Activation Of Hsp70 Promoter Research Articles

Yeast Two-Hybrid and One-Hybrid Screenings Identify Regulators of hsp70 Gene Expression.

The mammalian stress protein Hsp105β, which is specifically expressed during mild heat shock and localizes to the nucleus, induces the major stress protein Hsp70. In the present study, we performed yeast two-hybrid and one-hybrid screenings to identify the regulators of Hsp105β-mediated hsp70 gene expression. Six and two proteins were detected as Hsp105β- and hsp70 promoter-binding proteins, respectively. A luciferase reporter gene assay revealed that hsp70 promoter activation is enhanced by the transcriptional co-activator AF9 and splicing mediator SNRPE, but suppressed by the coiled-coil domain-containing protein CCDC127. Of these proteins, the knockdown of SNRPE suppressed the expression of Hsp70 irrespective of the presence of Hsp105β, indicating that SNRPE essentially functions as a transcriptional activator of hsp70 gene expression. The overexpression of HSP70 in tumor cells has been associated with cell survival and drug resistance. We here identified novel regulators of Hsp70 expression in stress signaling and also provided important insights into Hsp70-targeted anti-cancer therapy. J. Cell. Biochem. 117: 2109-2117, 2016. © 2016 Wiley Periodicals, Inc.

BAG3 affects the nucleocytoplasmic shuttling of HSF1 upon heat stress

Bcl2-associated athoanogene (BAG) 3 is a member of the co-chaperone BAG family. It is induced by stressful stimuli such as heat shock and heavy metals, and it regulates cellular adaptive responses against stressful conditions. In this study, we identified a novel role for BAG3 in regulating the nuclear shuttling of HSF1 during heat stress. The expression level of BAG3 was induced by heat stress in HeLa cells. Interestingly, BAG3 rapidly translocalized to the nucleus upon heat stress. Immunoprecipitation assay showed that BAG3 interacts with HSF1 under normal and stressed conditions and co-translocalizes to the nucleus upon heat stress. We also demonstrated that BAG3 interacts with HSF1 via its BAG domain. Over-expression of BAG3 down-regulates the level of nuclear HSF1 by exporting it to the cytoplasm during the recovery period. Depletion of BAG3 using siRNA results in reduced nuclear HSF1 and decreased Hsp70 promoter activity. BAG3 in MEF(hsf1−/−) cells actively translocalizes to the nucleus upon heat stress suggesting that BAG3 plays a key role in the processing of the nucleocytoplasmic shuttling of HSF1 upon heat stress.

Activity of heat shock genes' promoters in thermally contrasting animal species.

Heat shock gene promoters represent a highly conserved and universal system for the rapid induction of transcription after various stressful stimuli. We chose pairs of mammalian and insect species that significantly differ in their thermoresistance and constitutive levels of Hsp70 to compare hsp promoter strength under normal conditions and after heat shock (HS). The first pair includes the HSPA1 gene promoter of camel (Camelus dromedarius) and humans. It was demonstrated that the camel HSPA1A and HSPA1L promoters function normally in vitro in human cell cultures and exceed the strength of orthologous human promoters under basal conditions. We used the same in vitro assay for Drosophila melanogaster Schneider-2 (S2) cells to compare the activity of the hsp70 and hsp83 promoters of the second species pair represented by Diptera, i.e., Stratiomys singularior and D. melanogaster, which dramatically differ in thermoresistance and the pattern of Hsp70 accumulation. Promoter strength was also monitored in vivo in D. melanogaster strains transformed with constructs containing the S. singularior hsp70 ORF driven either by its own promoter or an orthologous promoter from the D. melanogaster hsp70Aa gene. Analysis revealed low S. singularior hsp70 promoter activity in vitro and in vivo under basal conditions and after HS in comparison with the endogenous promoter in D. melanogaster cells, which correlates with the absence of canonical GAGA elements in the promoters of the former species. Indeed, the insertion of GAGA elements into the S. singularior hsp70 regulatory region resulted in a dramatic increase in promoter activity in vitro but only modestly enhanced the promoter strength in the larvae of the transformed strains. In contrast with hsp70 promoters, hsp83 promoters from both of the studied Diptera species demonstrated high conservation and universality.

Hsf4 counteracts Hsf1 transcription activities and increases lens epithelial cell survival in vitro

The interplay between Hsf4 and Hsf1 plays an important role in the regulation of lens homeostasis. However, the mechanism of the intermolecular association involved is still unclear. In this paper, we find that reconstitution of Hsf4b into Hsf4−/− lens epithelial (mLEC/Hsf4−/−) cells can simultaneously downregulate Hsp70 expression and upregulate the expression of small heat shock proteins Hsp25 and αB-crystallin at both RNA and protein levels. ChIP assay results indicate Hsf4b, which binds to the promoters of Hsp90α, Hsp70.3, Hsp25 and αB-crystallin but not Hsp70.1, can inhibit Hsf1 binding to Hsp70.3 promoter and the heat shock mediated Hsp70 promoter activity by reducing Hsf1 protein expression. Hsf4b N-terminal hydrophobic region can interact with Hsf1 N-terminal hydrophobic region. Their interaction impairs Hsf1's intramolecular interaction between the N- and C-terminal hydrophobic regions, leading to Hsf1's cytosolic retention and protein degradation. Both lysosome inhibitors (chloroquine, pepstatin A plus E64d) and proteasome inhibitor MG132 can inhibit Hsf4-mediated Hsf1 protein degradation, but MG132 can induce Hsf1 activation as well. Upregulation of Hsf4b can significantly inhibit cisplatin and staurosporine induced lens epithelial cell apoptosis through direct upregulation of Hsp25 and αB-crystallin expression. Taken together, our results imply that upregulation of Hsf4b modulates the expression pattern of heat shock proteins in lens tissue by either directly binding to their promoters or promoting Hsf1 protein degradation. Moreover, upregulation of Hsf4b protects lens cell survival by upregulating anti-apoptotic pathways. These studies reveal a novel regulatory mechanism between Hsf1 and Hsf4b in modulating lens epithelial cell homeostasis.

Dual-reporter in vivo imaging of transient and inducible heat-shock promoter activation

Gene promoter activity can be studied in vivo by molecular imaging methods using reporter gene technology. Transcription of the reporter and the reported genes occurs simultaneously. However, imaging depends on reporter protein translation, stability, and cellular fate that may differ among the various proteins. A double transgenic mouse strain expressing the firefly luciferase (lucF) and fluorescent mPlum protein under the transcriptional control of the thermo-inducible heat-shock protein (Hspa1b) promoter was generated allowing to follow up the reporter proteins by different and complementary in vivo imaging technologies. These mice were used for in vivo imaging by bioluminescence and epi fluorescence reflectance imaging (BLI & FRI) and as a source of embryonic fibroblast (MEF) for in vitro approaches. LucF, mPlum and endogenous Hsp70 mRNAs were transcribed simultaneously. The increase in mRNA was transient, peaking at 3 h and then returning to the basal level about 6 h after the thermal stimulations. The bioluminescent signal was transient and initiated with a 3 h delay versus mRNA expression. The onset of mPlum fluorescence was more delayed, increasing slowly up to 30 h after heat-shock and remaining for several days. This mouse allows for both bioluminescence imaging (BLI) and fluorescence reflectance imaging (FRI) of Hsp70 promoter activation showing an early and transient lucF activity and a retrospective and persistent mPlum fluorescence. This transgenic mouse will allow following the transient local induction of Hsp-70 promoter beyond its induction time-frame and relate into subsequent dynamic biological effects of the heat-shock response.

Characterization of four heat-shock protein genes from Nile tilapia (Oreochromis niloticus) and demonstration of the inducible transcriptional activity of Hsp70 promoter

Heat-shock proteins (Hsps), known as stress proteins and extrinsic chaperones, play important roles in the folding, translocation, and refolding/degradation of proteins. In this study, we identified four Hsps in Nile tilapia (Oreochromis niloticus), which display conserved Hsp characteristics in their predicted amino acid sequences. Further analyses on the structures, homology, and phylogenetics revealed that the four Hsps belong to Hsp70 family. One of them does not contain introns and is named Hsp70, while all the other three contain introns and are named Hsc70-1, Hsc70-2, and Hsc70-3. Expressions of the four Hsp proteins were observed in all examined tissues. Six hours after infection of Streptococcus agalactiae in Nile tilapia, the expression of Hsp70 was significantly increased in the liver, head kidney, spleen and gill, while Hsc70s' expression was unchanged in all examined tissues except the head kidney that showed significantly reduced expression of both Hsc70-2 and Hsc70-3. These results suggest that Hsp70 may participate in the defense against S. agalactiae infection. We then isolated the promoter of Hsp70 gene and inserted it into the donor plasmid of Tgf2 transposon system containing green fluorescent protein (GFP) gene. The plasmid was microinjected into zebrafish embryos, where the expression of GFP was induced by heat shock, S. agalactiae immersion challenge, indicating that the isolated Hsp70 promoter has transcriptional activity and is inducible by both heat shock and bacterial challenge. This promoter may facilitate the future construction of disease-resistant transgenic fish. The work also contributes to the further study of immune response of tilapia after bacterial infection.

Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated IκBα degradation and NF-κB p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA’s anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells.

Basal and stress-induced Hsp70 are modulated by ataxin-3

Regulation of basal and induced levels of hsp70 is critical for cellular homeostasis. Ataxin-3 is a deubiquitinase with several cellular functions including transcriptional regulation and maintenance of protein homeostasis. While investigating potential roles of ataxin-3 in response to cellular stress, it appeared that ataxin-3 regulated hsp70. Basal levels of hsp70 were lower in ataxin-3 knockout (KO) mouse brain from 2 to 63 weeks of age and hsp70 was also lower in fibroblasts from ataxin-3 KO mice. Transfecting KO cells with ataxin-3 rescued basal levels of hsp70 protein. Western blots of representative chaperones including hsp110, hsp90, hsp70, hsc70, hsp60, hsp40/hdj2, and hsp25 indicated that only hsp70 was appreciably altered in KO fibroblasts and KO mouse brain. Turnover of hsp70 protein was similar in wild-type (WT) and KO cells; however, basal hsp70 promoter reporter activity was decreased in ataxin-3 KO cells. Transfecting ataxin-3 restored hsp70 basal promoter activity in KO fibroblasts to levels of promoter activity in WT cells; however, mutations that inactivated deubiquitinase activity or the ubiquitin interacting motifs did not restore full activity to hsp70 basal promoter activity. Hsp70 protein and promoter activity were higher in WT compared to KO cells exposed to heat shock and azetidine-2-carboxylic acid, but WT and KO cells had similar levels in response to cadmium. Heat shock factor-1 had decreased levels and increased turnover in ataxin-3 KO fibroblasts. Data in this study are consistent with ataxin-3 regulating basal level of hsp70 as well as modulating hsp70 in response to a subset of cellular stresses.

Arrhenius analysis of the relationship between hyperthermia and Hsp70 promoter activation: A comparison between ex vivo and in vivo data

Purpose: Tight regulation of gene expression in the region where therapy is necessary and for the duration required to achieve a therapeutic effect and to minimise systemic toxicity is very important for clinical applications of gene therapy. Hyperthermia in combination with a temperature sensitive heat shock protein (Hsp70) promoter presents a unique approach allowing non-invasive spatio-temporal control of transgene expression. In this study we investigated the in vivo and ex vivo relationship between temperature and duration of thermal stress with respect to the resulting gene expression using an Arrhenius analysis.Materials and methods: A transgenic mouse expressing the luciferase reporter gene under the transcriptional control of a thermosensitive promoter was used to assure identical genotype for in vivo (mouse leg) and ex vivo (bone marrow mononuclear and embryonic fibroblast cells) studies. The mouse leg and cells were heated at different temperatures and different exposure times. Bioluminescence imaging and in vitro enzymatic assay were used to measure the resulting transgene expression.Results: We showed that temperature-induced Hsp70 promoter activation was modulated by both temperature as well as duration of hyperthermia. The relationship between temperature and duration of hyperthermia and the resulting reporter gene expression can be modelled by an Arrhenius analysis for both in vivo as well as ex vivo.Conclusions: However, the increase in reporter gene expression after elevating the temperature of the thermal stress with 1°C is not comparable for in vivo and ex vivo situations. This information may be valuable for optimising clinical gene therapy protocols.

Effect of 60 Hz electromagnetic fields on the activity of hsp70 promoter: an in vivo study.

Exposure to EMFs (electromagnetic fields) results in a number of important biological changes, including modification of genetic expression. We have investigated the effect of 60 Hz sinusoidal EMFs at a magnetic flux density of 80 μT on the expression of the luciferase gene contained in a plasmid labelled as pEMF (EMF plasmid). This gene construct contains the specific sequences for the induction of hsp70 (heat-shock protein 70) expression by EMFs, as well as the reporter for the luciferase gene. The pEMF vector was electrotransferred into quadriceps muscles of BALB/c mice that were later exposed to EMFs. Increased luciferase expression was observed in mice exposed to EMFs 2 h daily for 7 days compared with controls (P<0.05). These data along with other reports in the literature suggest that EMFs can have far-reaching effects on the genome.

Tonicity enhancer binding protein (TonEBP) and hypoxia-inducible factor (HIF) coordinate heat shock protein 70 (Hsp70) expression in hypoxic nucleus pulposus cells: Role of Hsp70 in HIF-1α degradation

The objective of our study was to examine the regulation of hypoxic expression of heat shock protein 70 (Hsp70) in nucleus pulposus cells and to determine if Hsp70 promoted hypoxia-inducible factor (HIF)-1α degradation. Rat nucleus pulposus cells were maintained in culture in either 21% or 1% oxygen. To determine the regulation of Hsp70 expression by tonicity enhancer binding protein (TonEBP) and HIF-1/2, loss-of-function and gain-of-function experiments and mutational analysis of the Hsp70 promoter were performed. Hypoxia increased Hsp70 expression in nucleus pulposus cells. Noteworthy, hypoxia increased TonEBP transactivation and mutation of TonE motifs blocked hypoxic induction of the Hsp70 promoter. In contrast, mutation of hypoxia response element (HRE) motifs coupled with loss-of-function experiments suggested that HIF-1 and HIF-2 suppressed Hsp70 promoter activity and transcription. Interestingly, HIF-α interferes with TonEBP function and suppresses the inductive effect of TonEBP on the Hsp70 promoter. In terms of Hsp70 function, when treated with Hsp70 transcriptional inhibitor, KNK437, there was an increase in HIF-1α protein stability and transcriptional activity. Likewise, when Hsp70 was overexpressed, the stability of HIF-1α and its transcriptional activity decreased. Hsp70 interacted with HIF-1α under hypoxic conditions and evidenced increased binding when treated with MG132, a proteasomal inhibitor. These results suggest that Hsp70 may promote HIF-1α degradation through the proteasomal pathway in nucleus pulposus cells. In hypoxic and hyperosmolar nucleus pulposus cells, Hsp70, TonEBP, and HIFs form a regulatory loop. We propose that the positive regulation by TonEBP and negative regulation of Hsp70 by HIF-1 and HIF-2 may serve to maintain Hsp70 levels in these cells, whereas Hsp70 may function in controlling HIF-1α homeostasis.

4-Phenylbutyrate Stimulates Hsp70 Expression through the Elp2 Component of Elongator and STAT-3 in Cystic Fibrosis Epithelial Cells

Sodium 4-phenylbutyrate (4PBA) corrects trafficking of ΔF508-CFTR in Cystic Fibrosis (CF) epithelia, which is hypothesized to, at least in part, result from increased expression of Hsp70 (stress-induced 70 kDa heat shock protein). To identify other 4PBA-regulated proteins that may promote correction of ΔF508 trafficking, we performed differential display RT-PCR on mRNA from IB3-1 CF bronchiolar epithelial cells treated for 0-24 h with 1 mM 4PBA. In this screen, a STAT-3 (signal transducer and activator of transcription-3)-interacting protein, StIP-1 that regulates STAT-3 activation had transiently increased expression. StIP-1 is identical to Elongator protein 2 (Elp2), a component of the Elongator complex that regulates RNA polymerase II. Previous studies have suggested that Elongator regulates Hsp70 mRNA transcription, and that the Hsp70 promoter contains functional STAT-3-binding sites. We therefore tested the hypothesis that 4PBA increases Hsp70 expression by an Elongator- and STAT-3-dependent mechanism. 4PBA treatment of IB3-1 CF bronchiolar epithelial cells caused transiently increased expression of Hsp70 protein, as well as Elp2 protein and mRNA. Elp2 depletion by transfection of small interfering RNAs, reduced both Elp2 and Hsp70 protein expression. 4PBA also caused transient activation of STAT-3, and increased abundance of nuclear proteins that bind to the STAT-3-responsive element of the Hsp70 promoter. Luciferase reporter assays demonstrated that both Elp2 overexpression and 4PBA increase Hsp70 promoter activity, while Elp2 depletion blocked the ability of 4PBA to stimulate Hsp70 promoter activity. Together, these data suggest that Elp2 and STAT-3 mediate, at least in part, the stimulation of Hsp70 expression by 4PBA.

Heat shock factor 4a (HSF4a) represses HSF2 expression and HSF2‐mediated transcriptional activity

Heat shock factors (HSFs) are the main transcriptional regulators of the stress-induced expression of heat shock protein genes. HSF2, which is one of the HSFs, is activated during differentiation and development but it is unclear how they regulate during cellular processes. Here, we examined the role of HSF4a on the regulation of HSF2 in HEK 293 cells. We found that HSF2 levels are negatively correlated with HSF4a expression and that overexpression of HSF4a reduces hemin-induced HSF2 mRNA and protein levels. Moreover, hemin-induced activation of HSF2 was also markedly inhibited in HSF4a expressed cells. Immunoprecipitation assay showed that HSF2 binds to the oligomerization domain of HSF4a. Hemin treatment inhibited their interaction and induced localization of HSF2 and HSF4a in nuclear. In addition, we found that HSF4a or HSF4a DNA binding domain (117 aa) inhibited the activity of hemin-induced HSP70 promoter. Consequently, HSF4a inhibits HSF2 expression or transcriptional activity through negative regulation of HSF2 binding to the HSP70 promoter. In summary, our findings suggest novel mechanisms of HSF2 regulation controlled by HSF4a.

Tumor response to mTHPC-mediated photodynamic therapy exhibits strong correlation with extracellular release of HSP70

We investigated the relationship among heat shock protein 70 (hsp70) promoter activation, extracellular HSP70 protein levels, and tumor cure in an animal model of meso-tetrahydroxyphenyl chlorin (mTHPC; Foscan®)-mediated photodynamic therapy (PDT). Using Western blot analysis, we compared HSP70 protein levels in control and PDT-treated EMT6 cells with the amplitude of hsp70-promoter driven green fluorescent protein (GFP) expression in identically treated, stably transfected hsp70-GFP/EMT6 cells. A clonogenic survival assay was performed to assess the relationship among promoter activation, HSP70 levels, and cell survival in vitro. Tumor growth studies with this transfected cell line were performed to examine responses to fluences from 0.1 to 10 J cm(-2) , which ranged from sub-curative to curative. In vivo stereofluorescence and confocal fluorescence imaging were used to assess the temporal kinetics in hsp70 activation in tumors subjected to these fluences and the intratumor spatial correlation between hsp70 induction and extracellular levels of HSP70, respectively. Maximum GFP expression and HSP protein levels in cells were observed at PDT doses that corresponded to 30% cell survival. The relative changes in GFP and HSP70 protein accumulation as analyzed using Western immunoblots agreed very well, thereby confirming the validity of fluorescent reporter assessment of gene expression in our studies. In vivo imaging revealed that hsp70 promoter-driven GFP expression and accumulation of extracellular HSP70 in PDT-treated tumors subjected to non-curative doses exhibit minimal spatial correlation. There is a strong correlation between mTHPC-PDT doses that result in long-term tumor cure and those that cause high levels of surface exposed or extracellularly released HSP70s. Treatment conditions that induce strong promoter activation do not correspond to tumor cure. PDT doses that result in long-term tumor growth control also produce significant accumulation of extracellular HSP70.

Sensing the Heat Stress by Mammalian Cells

BackgroundThe heat-shock response network controls the adaptation and survival of the cell against environmental stress. This network is highly conserved and is connected with many other signaling pathways. A key element of the heat-shock network is the heat-shock transcription factor-1 (HSF), which is transiently activated by elevated temperatures. HSF translocates to the nucleus upon elevated temperatures, forming homotrimeric complexes. The HSF homotrimers bind to the heat shock element on the DNA and control the expression of the hsp70 gene. The Hsp70 proteins protect cells from thermal stress. Thermal stress causes the unfolding of proteins, perturbing thus the pathways under their control. By binding to these proteins, Hsp70 allows them to refold and prevents their aggregation. The modulation of the activity of the hsp70-promoter by the intensity of the input stress is thus critical for cell's survival. The promoter activity starts from a basal level and rapidly increases once the stress is applied, reaches a maximum level and attenuates slowely back to the basal level. This phenomenon is the hallmark of many experimental studies and of all computational network analysis.ResultsThe molecular construct used as a measure of the response to thermal stress is a Hsp70-GFP fusion gene transfected in Chinese hamster ovary (CHO) cells. The time profile of the GFP protein depends on the transient activity, Transient(t), of the heat shock system. The function Transient(t) depends on hsp70 promoter activity, transcriptional regulation and the translation initiation effects elicited by the heat stress. The GFP time profile is recorded using flow cytometry measurements, a technique that allows a quantitative measurement of the fluorescence of a large number of cells (104). The GFP responses to one and two heat shocks were measured for 261 conditions of different temperatures and durations. We found that: (i) the response of the cell to two consecutive shocks (i.e., no recovery time in between shocks) depends on the order of the input shocks, that is the shocks do not commute; (ii) the responses may be classified as mild or severe, depending on the temperature level and the duration of the heat shock and (iii) the response is highly sensitive to small variations in temperature.ConclusionsWe propose a mathematical model that maps temperature into the transient activity using experimental data that describes the time course of the response to input thermal stress. The model is built on thermotolerance without recovery time, sharp sensitivity to small variations in temperature and the existence of mild and severe classes of stress responses. The theoretical predictions are tested against experimental data using a series of double-shock inputs. The theoretical structure is represented by a sequence of three cascade processes that transform the input stress into the transient activity. The structure of the cascade is nonlinear-linear-nonlinear (NLN). The first nonlinear system (N) from the NLN structure represents the amplification of small changes in the environmental temperature; the linear system (L) represents the thermotolerance without recovery time, whereas the last system (N) represents the transition of the cell's response from a mild to a severe shock.

Histone variant H3.3 stimulates HSP70 transcription through cooperation with HP1γ

Histone variant H3.3 and heterochromatin protein 1γ (HP1γ) are two functional components of chromatin with role in gene transcription. However, the regulations of their dynamics during transcriptional activation and the molecular mechanisms underlying their actions remain poorly understood. Here, we provide evidence that heat shock-induced transcription of the human HSP70 gene is regulated via the coordinated and interdependent action of H3.3 and HP1γ. H3.3 and HP1γ are rapidly co-enriched at the human HSP70 promoters upon heat shock in a manner that closely parallels the initiation of transcription. Knockdown of H3.3 prevents the stable recruitment of HP1γ, inhibits active histone modifications, and attenuates HSP70 promoter activity. Likewise, knockdown of HP1γ leads to the decreased levels of H3.3 in the promoter regions and the repression of HSP70 genes. HP1γ selectively recognizes particular modification states of H3.3 in the nucleosome for its action. Moreover, HP1γ is overexpressed in three representative cancer cell lines, and its knockdown leads to reduction in HSP70 gene transcription and inhibition of cancer cell proliferation. We conclude that the physical and functional interactions between H3.3 and HP1γ make a unique contribution to acute HSP70 transcription and cancer development related to the misregulation of this transcription event.

Functional analysis of the HS185 regulatory element in the rice HSP70 promoter

A series of HSP70 promoter deletion constructs was established. Analysis of beta-glucuronidase activities from the promoter deletion constructs in transient expression assays identified a cis-element, located from -493 to -308 bp upstream of the ATG start site. This element was designated as HS185 and has a crucial role in HSP70 promoter activity. HS185 has some characteristics of a miniature inverted-repeat transposable element (MITE), such as terminal inverted repeats (TIRs) (GGTCCCACA) and a putative target site duplication. There are 362 copies of homologous sequences of HS185 in the rice genome, which are preferentially distributed to non-coding regions. Based on these sequence features, we propose that HS185 is an uncharacterized rice MITE, possibly derived from the rice transposon Mutator-like element VIII family. Further transient expression assays showed that HS185 inhibited the enhancer activity of the cauliflower mosaic virus 35S promoter. These results demonstrate that not only is HS185 necessary for HSP70 promoter activity, but it also has a functional role as an insulator. This study explored new regulatory functions of non-coding repeat sequences in rice.

Non-Invasive Monitoring of Hsp70 Promoter Activity Using Firefly Luciferase and Cell Viability of Three Tumor Cell Lines Exposed to Different Levels of Heat

To better understand the temporal pattern of hsp70 transcription, three tumor cell lines (M21, SCCVII, NIH3T3) were transfected with a Hsp70A.1 promoter to express luciferase (luc). The cells were exposed to a range of temperatures (38-52°C) for 20 minutes and luciferase activity was measured using a CCD camera. Luciferase activity was assessed every 2 h for 12 h, then every 6 h for another 12 h, and finally every 12 h for an additional 84 h post stress. Each cell line had its own distinct temporal pattern of luciferase activity. The NIH3T3 cells had the highest luciferase activity at 46°C 12 h post-stress. The SCCVII line was the least heat resistant cell line. The human M21 line was the most heat resistant cell line, maximum luciferase activity occurred at 48°C and 48 h post-stress. At the highest level of luciferase activity, the SCCVII and the NIH3T3 line had 54% and 58% viable cells whereas the M21 line had 94%. The combination of assessing the luciferase activity and the viability of cells may be useful for gene therapy applications and for evaluating collateral thermal damage generated during treatment of biological tissue with heat. Keywords: Tumor cell lines, luciferase, hsp70, thermal stress, viability, Promoter Activity, biological tissue, Heat shock proteins, bioluminescence imaging technology, Cell Culture, Transfection, Immunoblot

Effect of 60 Hz magnetic fields on the activation of hsp70 promoter in cultured INER-37 and RMA E7 cells

It has been reported that 50-60 Hz magnetic fields (MF) with flux densities ranging from microtesla to millitesla are able to induce heat shock factor or heat shock proteins in various cells. In this study, we investigated the effect of 60 Hz sinusoidal MF at 8 and 80 μT on the expression of the luciferase gene contained in a plasmid labeled as electromagnetic field-plasmid (pEMF). This gene construct contains the specific sequences previously described for the induction of hsp70 expression by MF, as well as the reporter for the luciferase gene. The pEMF vector was transfected into INER-37 and RMA E7 cell lines that were later exposed to either MF or thermal shock (TS). Cells that received the MF or TS treatments and their controls were processed according to the luciferase assay system for evaluate luciferase activity. An increased luciferase gene expression was observed in INER-37 cells exposed to MF and TS compared with controls (p < 0.05), but MF exposure had no effect on the RMA E7 cell line.

The effect of 100 mT SMF on activation of the hsp70 promoter in a heat shock/luciferase reporter system

Human exposure to magnetic fields, increased through use of new technologies like magnetic resonance imaging (MRI), has prompted investigations into possible effects of static magnetic fields (SMFs) on cellular processes. However, controversy still remains between many studies, which likely results from a lack of uniformity across experimental parameters, including the length of magnetic field exposure, the strength of the magnetic field, and the cell type or organism under investigation. The purpose of this research was to monitor effects of SMF exposure using real-time luminescence photometry. The study investigated the potential interaction of a 100 mT SMF on a heat shock protein (hsp70)/luciferase reporter construct in stably transfected NIH3T3 cells. Changes in heat shock promoter activation following 100 mT SMF exposure were analyzed and detected as bioluminescence in real-time. Two heat parameters were considered in combination with sham- and 100 mT-exposed experiments: no heat or 1,800 s heat. As expected, there was a significant increase in bioluminescence in response to 1,800 s of heat alone. However, no significant difference in average hsp70 promoter activation between sham and 100 mT experiments was observed for no heat or 1,800 s heat experiments. Therefore, a 100 mT SMF was shown to have no effect on the activation of the heat shock protein promoter during SMF exposure or when SMF exposure was combined with a heat insult.