HSF1 Binding Research Articles

Abstract 6441: Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer

Abstract Background. HSF1 helps cancer cells cope with multiple stresses caused by oncogene activation. Methods. See details below. Results. We discovered the bisamide NXP800 as an inhibitor of HSF1-mediated transcription through phenotypic screening and med chem optimization. Gene expression microarray analysis of human cancer cell lines and tumor xenografts treated with bisamide inhibitors indicated activation of the ATF4 axis of the ISR as a key mechanism of action. In SK-OV-3 ovarian cancer xenografts, NXP800 showed clear PK/PD relationships with increased expression of ATF4 transcriptional target genes alongside decreased expression of HSF1 transcriptional targets. NXP800 also caused tumor regressions in this model. Expanding in additional human ovarian cancer xenograft models, we observed efficacy in five and a complete lack of response in three others. The sensitive models all had homozygous deleterious mutations in the ARID1A gene, whereas the non-responding xenografts were all wild type (WT). ARID1A is a component of the SWI/SNF chromatin remodelling complex involved in repression and activation of target genes. Subsequent screening of the large Sanger human cancer cell line panel confirmed ARID1A as the most significant common disease-related alteration predicting sensitivity to NXP800 in ovarian cancer cell lines. This predictive relationship was confirmed in an ARID1A isogenic HCT-116 cell line pair; sensitivity was greater in the homozygous ARID1A mutant cells compared to WT, resulting in PARP cleavage in the mutant cells only. In vivo there was no effect in WT HCT-116 xenografts whereas growth inhibition was observed in the homozygous mutant cells, resulting in significantly smaller tumors. In addition, the induction of ATF4 target genes was stronger and more prolonged in the mutant cells. It is known that whereas short term ATF4 activation is adaptive, persistent activation can promote the induction of apoptosis. CHIP-seq analysis confirmed clear relationships between ARID1A status, ATF4 and HSF1 promoter occupancy, and the distribution of BRG1 and RNA pol II at target sites, although these were often gene-specific and complex. A relatively simple example is the regulation of the INHBE gene (expression of which is a PD biomarker). In untreated samples there is no binding of ATF4, BRG1 and RNA pol II at the INHBE promoter. NXP800 treatment results in the recruitment of BRG1, ATF4 and RNA pol II in ARID1A mutant TOV-21G cells but not in the RMGI WT cells, leading to increased expression in the mutant cells only. Conclusions. We propose that ARID1A loss alters the binding and recruitment of ATF4 and HSF1 leading to the altered and prolonged expression of ATF4 target genes and increased sensitivity to NXP800. NXP800 is currently in phase Ib for the treatment of ARID1A mutant platinum resistant ovarian cancer (NCT05226507). Citation Format: Robert H. te Poele, Marissa Powers, Swee Sharp, Emmanuel de Billy, Maria Taskinen, Loredana Pellegrino, Sharon Gowan, Asadh Miah, Angela Hayes, Matthew Cheeseman, Keith Jones, Suzanne Eccles, Florence Raynaud, Paul Clarke, Paul Workman. Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6441.

Transcriptional responses of cancer cells to heat shock-inducing stimuli involve amplification of robust HSF1 binding

Responses of cells to stimuli are increasingly discovered to involve the binding of sequence-specific transcription factors outside of known target genes. We wanted to determine to what extent the genome-wide binding and function of a transcription factor are shaped by the cell type versus the stimulus. To do so, we induced the Heat Shock Response pathway in two different cancer cell lines with two different stimuli and related the binding of its master regulator HSF1 to nascent RNA and chromatin accessibility. Here, we show that HSF1 binding patterns retain their identity between basal conditions and under different magnitudes of activation, so that common HSF1 binding is globally associated with distinct transcription outcomes. HSF1-induced increase in DNA accessibility was modest in scale, but occurred predominantly at remote genomic sites. Apart from regulating transcription at existing elements including promoters and enhancers, HSF1 binding amplified during responses to stimuli may engage inactive chromatin.

The CSN5/HSF/SPI1/PU.1 Axis Regulates Cell Proliferation in Hypocellular Myelodysplastic Syndrome Patients.

This study explored the relationship between the activation of the jak/stat3 signaling pathway and the CSN5 gene transcript and protein expression levels in the hematopoietic stem cells of patients with myelodysplastic syndromes (MDSs). This study also aimed to investigate the correlation between the expression level of CSN5 and the deubiquitination of HSF1, as well as the transcript level of the spi1/pu.1 genes to explore the pathogenesis of MDS. We isolated cells from normal individuals and MDS patients, and the mRNA and protein expression levels of spi1/pu.1 in cd34+ cells (hematopoietic stem cells) were measured by PCR and western blotting, respectively. A ChIP assay was used to detect the binding of HSF1 to the spi1/pu.1 promoter in cd34+ cells. The ubiquitination of HSF1 in cd34+ cells was detected by CO-IP. The binding of HSF1 and Fbxw7α was detected in in cd34+ cells by CO-IP. The binding of HSF1 and CSN5 was evaluated. A luciferase reporter assay was used to detect the effect of STAT3 on CSN5 promoter activation in cd34+ cells. Western blotting was used to detect the phosphorylation of STAT3 in cd34+ cells of MDS patients. The binding of STAT3 and C/EBP beta in cd34+ cells was detected by CO-IP. Inhibition of SPI1/PU.1 expression was observed in MDS samples with low proliferation ability. Further experiments proved that phosphorylation of STAT3 affected CSN5 function and mediated the ubiquitination of HSF, the upstream regulator of SPI1/PU.1 transcription, which led to the inhibition of SPI1/PU.1 expression. Restoration of CSN5 rescued the inhibition of HSF1 ubiquitination, causing SPI1/PU.1 transcription to resume and increasing SPI1/PU.1 expression, promoting the recovery of cell proliferation in hypocellular MDS. Our research revealed the regulatory role of the CSN5/HSF/SPI1/PU.1 axis in hypocellular MDS, providing a probable target for clinical intervention.

Abstract 620: Hsf-1 Enhances Cardioprotective Potential Of Stem Cells Via Exosome Biogenesis And Their Mirna Cargo Enrichment.

Introduction: Stem cells work via different mechanisms of which paracrine mechanism is able to justify most of the effects. Therefore, identifying the control arms for paracrine cocktail production is necessary to tailor stem cell functions. Objective: This study shows how HSF1 determines stem cell potential to repair ischemic heart via affecting production of paracrine factors including exosome and their miRNA cargo. Methods and Results: CRISPR-Cas9 gene editing tool was used to knock out HSF1 gene in nMSCs while lentiviral hsRNA constructs for HSF1 were used to overexpress it in aMSCs. We identified that HSF1 knockout resulted in reduced cardiac regeneration abilities in nMSCs while its overexpression in aMSCs had opposite effects as depicted by functional characteristics including LVEF, LVEDV, LVESV and LVFS in our rat ischemic model. Further, we found that exosome number in nMSCs decreased significantly after HSF1 knockout while it went up in aMSCs after HSF1’s overexpression as determined by NanoSite particle tracking analysis. The exosomes from nMSCs (nEXOs), aMSCs (aEXOs) and their genetically modified versions (nKO-EXOs) and aOE-EXOs) respectively) were analysed for miRNA cargo using RT-PCR. nKO-EXOs showed significantly reduced candidate miRNA expression (miR-145, miR-146, 199-3p, 199b and miR-590) than nEXOs while aOE-EXOs had increased candidate miRNA expression compared to aEXOs. Since HSF1 does not bind to RNA molecules, we hypothesised that YBX1, another protein highly expressed by nMSCs than aMSCs and has RNA binding nature, under the influence of HSF1 sorts miRNAs into exosomes. YBX1 expression was found to be controlled by HSF1 as shown by RT-PCR and western results. HSF1 controls YBX1 expression, at transcriptional level, is strongly supported by our finding that YBX1 promoter possesses heat shock elements (HSEs) as HSF1 binding sites. We further went on to see whether YBX1 alteration affects miRNA loading of exosomes and it was demonstrated that absence of YBX1 resulted in accumulation of candidate miRNAs into the cells. Conclusion: Together, our data shows that HSF1 plays a critical role in determining the regenerative potential of stem cells. HSF1 does that by affecting exosome biogenesis and miRNA cargo sorting via YBX1 protein.

Heat shock transcription factors demonstrate a distinct mode of interaction with mitotic chromosomes.

A large number of transcription factors have been shown to bind and interact with mitotic chromosomes, which may promote the efficient reactivation of transcriptional programs following cell division. Although the DNA-binding domain (DBD) contributes strongly to TF behavior, the mitotic behaviors of TFs from the same DBD family may vary. To define the mechanisms governing TF behavior during mitosis in mouse embryonic stem cells, we examined two related TFs: Heat Shock Factor 1 and 2 (HSF1 and HSF2). We found that HSF2 maintains site-specific binding genome-wide during mitosis, whereas HSF1 binding is somewhat decreased. Surprisingly, live-cell imaging shows that both factors appear excluded from mitotic chromosomes to the same degree, and are similarly more dynamic in mitosis than in interphase. Exclusion from mitotic DNA is not due to extrinsic factors like nuclear import and export mechanisms. Rather, we found that the HSF DBDs can coat mitotic chromosomes, and that HSF2 DBD is able to establish site-specific binding. These data further confirm that site-specific binding and chromosome coating are independent properties, and that for some TFs, mitotic behavior is largely determined by the non-DBD regions.

Trimetazidine enhances myocardial angiogenesis in pressure overload-induced cardiac hypertrophy mice through directly activating Akt and promoting the binding of HSF1 to VEGF-A promoter

Latest clinical research shows that trimetazidine therapy during the perioperative period relieves endothelial dysfunction in patients with unstable angina induced by percutaneous coronary intervention. In this study we investigated the effects of TMZ on myocardial angiogenesis in pressure overload-induced cardiac hypertrophy mice. Cardiac hypertrophy was induced in mice by transverse aortic constriction (TAC) surgery. TAC mice were administered trimetazidine (2.8 mg/100 µL, i.g.) for 28 consecutive days. We showed that trimetazidine administration significantly increased blood vessel density in the left ventricular myocardium and abrogated cardiac dysfunction in TAC mice. Co-administration of a specific HSF1 inhibitor KRIBB11 (1.25 mg/100 µL, i.h.) abrogated the angiogenesis-promoting effects of trimetazidine in TAC mice. Using luciferase reporter and electrophoretic mobility shift assays we demonstrated that the transcription factor HSF1 bound to the promoter region of VEGF-A, and the transcriptional activity of HSF1 was enhanced upon trimetazidine treatment. In molecular docking analysis we found that trimetazidine directly bound to Akt via a hydrogen bond with Asp292 and a pi–pi bond with Trp80. In norepinephrine-treated HUVECs, we showed that trimetazidine significantly increased the phosphorylation of Akt and the synergistic nuclear translocation of Akt and HSF1, as well as the binding of Akt and HSF1 in the nucleus. These results suggest that trimetazidine enhances myocardial angiogenesis through a direct interaction with Akt and promotion of nuclear translocation of HSF1, and that trimetazidine may be used for the treatment of myocardial angiogenic disorders in hypertensive patients.

FgSfl1 and Its Conserved PKA Phosphorylation Sites Are Important for Conidiation, Sexual Reproduction, and Pathogenesis in Fusarium graminearum

The fungal plant pathogen, Fusarium graminearum, contains two genes, FgCPK1 and FgCPK2, encoding the catalytic subunits of cAMP-dependent protein kinase A. FgCPK1 and FgCPK2 are responsible for most of the PKA activities and have overlapping functions in various cellular processes in F. graminearum. The cpk1 cpk2 double mutant was significantly reduced in growth, rarely produced conidia, and was non-pathogenic. In this study, we found that the cpk1 cpk2 double mutant was unstable and produced fast-growing spontaneous sectors that were defective in plant infection. All spontaneous suppressor strains had mutations in FgSFL1, a transcription factor gene orthologous to SFL1 in yeast. Thirteen suppressor strains had non-sense mutations at Q501, three suppressor strains had frameshift mutations at W198, and five suppressor strains had mutations in the HSF binding domain of FgSfl1. Only one suppressor strain had both a non-synonymous mutation at H225 and a non-sense mutation at R490. We generated the SFL1 deletion mutant and found that it produced less than 2% of conidia than that of the wild-type strain PH-1. The sfl1 mutant was significantly reduced in the number of perithecia on carrot agar plates at 7 days post-fertilization (dpf). When incubated for more than 12 days, ascospore cirrhi were observed on the sfl1 mutant perithecia. The infection ability of the sfl1 deletion mutant was also obviously defective. Furthermore, we found that in addition to the S223 and S559 phosphorylation sites, FgSFL1 had another predicted phosphorylation site: T452. Interestingly, the S223 phosphorylation site was responsible for sexual reproduction, and the T452 phosphorylation site was responsible for growth and sexual reproduction. Only the S559 phosphorylation site was found to play an important role in conidiation, sexual reproduction, and infection. Overall, our results indicate that FgSFL1 and its conserved PKA phosphorylation sites are important for vegetative growth, conidiation, sexual reproduction, and pathogenesis in F. graminearum.

Structures of heat shock factor trimers bound to DNA

SummaryHeat shock factor 1 (HSF1) and 2 (HSF2) play distinct but overlapping regulatory roles in maintaining cellular proteostasis or mediating cell differentiation and development. Upon activation, both HSFs trimerize and bind to heat shock elements (HSEs) present in the promoter region of target genes. Despite structural insights gained from recent studies, structures reflecting the physiological architecture of this transcriptional machinery remains to be determined. Here, we present co-crystal structures of human HSF1 and HSF2 trimers bound to DNA, which reveal a triangular arrangement of the three DNA-binding domains (DBDs) with protein-protein interactions largely mediated by the wing domain. Two structural properties, different flexibility of the wing domain and local DNA conformational changes induced by HSF binding, seem likely to contribute to the subtle differential specificity between HSF1 and HSF2. Besides, two more structures showing DBDs bound to “two-site” head-to-head HSEs were determined as additions to the published tail-to-tail dimer-binding structures.

Insulin/IGF-1 signaling and heat stress differentially regulate HSF1 activities in germline development.

SUMMARYGermline development is sensitive to nutrient availability and environmental perturbation. Heat shock transcription factor 1 (HSF1), a key transcription factor driving the cellular heat shock response (HSR), is also involved in gametogenesis. The precise function of HSF1 (HSF-1 in C. elegans) and its regulation in germline development are poorly understood. Using the auxin-inducible degron system in C. elegans, we uncovered a role of HSF-1 in progenitor cell proliferation and early meiosis and identified a compact but important transcriptional program of HSF-1 in germline development. Interestingly, heat stress only induces the canonical HSR in a subset of germ cells but impairs HSF-1 binding at its developmental targets. Conversely, insulin/insulin growth factor 1 (IGF-1) signaling dictates the requirement for HSF-1 in germline development and functions through repressing FOXO/DAF-16 in the soma to activate HSF-1 in germ cells. We propose that this non-cell-autonomous mechanism couples nutrient-sensing insulin/IGF-1 signaling to HSF-1 activation to support homeostasis in rapid germline growth.

Transcription Factor HSF1 Suppresses the Expression of Surfactant Protein D in Cells Infected with Aspergillus fumigatus.

Aspergillosis is a life-threatening disease in patients with compromised immune systems. The process of fungal invasion is an important step during host cell infection. We investigated the transcription factor and promoter region of SFTPD, which is activated during the infection process in conidia-treated cells. To investigate the promoter activity of SFTPD in fungal-infected cells, we cloned various lengths of the promoter region (−1000 to +1) of SFTPD and examined its activity in A549 cells treated with Aspergillus fumigatus conidia. We determined the location within the promoter region of SFTPD that exhibits a response to conidia infection. AliBaba 2.1 software was used to predict the transcription factor involved as well as the binding sites in the SFTPD promoter region. The results of a decoy assay show that the HSF1 transcription factor is sufficient to decrease the SFTPD expression. Using chromatin immunoprecipitation, we confirmed that HSF1 directly binds to the selected sequence, which is located in the response region (−142 to −134 bp). These findings suggest that inhibiting the binding of HSF1 to the promoter region of SFTPD is an important step to prevent conidia infection.

Heme oxygenase-1 induction by heat shock in rat hepatoma cell line is regulated by the coordinated function of HSF1, NRF2 and BACH1.

The mechanism of heme oxygenase-1 (HO-1) induction by heat shock (HS) loading remains unclear. Here, we investigated the contribution of transcription factors to HS-induced HO-1 expression, using a rat hepatoma cell line (H-4-II-E). Our results demonstrated that HS treatment resulted in a marked induction of HO-1. Immunohistochemical analysis showed a slight mismatch in the expression levels of HO-1 and HSP70 by HS among cells, suggesting a conflict between multiple induction mechanisms. We observed HS-induced nuclear localization of, not only phosphorylated HSF1 but also NRF2, which is a typical transcription factor activated by oxidative stress. HSF1 knockdown in H-4-II-E markedly reduced HO-1 induction by HS, while NRF2 knockdown resulted in a partial effect. The chromatin immunoprecipitation assay demonstrated that HS loading resulted in significant binding of HSF1 to the HSE in the promoter proximal region of HO-1 gene and another HSE located close to the Maf recognition element (MARE) in the -4 kb upstream enhancer region 1, where NRF2 also bound, together with basic leucine zipper transcription factor 1, a negative transcription factor of HO-1. These observations indicate that HO-1 induction by HS is mainly mediated by HSF1 binding to the proximal HSE. NRF2 binding to MARE by HS is predominantly suppressed by an increased binding of BACH1.

Structures of Heat Shock Factor Trimers Bound to DNA Reveal the Panoramic Architecture of the HSF-Regulated Transcriptional Machinery

Heat shock factor 1 (HSF1) and 2 (HSF2) play distinct but overlapping regulatory roles in maintaining cellular protein homeostasis or mediating cell differentiation anddevelopment. Upon activation, both HSFs trimerize and bind to heat shock elements(HSEs) present in the promoter region of target genes. Despite innovative insights gained from recent structural studies, the panoramic architecture of this transcriptional machinery remains to be determined. Here, we present crystal structures of humanHSF1 and HSF2 trimers bound to DNA, which reveal a triangular arrangement of thethree DNA-binding domains (DBDs) with protein-protein interactions largely mediated by the wing domain. Two structural properties, the different flexibility of the wing domain and local DNA conformational changes induced by HSF binding, seem likelyto contribute to the subtle differential specificity between HSF1 and HSF2. Besides, two more structures showing DBDs bound to ‘two-site’ head-to-head HSEs weredetermined as additions to the published tail-to-tail dimer-binding structures. Our structures lay a foundation for further mechanistic study on how HSFs fulfill complex regulation on different target genes.

A simplified and sensitive immunoprecipitation approach for the analysis of HSF1 in murine liver tissue

Heat shock factor 1, HSF1, is one of several family members that recognize repeated nGAAn sequences associated with the heat shock element of heat shock and other genes. This transactivator is activated from a monomeric to trimeric form by oxidative, thermal and other stressors. Various studies show that HSF1 levels increase with cancer and decrease with aging and neurodegenerative disorders. It has a role in development as well as infections and inflammation.HSF1 is regulated by post-translational modifications and interactions with other proteins such as HSBP-1. Given its central importance in stress responsivity, various methods have been developed to identify HSF1 and its interacting partners. To date, multiple studies use conventional immunoprecipitation of HSF1 with commercially available antibodies which work well in cell lines but not whole tissue extracts. To remedy this shortfall, we developed a technique to retrieve activated HSF1 with an oligonucleotide link to a magnetic bead. The method captures HSF1 using a DNA sequence specific for HSF1 binding sites on promoter of heat shock genes. Confirmation of tissue derived HSF1 is identified using antibody against HSF1. The magnetic beads conjugated with DNA sequence specific to HSF1 binding was capable of yielding a reproducible band of high signal intensity with low background after native gel electrophoresis and ECL. Thus, the trimeric form of HSF1 can be isolated from tissue with magnetic beads conjugated with a short DNA sequence specific to HSF1 binding. This new method to identify HSF1 is economic, easy, and reproducible and does not require specialized equipment. It overcomes limitations of HSF1 tissue extraction by conventional immunoprecipitation, thus allowing for new approaches to understand HSF1 function in animal and human tissue.•HSF1 is a transcription factor that homotrimerize and binds to a conserved regulatory site, the heat shock element (HSE), consists of repeats of pentameric sequence ‘5-nGAAn-3’ present in the promoters of inducible heat shock protein genes.•This protocol allows isolation of trimeric forms of HSF1 from tissue lysate using magnetic beads conjugated with a short DNA sequence with specific binding to HSF1.•This method is easy, economic and does not require unique instrumentation.

Insulin/IGF-1 Signaling and Heat Stress Differentially Regulate HSF1 Activities in Germline Development

Germline development is sensitive to nutrient availability and environmental perturbation. HSF1, a key transcription factor driving the cellular heat shock response (HSR), is also involved in gametogenesis. However, the precise function of HSF1 and its regulation in germline development are poorly understood. Using the auxin-inducible degron system in C. elegans, we uncovered a role for HSF-1 in germline progenitor cell proliferation and early meiosis, and identified a compact yet important HSF-1 transcriptional program in germline development. Interestingly, heat stress only induces the canonical HSR in a subset of germ cells, but impairs HSF-1 binding at its developmental targets. Conversely, we found that insulin/IGF-1 signaling dictates the requirement for HSF-1 in germline development, and functions through repressing FOXO/DAF-16 in the soma to activate HSF-1 in germ cells. We propose that this cell-non-autonomous mechanism couples the nutrient sensing, insulin/IGF-1 signaling pathway to HSF-1 activation to support homeostasis and development of the germline.

Co-enzyme Q10 protects chicken hearts from in vivo heat stress via inducing HSF1 binding activity and Hsp70 expression.

In this report, we investigated the protective function of co-enzyme Q10 on chicken hearts during in vivo heat stress (HS) and the relationship with Hsp70 expression. The concentration of co-enzyme Q10 (Q10) in the serum indicated that Q10 exogenously added prior HS was fully absorbed by chickens and is maintained at high levels during HS. The level of heart and oxidative damage-associated enzymes in the serum revealed that treatment with Q10 decreased the activity of CK-MB, CK, and LDH compared with the HS group; moreover, oxidative injury was also alleviated by Q10 according to the level of SOD, MDA, and T-AOC in the serum compared with HS group during heat stress. A pathological examination indicated that the chicken hearts suffered serious damage during HS, including hemorrhage, granular changes, karyopyknosis, and cardiac muscle fiber disorder; however, the extent of heart damage was reduced in HS + Q10 group. Our results indicated that the addition of Q10 could upregulate the expression of Hsp70 during HS compared with the HS group. Compared with the HS group, the addition of Q10 significantly increased the gene expression of hsf1 during HS and hsf3 at 5h of HS. The expression of hsf2 and hsf4 was not influenced by HS. Q10 could only accelerate the trimerization of HSF1 as well binding activities to Hsp70 HSE according to native page and ChIP assays. These findings suggest that co-enzyme Q10 can protect chicken hearts from in vivo HS by inducing HSF1 binding activity and Hsp70 expression.

Genetic and epigenetic determinants establish a continuum of Hsf1 occupancy and activity across the yeast genome.

Heat shock factor 1 is the master transcriptional regulator of molecular chaperones and binds to the same cis-acting heat shock element (HSE) across the eukaryotic lineage. In budding yeast, Hsf1 drives the transcription of ∼20 genes essential to maintain proteostasis under basal conditions, yet its specific targets and extent of inducible binding during heat shock remain unclear. Here we combine Hsf1 chromatin immunoprecipitation sequencing (seq), nascent RNA-seq, and Hsf1 nuclear depletion to quantify Hsf1 binding and transcription across the yeast genome. We find that Hsf1 binds 74 loci during acute heat shock, and these are linked to 46 genes with strong Hsf1-dependent expression. Notably, Hsf1’s induced DNA binding leads to a disproportionate (∼7.5-fold) increase in nascent transcription. Promoters with high basal Hsf1 occupancy have nucleosome-depleted regions due to the presence of “pioneer factors.” These accessible sites are likely critical for Hsf1 occupancy as the activator is incapable of binding HSEs within a stably positioned, reconstituted nucleosome. In response to heat shock, however, Hsf1 accesses nucleosomal sites and promotes chromatin disassembly in concert with the Remodels Structure of Chromatin (RSC) complex. Our data suggest that the interplay between nucleosome positioning, HSE strength, and active Hsf1 levels allows cells to precisely tune expression of the proteostasis network.

Identification of genome-wide binding sites of heat shock factor 1, Hsf1, under basal conditions in the human pathogenic yeast, Candida albicans

The master regulator of thermal stress response, Hsf1, is also an essential determinant for viability and virulence in Candida albicans. Our recent studies highlighted that apart from ubiquitous roles of Hsf1 at higher temperatures, it also has myriad non-heat shock responsive roles essential under iron deprivation and drug defense. Here, we further explored its implications in the normal cellular functioning, by profiling its genome-wide occupancy using chromatin immuno-precipitation coupled to high-density tiling arrays under basal and iron deprived conditions. Hsf1 recruitment profiles revealed that it binds to promoters of 660 genes of varied functions, under both the conditions, however, elicited variability in intensity of binding. For instance, Hsf1 binding was observed on several genes of oxidative and osmotic stress response, cell wall integrity, iron homeostasis, mitochondrial, hyphal and multidrug transporters. Additionally, the present study divulged a novel motif under basal conditions comprising, -GTGn3GTGn3GTG- where, Hsf1 displays strong occupancy at significant number of sites on several promoters distinct from the heat induced motif. Hence, by binding to and regulating major chaperones, stress responsive genes and drug resistance regulators, Hsf1 is imperative in regulating various cellular machineries. The current study provides a framework for understanding novel aspects of how Hsf1 coordinates diverse cellular functions.

Fibronectin is a stress responsive gene regulated by HSF1 in response to geldanamycin

Fibronectin is an extracellular matrix glycoprotein with key roles in cell adhesion and migration. Hsp90 binds directly to fibronectin and Hsp90 depletion regulates fibronectin matrix stability. Where inhibition of Hsp90 with a C-terminal inhibitor, novobiocin, reduced the fibronectin matrix, treatment with an N-terminal inhibitor, geldanamycin, increased fibronectin levels. Geldanamycin treatment induced a stress response and a strong dose and time dependent increase in fibronectin mRNA via activation of the fibronectin promoter. Three putative heat shock elements (HSEs) were identified in the fibronectin promoter. Loss of two of these HSEs reduced both basal and geldanamycin-induced promoter activity, as did inhibition of the stress-responsive transcription factor HSF1. Binding of HSF1 to one of the putative HSE was confirmed by ChIP under basal conditions, and occupancy shown to increase with geldanamycin treatment. These data support the hypothesis that fibronectin is stress-responsive and a functional HSF1 target gene. COLA42 and LAMB3 mRNA levels were also increased with geldanamycin indicating that regulation of extracellular matrix (ECM) genes by HSF1 may be a wider phenomenon. Taken together, these data have implications for our understanding of ECM dynamics in stress-related diseases in which HSF1 is activated, and where the clinical application of N-terminal Hsp90 inhibitors is intended.

Mitochondrial Stress Restores the Heat Shock Response and Prevents Proteostasis Collapse during Aging

In Caenorhabditis elegans, the programmed repression of the heat shock response (HSR) accompanies the transition to reproductive maturity, leaving cells vulnerable to environmental stress and protein aggregation with age. To identify the factors driving this event, we performed an unbiased genetic screen for suppressors of stress resistance and identified the mitochondrial electron transport chain (ETC) as a central regulator of the age-related decline of the HSR and cytosolic proteostasis. Mild downregulation of ETC activity, either by genetic modulation or exposure to mitochondria-targeted xenobiotics, maintained the HSR in adulthood by increasing HSF-1 binding and RNA polymerase II recruitment at HSF-1 target genes. This resulted in a robust restoration of cytoplasmic proteostasis and increased vitality later in life, without detrimental effects on fecundity. We propose that low levels of mitochondrial stress regulate cytoplasmic proteostasis and healthspan during aging by coordinating the long-term activity of HSF-1 with conditions preclusive to optimal fitness.

Preheating mitigates cadmium toxicity in zebrafish livers: Evidence from promoter demethylation, gene transcription to biochemical levels

The working hypothesis for this study was that moderate heat stress would alleviate the deleterious effects of subsequent cadmium (Cd) exposure on fish. Thus, zebrafish (Danio rerio) were subjected to water maintained at 26°C and 34°C for 4days, and then exposed to 0 or 200μg/L Cd for 1 week at 26°C. Multiple indicators were measured from livers of zebrafish at different levels, including DNA, RNA, protein and enzymatic activity associated with oxidative stress, inflammation and metal transport. The ameliorative effect of preheatinging on Cd toxicity was demonstrated. In the Cd-exposed groups, preheating decreased mortality and lipid peroxidation, increased activity levels of catalase (CAT) and copper/zinc-superoxide dismutase (Cu/Zn-SOD), and up-regulated mRNA levels of heat shock protein 70 (HSP70) and heat shock factor 2 (HSF2). Preheating also mitigated Cd-induced increases in protein and mRNA levels of metallothioneins (MTs), and mRNA levels of several inflammation-related genes. Furthermore, preheating alone dramatically up-regulated mRNA levels of genes related to antioxidant and immune defenses, zinc and copper transporters, protein folding, and reduced methylation levels in the HSF binding motif of the HSP70 promoter. Overall, preheating-induced accumulation of transcripts via demethylation might support the rapid defense responses at post-transcriptional levels caused by subsequent Cd exposure, indicating an adaptive mechanism for organisms exposed to one mild stressor followed by another.