF1 from the chaperone. Taken our results together, we conclude that latent HSF1 populate dynamic complexes with HSP70, which are sensitive to increased levels of misfolded proteins that compete for binding to the HSP70 substrate binding domain. Thus, human HSF1 is activated by various stress conditions that all titrate available HSP70.

Intermolecular Interactions between Cysteine and Aromatic Amino Acids with a Phenyl Moiety in the DNA-Binding Domain of Heat Shock Factor 1 Regulate Thermal Stress-Induced Trimerization.

In this study, we investigated the trimerization mechanism and structure of heat shock factor 1 (HSF1) using western blotting, tryptophan (Trp) fluorescence spectroscopy, and molecular modeling. First, we examined the DNA-binding domains of human (Homo sapiens), goldfish (Carassius auratus), and walleye pollock (Gadus chalcogrammus) HSF1s by mutating key residues (36 and 103) that are thought to directly affect trimer formation. Human, goldfish, and walleye pollock HSF1s contain cysteine at residue 36 but cysteine (C), tyrosine (Y), and phenylalanine (F), respectively, at residue 103. The optimal trimerization temperatures for the wild-type HSF1s of each species were found to be 42, 37, and 20 °C, respectively. Interestingly, a mutation experiment revealed that trimerization occurred at 42 °C when residue 103 was cysteine, at 37 °C when it was tyrosine, and at 20 °C when it was phenylalanine, regardless of the species. In addition, it was confirmed that when residue 103 of the three species was mutated to alanine, trimerization did not occur. This suggests that in addition to trimerization via disulfide bond formation between the cysteine residues in human HSF1, trimerization can also occur via the formation of a different type of bond between cysteine and aromatic ring residues such as tyrosine and phenylalanine. We also confirmed that at least one cysteine is required for the trimerization of HSF1s, regardless of its position (residue 36 or 103). Additionally, it was shown that the trimer formation temperature is related to growth and survival in fish.

PH Dependence of HSF1 trimerization is shaped by intramolecular interactions

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. Previous studies have shown that low pH within the physiological range directly activates HSF1 function in vitro. However, the detailed molecular mechanisms remain unclear. This study proposes a molecular mechanism based on the trimerization behavior of HSF1 at different pH values. Extensive mutagenesis of human and goldfish HSF1 revealed that the optimal pH for trimerization depended on the identity of residue 103. In particular, when residue 103 was occupied by tyrosine, a significant increase in the optimal pH was observed, regardless of the rest of the sequence. This behavior can be explained by the protonation state of the neighboring histidine residues, His101 and His110. Residue 103 plays a key role in trimerization by forming disulfide or non-covalent bonds with Cys36. If tyrosine resides at residue 103 in an acidic environment, its electrostatic interactions with positively charged histidine residues prevent effective trimerization. His101 and His110 are neutralized at a higher pH, which releases Tyr103 to interact with Cys36 and drives the effective trimerization of HSF1. This study showed that the protonation state of a histidine residue can regulate the intramolecular interactions, which consequently leads to a drastic change in the oligomerization behavior of the entire protein.

An 85-amino-acid polypeptide from Myrmeleon bore larvae (antlions) homologous to heat shock factor binding protein 1 with antiproliferative activity against MG-63 osteosarcoma cells in vitro.

Venomous arthropods have substances in their venom with antiproliferative potential for neoplastic cells. To identify a polypeptide from Myrmeleon bore (antlion) with antiproliferative activity against neoplastic cells, and to elucidate the molecular mechanism of the activity. We used gel filtration and ion exchange chromatography to purify a polypeptide with antiproliferative activity against MG-63 human osteosarcoma cells from a proteinaceous extract of antlion. The polypeptide was sequenced and the stability of its antiproliferative activity was tested under a range of conditions in vitro. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the antiproliferative activity of the polypeptide against the MG-63 osteosarcoma cells and MC3T3-E1 mouse calvarial osteoblasts, which were used as a non-neoplastic control. We used western blotting to compare the levels of expression of heat shock transcription factor 1 (HSF1), heat shock protein 90 (HSP90), cyclin-dependent kinase 4 (CDK4), and protein kinase B alpha (ATK1) in MG-63 osteosarcoma cells and their mouse homologs in MC3T3-E1 osteoblasts after their treatment with the antlion antiproliferative polypeptide (ALAPP). The 85-amino-acid ALAPP has a 56% sequence identity with the human heat shock factor binding protein 1 (HSBP1). The antiproliferative activity of the polypeptide is relatively insensitive to temperature, pH, and metal ions. ALAPP has a strong concentration-dependent antiproliferative activity against MG-63 osteosarcoma cells compared with its effect on MC3T3-E1 osteoblasts. ALAPP significantly upregulates the expression of HSF1 in MC3T3-EL osteoblasts, but not in MG-63 osteosarcoma. ALAPP significantly downregulated the expression of HSP90, CDK4, and AKT1 expression in MG-63 osteosarcoma, but not in the osteoblasts. ALAPP has significant antiproliferative activity against MG-63 osteosarcoma cells, but not nonneoplastic MC3T3-E1 osteoblasts. We speculate that non-neoplastic cells may evade the antiproliferative effect of ALAPP by upregulating HSF1 to maintain their HSP90, CDK4, and AKT1 expression at a relatively constant level.

Detailed protocol for optimised expression and purification of functional monomeric human Heat Shock Factor 1

Heat Shock Factor 1 (HSF1) is the master regulator of the heat shock response, a universal survival mechanism throughout eukaryotic species used to buffer potentially lethal proteotoxic conditions. HSF1's function in vivo is regulated by several factors, including post translational modifications and elevated temperatures, whereupon it forms trimers to bind with heat shock elements in DNA. Unsurprisingly, HSF1 is also extremely sensitive to elevated temperatures in vitro, which poses specific technical challenges when producing HSF1 using a recombinant expression system. Although there are several useful publications which outline steps taken for HSF1 expression and purification, studies that describe specific strategies and detailed protocols to overcome HSF1 trimerisation and degradation are currently lacking. Herein, we have reported our detailed experimental protocol for the expression and purification of monomeric human HSF1 (HsHSF1) as a major species. We also propose a refined method of inducing HsHSF1 activation in vitro, that we consider more accurately mimics HsHSF1 activation in vivo and is therefore more physiologically relevant.

RNA aptamer capture of macromolecular complexes for mass spectrometry analysis.

Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA–MS (aptamer affinity–mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA–MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.

FAM3C-YY1 axis is essential for TGFβ-promoted proliferation and migration of human breast cancer MDA-MB-231 cells via the activation of HSF1.

Family with sequence similarity three member C (FAM3C) (interleukin‐like EMT inducer [ILEI]), heat shock factor 1 (HSF1) and Ying‐Yang 1 (YY1) have been independently reported to be involved in the pathogenesis of various cancers. However, whether they are coordinated to trigger the development of cancer remains unknown. This study determined the role and mechanism of YY1 and HSF1 in FAM3C‐induced proliferation and migration of breast cancer cells. In human MDA‐MB‐231 breast cancer cell line, transforming growth factor‐β (TGFβ) up‐regulated FAM3C, HSF1 and YY1 expressions. FAM3C overexpression promoted the proliferation and migration of MDA‐MB‐231 cells with YY1 and HSF1 up‐regulation, whereas FAM3C silencing exerted the opposite effects. FAM3C inhibition repressed TGFβ‐induced HSF1 activation, and proliferation and migration of breast cancer cells. YY1 was shown to directly activate HSF1 transcription to promote the proliferation and migration of breast cancer cells. YY1 silencing blunted FAM3C‐ and TGFβ‐triggered activation of HSF1‐Akt‐Cyclin D1 pathway, and proliferation and migration of breast cancer cells. Inhibition of HSF1 blocked TGFβ‐, FAM3C‐ and YY1‐induced proliferation and migration of breast cancer cells. YY1 and HSF1 had little effect on FAM3C expression. Similarly, inhibition of HSF1 also blunted FAM3C‐ and TGFβ‐promoted proliferation and migration of human breast cancer BT‐549 cells. In human breast cancer tissues, FAM3C, YY1 and HSF1 protein expressions were increased. In conclusion, FAM3C activated YY1‐HSF1 signalling axis to promote the proliferation and migration of breast cancer cells. Furthermore, novel FAM3C‐YY1‐HSF1 pathway plays an important role in TGFβ‐triggered proliferation and migration of human breast cancer MDA‐MB‐231 cells.

HSF1 mediated TNF-α production during proteotoxic stress response pioneers proinflammatory signal in human cells.

The proteotoxic stress response that safeguards the cellular proteome from various stressors was shown to activate NF-κB signaling pathways (NκBS) with an underlying mechanism that is poorly understood. We show here that the TNF-α gene, a pleiotropic NκBS inducer, is a direct target of heat shock factor 1 (HSF1). Human HSF1 drives this process by assembling a multiprotein activation complex at a heat shock element (HSE) located at the 3'-UTR of the TNF-α gene (HSE5). HSF1 associated with the HSE5 at the TNF-α 3'-UTR communicates with the promoter through chromatin looping by recruiting lymphoid enhancer-binding factor 1 at an adjacent Wnt-responsive element through its transactivation domain. TNF-α thus produced guides the activation of NκBS by acting through TNF-α receptor 1 (TNFR1). Notably, cells with TNFR1-/- background or masked HSE5 through Clustered Regularly Interspaced Short Palindromic Repeats/dead CRISPR-associated protein 9 were defective in NκBS and exhibited marked alteration in cellular biology, which includes loss of ability of cancer cells to migrate, to clear the protein aggregates, and associated toxicity upon heat shock. For the first time, our results suggest that TNF-α thus produced pioneers the proinflammatory signal during proteotoxic stress response with an important implication for inflammation and cancer.-Ali, A., Biswas, A., Pal, M. HSF1 mediated TNF-α production during proteotoxic stress response pioneers proinflammatory signal in human cells.

붕어와 마우스의 간세포 배양에서 열 스트레스에 의해 유도되는 heat shock factor1 (HSF1)의 비교

Heat shock proteins (HSPs) are induced in response to various physiological or environmental stressors. However, the transcriptional activation of HSPs is regulated by a family of heat shock factors (HSFs). Fish models provide an ideal system for examining the biochemical and molecular mechanisms of adaptation to various temperatures and water environments. In this study, we examined the pattern differentials of heat shock factor 1 (HSF1) and expression of heat shock protein 70 (HSP70) in response to thermal stress in goldfish and mouse hepatocyte cultures by immune-blot analysis. Goldfish HSF1 (gfHSF1) changed from a monomer to a trimer at 33°C and showed slightly at 37°C, whereas mouse HSF1 (mHSF1) did so at 42°C. This experiment showed similar results to a previous study, indicating that gfHSF1 and mHSF1 play different temperature in the stress response. We also examined the activation conditions of the purified recombinant proteins in human HSF1 (hmHSF1) and gfHSF1 using CD spectroscopy and immune-blot analysis. The purified recombinant HSF1s were treated from 25°C to 42°C. Structural changes were observed in hmHSF1 and gfHSF1 according to the heat-treatment conditions. These results revealed that both mammal HSF1 (human and mouse HSF1) and fish HSF1 exhibited temperature-dependent changes; however, their optimal activation temperatures differed.

Azadiradione ameliorates polyglutamine expansion disease inDrosophilaby potentiating DNA binding activity of heat shock factor 1

Aggregation of proteins with the expansion of polyglutamine tracts in the brain underlies progressive genetic neurodegenerative diseases (NDs) like Huntington's disease and spinocerebellar ataxias (SCA). An insensitive cellular proteotoxic stress response to non-native protein oligomers is common in such conditions. Indeed, upregulation of heat shock factor 1 (HSF1) function and its target protein chaperone expression has shown promising results in animal models of NDs. Using an HSF1 sensitive cell based reporter screening, we have isolated azadiradione (AZD) from the methanolic extract of seeds of Azadirachta indica, a plant known for its multifarious medicinal properties. We show that AZD ameliorates toxicity due to protein aggregation in cell and fly models of polyglutamine expansion diseases to a great extent. All these effects are correlated with activation of HSF1 function and expression of its target protein chaperone genes. Notably, HSF1 activation by AZD is independent of cellular HSP90 or proteasome function. Furthermore, we show that AZD directly interacts with purified human HSF1 with high specificity, and facilitates binding of HSF1 to its recognition sequence with higher affinity. These unique findings qualify AZD as an ideal lead molecule for consideration for drug development against NDs that affect millions worldwide.

Purification, crystallization and X-ray diffraction analysis of the DNA-binding domain of human heat-shock factor 2.

Cells respond to various proteotoxic stimuli and maintain protein homeostasis through a conserved mechanism called the heat-shock response, which is characterized by the enhanced synthesis of heat-shock proteins. This response is mediated by heat-shock factors (HSFs). Four genes encoding HSF1-HSF4 exist in the genome of mammals. In this protein family, HSF1 is the orthologue of the single HSF in lower eukaryotic organisms and is the major regulator of the heat-shock response, while HSF2, which shows low sequence homology to HSF1, serves as a developmental regulator. Increasing evidence has revealed biochemical properties and functional roles that are unique to HSF2, such as its DNA-binding preference and sumoylation patterns, which are distinct from those of HSF1. The structural basis for such differences, however, is poorly understood owing to the lack of available mammalian HSF structures. The N-terminal DNA-binding domain (DBD) is the most conserved functional module and is the only crystallizable domain in HSFs. To date, only HSF1 homologue structures from yeast and fruit fly have been determined. Along with extensive studies of the HSF family, more structural information, particularly from members with a remoter phylogenic relationship to the reported structures, e.g. HSF2, is needed in order to better understand the detailed mechanisms of HSF biology. In this work, the recombinant DBD (residues 7-112) from human HSF2 was produced in Escherichia coli and crystallized. An X-ray diffraction data set was collected to 1.32 Å resolution from a crystal belonging to space group P212121 with unit cell-parameters a = 65.66, b = 67.26, c = 93.25 Å. The data-evaluation statistics revealed good quality of the collected data, thus establishing a solid basis for the determination of the first structure at atomic resolution in this protein family.

High-throughput screening system for inhibitors of human Heat Shock Factor 2.

Development of novel anti-cancer drug leads that target regulators of protein homeostasis is a formidable task in modern pharmacology. Finding specific inhibitors of human Heat Shock Factor 1 (hHSF1) has proven to be a challenging task, while screening for inhibitors of human Heat Shock Factor 2 (hHSF2) has never been described. We report the development of a novel system based on an in vivo cell growth restoration assay designed to identify specific inhibitors of human HSF2 in a high-throughput format. This system utilizes a humanized yeast strain in which the master regulator of molecular chaperone genes, yeast HSF, has been replaced with hHSF2 with no detrimental effect on cell growth. This replacement preserves the general regulatory patterns of genes encoding major molecular chaperones including Hsp70 and Hsp90. The controlled overexpression of hHSF2 creates a slow-growth phenotype, which is the basis of the growth restoration assay used for high-throughput screening. The phenotype is most robust when cells are cultured at 25 °C, while incubation at temperatures greater than 30 °C leads to compensation of the phenotype. Overexpression of hHSF2 causes overexpression of molecular chaperones which is a likely cause of the slowed growth. Our assay is characterized by two unique advantages. First, screening takes place in physiologically relevant, in vivo conditions. Second, hits in our screen will be of medically relevant potency, as compounds that completely inhibit hHSF2 function will further inhibit cell growth and therefore will not be scored as hits. This caveat biases our screening system for compounds capable of restoring hHSF2 activity to a physiologically normal level without completely inhibiting this essential system.

Function of Hsf1 in SV40 T-antigen-transformed HEK293T cells

Heat shock factor 1 (HSF1), a main regulator of the heat shock response in eukaryotes, increases cell survival in numerous pathophysiological conditions. The aim of the present study was to o bserve the function of defective HSF1 expression in HEK293T cells. shRNA of human HSF1 was constructed into the retroviral vector pLTHR generating pLTHR‑shRNA‑HSF1. The shRNA was transiently transfected into HEK293T cells to silence the expression of the HSF1 gene. Cell colony formation, MTT and cell cycle assays were used to analyze the SV40 T‑antigen (Ag)‑transformed cell proliferation rate. Immunoblotting was used to study the protein expression of HSF1, SV40 T‑Ag, p53, p21, heat shock protein 90 (Hsp90), Hsp70 and Hsp25. The results revealed that a deficiency in HSF1 expression inhibited cellular growth. Defective HSF1 upregulated the protein expression of p53, retinoblastoma protein (Rb) and SV40 T‑Ag, and reduced the association between SV40 T‑Ag and p53/Rb, which resulted in growth inhibition of SV40 T‑Ag‑transformed cells. In conclusion, HSF1 is involved in the regulation of SV40 T‑Ag‑induced cell growth and modulates the expression of p53 and Rb proteins.

Genomic Heat Shock Element Sequences Drive Cooperative Human Heat Shock Factor 1 DNA Binding and Selectivity

The heat shock transcription factor 1 (HSF1) activates expression of a variety of genes involved in cell survival, including protein chaperones, the protein degradation machinery, anti-apoptotic proteins, and transcription factors. Although HSF1 activation has been linked to amelioration of neurodegenerative disease, cancer cells exhibit a dependence on HSF1 for survival. Indeed, HSF1 drives a program of gene expression in cancer cells that is distinct from that activated in response to proteotoxic stress, and HSF1 DNA binding activity is elevated in cycling cells as compared with arrested cells. Active HSF1 homotrimerizes and binds to a DNA sequence consisting of inverted repeats of the pentameric sequence nGAAn, known as heat shock elements (HSEs). Recent comprehensive ChIP-seq experiments demonstrated that the architecture of HSEs is very diverse in the human genome, with deviations from the consensus sequence in the spacing, orientation, and extent of HSE repeats that could influence HSF1 DNA binding efficacy and the kinetics and magnitude of target gene expression. To understand the mechanisms that dictate binding specificity, HSF1 was purified as either a monomer or trimer and used to evaluate DNA-binding site preferences in vitro using fluorescence polarization and thermal denaturation profiling. These results were compared with quantitative chromatin immunoprecipitation assays in vivo. We demonstrate a role for specific orientations of extended HSE sequences in driving preferential HSF1 DNA binding to target loci in vivo. These studies provide a biochemical basis for understanding differential HSF1 target gene recognition and transcription in neurodegenerative disease and in cancer.

Polymorphisms in human heat shock factor-1 and analysis of potential biological consequences.

The stress-activated transcription factor, heat shock factor-1 (HSF1), regulates many genes including cytoprotective heat shock proteins (HSPs). We hypothesized that polymorphisms in HSF1 may alter the level or function of HSF1 protein accounting for interindividual viability in disease susceptibility or prognosis. We searched for exomic variants in HSF1 by querying human genome databases and directly sequencing DNA from 80 anonymous genomic DNA samples. Overall, HSF1 sequence was highly conserved, with no common variations. We found 31 validated deviations from a reference sequence in the dbSNP database and an additional 5 novel variants by sequencing, with allele frequencies that were 0.06 or less. Of these 36, 2 were in 5'-untranslated region (5'UTR), 10 in 3'UTR, and 24 in the coding region. The potential effects of 5'UTR on secondary structure, protein structure/function, and 3'UTR targets of microRNAs were analyzed using RNAFold, PolyPhen-2, SIFT, and MicroSNiper. One of the 5'UTR variants was predicted to strengthen secondary structure. Eight of 3'UTR variants were predicted to modify microRNA target sequences. Eight of the coding region variants were predicted to modify HSF1 structure/function. Reducing HSF1 levels in A549 cells using short hairpin RNA (shRNA) increased sensitivity to heat-induced killing demonstrating the impact that genetic variants that reduce HSF1 levels might have. Using the pmirGLO expression system, we found that the wild-type HSF1 3'UTR suppressed translation of a firefly luciferase reporter plasmid by 65%. Introducing two of four 3'UTR single nucleotide polymorphisms (SNPs) increased HSF1 3'UTR translational suppression by 27-44% compared with the wild-type HSF1 3'UTR sequence while a third SNP reduced suppression by 25%. HSF1 variants may alter HSF1 protein levels or function with potential effects on cell functions, including sensitivity to stress.

High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn device

We describe a versatile 96-well microplate-based device that utilizes affinity microcolumn chromatography to complement downstream plate-based processing in aptamer selections. This device is reconfigurable and is able to operate in serial and/or parallel mode with up to 96 microcolumns. We demonstrate the utility of this device by simultaneously performing characterizations of target binding using five RNA aptamers and a random library. This was accomplished through 96 total selection tests. Three sets of selections tested the effects of target concentration on aptamer binding compared to the random RNA library using aptamers to the proteins green fluorescent protein (GFP), human heat shock factor 1 (hHSF1), and negative elongation factor E (NELF-E). For all three targets, we found significant effects consistent with steric hindrance with optimum enrichments at predictable target concentrations. In a fourth selection set, we tested the partitioning efficiency and binding specificity of our three proteins’ aptamers, as well as two suspected background binding sequences, to eight targets running serially. The targets included an empty microcolumn, three affinity resins, three specific proteins, and a non-specific protein control. The aptamers showed significant enrichments only on their intended targets. Specifically, the hHSF1 and NELF-E aptamers enriched over 200-fold on their protein targets, and the GFP aptamer enriched 750-fold. By utilizing our device’s plate-based format with other complementary plate-based systems for all downstream biochemical processes and analysis, high-throughput selections, characterizations, and optimization were performed to significantly reduce the time and cost for completing large-scale aptamer selections. FigureSchematic breakdown of a microplate-based enrichment device for the selection of aptamers (MEDUSA), which can be customized and assembled in both parallel and serial configurations. Up to 96 selections can be performed simultaneously. Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-7661-7) contains supplementary material, which is available to authorized users.

Histidine (His83) is Essential for Heat Shock Factor 1 (HSF1) Activation in Protecting against Acid pH Stress

The activation of heat shock factor 1 (HSF1) can be induced by the changes in environmental pH, but the mechanism of HSF1 activation by acidification is not completely understood. This paper reports that a low pH (pH~6.0) can trigger human HSF1 activation. Considering the involvement of the imidazole group of histidine residues under acid pH stress, an in vitro EMSA experiment, Trp-fluorescence spectroscopy, and protein structural analysis showed that the residue, His83, is the essential for pH-dependent human HSF1-activation. To determine the roles of His83 in the HSF1-mediated stress response affecting the cellular acid resistance, mouse embryo fibroblasts with normal wild-type or mutant mouse HSF1 expression were preconditioned by heating or pH stress. The results suggest that His83 is essential for HSF1 activation or the HSF1-mediated transcription of heat shock proteins, in protecting cells from acid pH stress.

Heavy metal response of the heat shock protein 70 gene is mediated by duplicated heat shock elements and heat shock factor 1

Heavy metals induce transcription of a number of mammalian genes, but in most cases the mechanism of induction has not been well characterized. The human heat shock protein 70 gene (hsp70) is activated by several heavy metals such as Cd and Zn, and the heat shock element (HSE) has been proposed to mediate metal response by previous studies. However, it was observed that the lack of further upstream sequences rendered the hsp70 promoter unresponsive to metals. A detailed deletion analysis of the promoter revealed that the distal HSE-like sequence (dHSE) is required for heavy metal response. A supershift assay showed that the heat shock factor 1 (HSF1) recognized dHSE, as well as the proximal HSE (pHSE). An hsp70 promoter variant with a dHSE mutation that blocks HSF1 binding completely lost metal response, demonstrating that the HSF1/dHSE interaction is crucial. Another promoter variant with a similar pHSE mutation partially lost metal response, indicating that both HSEs are required for full activity. Knockdown of HSF1 dramatically reduced the metal response of hsp70, demonstrating its essential role. Furthermore, a reporter gene regulated by the human hsp70 promoter, which lacked metal response in HSF1-null mouse cells, acquired the response upon over-expression of a recombinant human HSF1. These results demonstrate that the duplicated HSEs and HSF1 constitute the mechanism for the heavy metal response of hsp70, which is distinct from the known metal regulatory system for the metallothionein genes.

Heat Shock Factor 1 (HSF1) Controls Chemoresistance and Autophagy through Transcriptional Regulation of Autophagy-related Protein 7 (ATG7)

Heat shock factor 1 (HSF1), a master regulator of heat shock responses, plays an important role in tumorigenesis. In this study we demonstrated that HSF1 is required for chemotherapeutic agent-induced cytoprotective autophagy through transcriptional up-regulation of autophagy-related gene ATG7. Interestingly, this is independent of the HSF1 heat shock response function. Treatment of cancer cells with the FDA-approved chemotherapeutic agent carboplatin induced autophagy and growth inhibition, which were significantly increased upon knockdown of HSF1. Mechanistic studies revealed that HSF1 regulates autophagy by directly binding to ATG7 promoter and transcriptionally up-regulating its expression. Significantly, breast cancer patient sample study revealed that a higher ATG7 expression level is associated with poor patient survival. This novel finding was further confirmed by analysis of two independent patient databases, demonstrating a prognostic value of ATG7. Furthermore, a strong positive correlation was observed between levels of HSF1 and ATG7 in triple-negative breast cancer patient samples, thus validating our in vitro findings. This is the first study identifying a critical role for HSF1 in controlling cytoprotective autophagy through regulation of ATG7, which is distinct from the HSF1 function in the heat shock response. This is also the first study demonstrating a prognostic value of ATG7 in breast cancer patients. These findings strongly argue that combining chemotherapeutic agents with autophagy inhibition by repressing HSF1/ATG7 axis represents a promising strategy for future cancer treatment.

Multiplexed Microcolumn-Based Process for Efficient Selection of RNA Aptamers

We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin—a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 108 with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.