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Heat shock can induce either cytoprotective mechanisms or cell death. We found that in certain human and mouse cells, including spermatocytes, activated heat shock factor 1 (HSF1) binds to sequences located in the intron(s) of the PMAIP1 (NOXA) gene and upregulates its expression which induces apoptosis. Such a mode of PMAIP1 activation is not dependent on p53. Therefore, HSF1 not only can activate the expression of genes encoding cytoprotective heat shock proteins, which prevents apoptosis, but it can also positively regulate the proapoptotic PMAIP1 gene, which facilitates cell death. This could be the primary cause of hyperthermia-induced elimination of heat-sensitive cells, yet other pro-death mechanisms might also be involved.

The p53 and HSF1 transcription factors are key players in cellular responses to stress. They activate important signaling pathways triggering adaptive mechanisms that maintain cellular homeostasis. HSF1 is mainly activated by proteotoxic stress, and its induction leads to the synthesis of chaperones that provide proteome integrity. The p53 protein, which is primarily activated in response to DNA damage, causes cell cycle arrest allowing for DNA repair or directs cells to apoptosis, thereby maintaining genome integrity. Both signaling pathways are also involved in neoplastic transformation and tumor progression. Loss of tumor suppressor abilities of the wild-type p53 protein results in oncogenesis, whereas proper HSF1 action, though non-oncogenic itself, actively supports this process. Here, we describe in detail the interplay between the p53 and HSF1 signaling pathways, with particular emphasis on the molecular mechanisms involved, as well as their importance for normal cellular behavior, cancer development, the effectiveness of anti-cancer therapies and their toxicity. Detailed knowledge of the complex interplay between HSF1 and p53 may form a basis for the design of new protocols for cancer treatment.

Heat shock factor 1 (HSF1), a key regulator of transcriptional responses to proteotoxic stress, was recently linked to estrogen (E2) signaling through ESR1. We found that an HSF1 deficiency could lead to the inhibition of the mitogenic action of E2 in breast cancer cells. The stimulatory effect of E2 on the transcriptome is weaker in HSF1-deficient cells, in part due to the higher basal expression of E2-dependent genes, which correlates with the enhanced binding of unliganded ESR1 to chromatin. HSF1 and ESR1 can cooperate directly in E2-stimulated regulation of transcription, and HSF1 potentiates the action of ESR1 through a mechanism involving chromatin reorganization. Analyses of data from the TCGA database indicate that HSF1 increases the transcriptome diversity in ER-positive breast cancer and can enhance the genomic action of ESR1. Moreover, ESR1 and HSF1 are only prognostic when analyzed together (the worst prognosis for ER−/HSF1high cancers).

Heat shock factor 1 (HSF1), a key regulator of transcriptional responses to proteotoxic stress, was linked to estrogen (E2) signaling through estrogen receptor α (ERα). We found that an HSF1 deficiency may decrease ERα level, attenuate the mitogenic action of E2, counteract E2-stimulated cell scattering, and reduce adhesion to collagens and cell motility in ER-positive breast cancer cells. The stimulatory effect of E2 on the transcriptome is largely weaker in HSF1-deficient cells, in part due to the higher basal expression of E2-dependent genes, which correlates with the enhanced binding of unliganded ERα to chromatin in such cells. HSF1 and ERα can cooperate directly in E2-stimulated regulation of transcription, and HSF1 potentiates the action of ERα through a mechanism involving chromatin reorganization. Furthermore, HSF1 deficiency may increase the sensitivity to hormonal therapy (4-hydroxytamoxifen) or CDK4/6 inhibitors (palbociclib). Analyses of data from The Cancer Genome Atlas database indicate that HSF1 increases the transcriptome disparity in ER-positive breast cancer and can enhance the genomic action of ERα. Moreover, only in ER-positive cancers an elevated HSF1 level is associated with metastatic disease., eLife digest About 70% of breast cancers rely on supplies of a hormone called estrogen – which is the main hormone responsible for female physical characteristics – to grow. Breast cancer cells that are sensitive to estrogen possess proteins known as estrogen receptors and are classified as estrogen-receptor positive. When estrogen interacts with its receptor in a cancer cell, it stimulates the cell to grow and migrate to other parts of the body. Therefore, therapies that decrease the amount of estrogen the body produces, or inhibit the receptor itself, are widely used to treat patients with estrogen receptor-positive breast cancers. When estrogen interacts with an estrogen receptor known as ERα it can also activate a protein called HSF1, which helps cells to survive under stress. In turn, HSF1 regulates several other proteins that are necessary for ERα and other estrogen receptors to work properly. Previous studies have suggested that high levels of HSF1 may worsen the outcomes for patients with estrogen receptor-positive breast cancers, but it remains unclear how HSF1 acts in breast cancer cells. Vydra, Janus, Kuś et al. used genetics and bioinformatics approaches to study HSF1 in human breast cancer cells. The experiments revealed that breast cancer cells with lower levels of HSF1 also had lower levels of ERα and responded less well to estrogen than cells with higher levels of HSF1. Further experiments suggested that in the absence of estrogen, HSF1 helps to keep ERα inactive. However, when estrogen is present, HSF1 cooperates with ERα and enhances its activity to help cells grow and migrate. Vydra, Janus, Kuś et al. also found that cells with higher levels of HSF1 were less sensitive to two drug therapies that are commonly used to treat estrogen receptor-positive breast cancers. These findings reveal that the effect HSF1 has on ERα activity depends on the presence of estrogen. Therefore, cancer therapies that decrease the amount of estrogen a patient produces may have a different effect on estrogen receptor-positive tumors with high HSF1 levels than tumors with low HSF1 levels.

Modern immunotherapy together with targeted therapy has revolutionized the treatment of advanced melanoma. Inhibition of immune checkpoints significantly improved the median overall survival and gave hope to many melanoma patients. However, this treatment has three serious drawbacks: high cost, serious side effects, and an effectiveness limited only to approximately 50% of patients. Some patients do not derive any or short-term benefit from this treatment due to primary or secondary resistance. The response to immunotherapy depends on many factors that fall into three main categories: those associated with melanoma cells, those linked to a tumor and its microenvironment, and those classified as individual ontogenic and physiological features of the patient. The first category comprises expression of PD-L1 and HLA proteins on melanoma cells as well as genetic/genomic metrics such as mutational load, (de)activation of specific signaling pathways and epigenetic factors. The second category is the inflammatory status of the tumor: “hot” versus “cold” (i.e., high versus low infiltration of immune cells). The third category comprises metabolome and single nucleotide polymorphisms of specific genes. Here we present up-to-date data on those biological factors influencing melanoma response to immunotherapy with a special focus on signaling pathways regulating the complex process of anti-tumor immune response. We also discuss their potential predictive capacity.

Background Ability to adapt to temperature changes trough the Heat Shock Response (HSR) pathways is one of the most fundamental and clinically relevant cellular response systems. Heat Shock (HS) affects the signalling and gene expression responses of the Nuclear Factor κB (NF-κB) transcription factor, a critical regulator of proliferation and inflammation, however, our quantitative understanding of how cells sense and adapt to temperature changes is limited. Methods We used live-cell time-lapse microscopy and mathematical modelling to understand the signalling of the NF-κB system in the human MCF7 breast adenocarcinoma cells in response to pro-inflammatory Interleukin 1β (IL1β) and Tumour Necrosis Factor α (TNFα) cytokines, following exposure to a 37–43 °C range of physiological and clinical temperatures. Results We show that exposure to 43 °C 1 h HS inhibits the immediate NF-κB signalling response to TNFα and IL1β stimulation although uptake of cytokines is not impaired. Within 4 h after HS treatment IL1β-induced NF-κB responses return to normal levels, but the recovery of the TNFα-induced responses is still affected. Using siRNA knock-down of Heat Shock Factor 1 (HSF1) we show that this stimulus-specificity is conferred via the Inhibitory κB kinase (IKK) signalosome where HSF1-dependent feedback regulates TNFα, but not IL1β-mediated IKK recovery post HS. Furthermore, we demonstrate that through the temperature-dependent denaturation and recovery of IKK, TNFα and IL1β-mediated signalling exhibit different temperature sensitivity and adaptation to repeated HS when exposed to a 37–43 °C temperature range. Specifically, IL1β-mediated NF-κB responses are more robust to temperature changes in comparison to those induced by TNFα treatment. Conclusions We demonstrate that the kinetics of the NF-κB system following temperature stress is cytokine specific and exhibit differential adaptation to temperature changes. We propose that this differential temperature sensitivity is mediated via the IKK signalosome, which acts as a bona fide temperature sensor trough the HSR cross-talk. This novel quantitative understanding of NF-κB and HSR interactions is fundamentally important for the potential optimization of therapeutic hyperthermia protocols.

Additional file1 : Figure S1. Analysis of NF-κB signalling responses in MCF7 cells. (A) Analysis of NF-κB p65-Ser536 phosphorylation in transformed cells. The level of p65-Ser536 phosphorylation was analyzed by Western blot in the whole MCF7 p65-EGFP cells lysates. Cells cultured in 37 °C were treated with TNFα for indicated times. β-actin was used as a loading control. (B) Nuclear NF-κB trajectories in MCF7 cells stably expressing p65-EGFP after 1 h 43 °C HS treatment. Individual single cell trajectories (n = 50 per condition) are depicted with colour lines; population average is depicted with a black line. (C) Quantitative RT-PCR analysis of TNFAIP3, NFKBIA, CCL2, and TNF mRNA abundance 90 min after TNFα or IL1b stimulation of cells cultured at 37 °C (no HS) or following HS treatment and indicated recovery time. Shownare mean changes in relation to unstimulated cells ± SDs based on three replicate experiments. Figure S2. Analysis of NF-κB responses in HSF1 knock-down cells. (A) Heat maps of nuclear NF-κB trajectories in response to TNFα in MCF7 cells stably expressing p65-EGFP. Cells were treated with scrambled siRNA control (scrambled) or HSF1-specific siRNA (KD HSF1) and stimulated with the cytokine under normal conditions (37 °C, no HS) or after 1 h HS at 43 °C and 4 h recovery (1 h HS + 4 h). Heat maps of trajectories were normalized across all conditions (represented on a 0–3 scale). Individual single cell trajectories are shown. (B) Heat maps of nuclear NF-κB trajectories in response to IL1β in MCF7 cells stably expressing p65-EGFP. Cells were treated and data are presented as in A. (C) Percentage of cells responding (yellow) and non-responding (blue) to stimulation with TNFα or IL1β (from data shown in A and B). Statistical difference was assessed with Chi-square test (****p 117 cells per condition). Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparisons test was used to assess differences between groups (****p

Background The cellular response to ionizing radiation involves activation of p53-dependent pathways and activation of the atypical NF-κB pathway. The crosstalk between these two transcriptional networks include (co)regulation of common gene targets. Here we looked for novel genes potentially (co)regulated by p53 and NF-κB using integrative genomics screening in human osteosarcoma U2-OS cells irradiated with a high dose (4 and 10 Gy). Radiation-induced expression in cells with silenced TP53 or RELA (coding the p65 NF-κB subunit) genes was analyzed by RNA-Seq while radiation-enhanced binding of p53 and RelA in putative regulatory regions was analyzed by ChIP-Seq, then selected candidates were validated by qPCR. Results We identified a subset of radiation-modulated genes whose expression was affected by silencing of both TP53 and RELA, and a subset of radiation-upregulated genes where radiation stimulated binding of both p53 and RelA. For three genes, namely IL4I1, SERPINE1, and CDKN1A, an antagonistic effect of the TP53 and RELA silencing was consistent with radiation-enhanced binding of both p53 and RelA. This suggested the possibility of a direct antagonistic (co)regulation by both factors: activation by NF-κB and inhibition by p53 of IL4I1, and activation by p53 and inhibition by NF-κB of CDKN1A and SERPINE1. On the other hand, radiation-enhanced binding of both p53 and RelA was observed in a putative regulatory region of the RRAD gene whose expression was downregulated both by TP53 and RELA silencing, which suggested a possibility of direct (co)activation by both factors. Conclusions Four new candidates for genes directly co-regulated by NF-κB and p53 were revealed. Electronic supplementary material The online version of this article (10.1186/s12864-018-5211-y) contains supplementary material, which is available to authorized users.

The NF-κB transcription factors are activated via diverse molecular mechanisms in response to various types of stimuli. A plethora of functions associated with specific sets of target genes could be regulated differentially by this factor, affecting cellular response to stress including an anticancer treatment. Here we aimed to compare subsets of NF-κB-dependent genes induced in cells stimulated with a pro-inflammatory cytokine and in cells damaged by a high dose of ionizing radiation (4 and 10 Gy). The RelA-containing NF-κB species were activated by the canonical TNFα-induced and the atypical radiation-induced pathways in human osteosarcoma cells. NF-κB-dependent genes were identified using the gene expression profiling (by RNA-Seq) in cells with downregulated RELA combined with the global profiling of RelA binding sites (by ChIP-Seq), with subsequent validation of selected candidates by quantitative PCR. There were 37 NF-κB-dependent protein-coding genes identified: in all cases RelA bound in their regulatory regions upon activation while downregulation of RELA suppressed their stimulus-induced upregulation, which apparently indicated the positive regulation mode. This set of genes included a few “novel” NF-κB-dependent species. Moreover, the evidence for possible negative regulation of ATF3 gene by NF-κB was collected. The kinetics of the NF-κB activation was slower in cells exposed to radiation than in cytokine-stimulated ones. However, subsets of NF-κB-dependent genes upregulated by both types of stimuli were essentially the same. Hence, one should expect that similar cellular processes resulting from activation of the NF-κB pathway could be induced in cells responding to pro-inflammatory cytokines and in cells where so-called “sterile inflammation” response was initiated by radiation-induced damage.

The mouse 3110001I22Rik gene located in the first intron of Bfar is considered as a Bfar variant coding for the BFARv3 protein. However, it differs from other BFAR isoforms and resembles periphilin 1 (PPHLN1) due to its two (Lge1 and serine-rich) conserved domains. We identified the BFARv3/EGFP-interacting proteins by co-immunoprecipitation coupled to mass spectrometry, which revealed 40S ribosomal proteins (RPS3, RPS14, RPS19, RPS25, RPS27), histones (H1.2, H1.4, H3.3C), proteins involved in RNA processing and splicing (SFPQ, SNRPA1, HNRNPA3, NONO, KHDRBS3), calcium signaling (HPCAL1, PTK2B), as well as HSD17B4, GRB14, POSTN, and MYO10. Co-immunoprecipitation revealed that both Lge1 and Ser-rich domains of BFARv3 were necessary for binding to RNA-interacting factors NONO and SFPQ, known to be components of paraspeckles. Reciprocal co-immunoprecipitation and the proximity ligation assay confirmed that both BFARv3 and PPHLN1 could interact with NONO and SFPQ, suggesting a new function for PPHLN1 as well. BFARv3 and its Lge1 or Ser-rich-deficient mutants preferentially localize in the nucleus. We found an accumulation of BFARv3/EGFP (but not its mutated forms) in the nuclear granules, which was enhanced in response to arsenite treatment and ionizing radiation. Although Bfar v3 is expressed ubiquitously in mouse tissues, its expression is the highest in metaphase II oocytes. The BFARv3 interactome suggests its role in RNA metabolism, which is critical for the transcriptionally silent MII oocyte. Mouse BFARv3 has no ortholog in the human genome, thus it may contribute to the differences between these two species observed in oocyte maturation and early embryonic development.

Next-generation sequencing (NGS) in liquid biopsies may contribute to the diagnosis, monitoring, and personalized therapy of cancer through the real-time detection of a tumor’s genetic profile. There are a few NGS platforms offering high-sensitivity sequencing of cell-free DNA (cfDNA) samples. The aim of this study was to evaluate the Ion AmpliSeq HD Technology for targeted sequencing of tumor and liquid biopsy samples from patients with fourth-stage melanoma. Sequencing of 30 samples (FFPE tumor and liquid biopsy) derived from 14 patients using the Oncomine™ Pan-Cancer Cell-Free Assay was performed. The analysis revealed high concordance between the qPCR and NGS results of the BRAF mutation in FFPE samples (91%), as well as between the FFPE and liquid biopsy samples (91%). The plasma-tumor concordance of the non-BRAF mutations was 28%. A total of 17 pathogenic variants in 14 genes (from 52-gene panel), including TP53, CTNNB1, CCND1, MET, MAP2K1, and GNAS, were identified, with the CTNNB1S45F variant being the most frequent. A positive correlation between the LDH level and cfDNA concentration as well as negative correlation between the LDH level and time to progression was confirmed in a 22-patient cohort. The analysis showed both the potential and limitations of liquid biopsy genetic profiling using HD technology and the Ion Torrent platform.

Spermatocytes are among the most heat-sensitive cells and the exposure of testes to heat shock results in their Heat Shock Factor 1 (HSF1)-mediated apoptosis. Several lines of evidence suggest that pleckstrin-homology-like domain family A, member 1 (PHLDA1) plays a role in promoting heat shock-induced cell death in spermatogenic cells, yet its precise physiological role is not well understood. Aiming to elucidate the hypothetical role of PHLDA1 in HSF1-mediated apoptosis of spermatogenic cells we characterized its expression in mouse testes during normal development and after heat shock. We stated that transcription of Phlda1 is upregulated by heat shock in many adult mouse organs including the testes. Analyzes of the Phlda1 expression during postnatal development indicate that it is expressed in pre-meiotic or somatic cells of the testis. It starts to be transcribed much earlier than spermatocytes are fully developed and its transcripts and protein products do not accumulate further in the later stages. Moreover, neither heat shock nor expression of constitutively active HSF1 results in the accumulation of PHLDA1 protein in meiotic and post-meiotic cells although both conditions induce massive apoptosis of spermatocytes. Furthermore, the overexpression of PHLDA1 in NIH3T3 cells leads to cell detachment, yet classical apoptosis is not observed. Therefore, our findings indicate that PHLDA1 cannot directly contribute to the heat-induced apoptosis of spermatocytes. Instead, PHLDA1 could hypothetically participate in death of spermatocytes indirectly via activation of changes in the somatic or pre-meiotic cells present in the testes.

Heat Shock Factor 1 (HSF1) is a key regulator of gene expression during acute environmental stress that enables the cell survival, which is also involved in different cancer-related processes. A high level of HSF1 in estrogen receptor (ER)-positive breast cancer patients correlated with a worse prognosis. Here we demonstrated that 17&beta, estradiol (E2), as well as xenoestrogen bisphenol A and ER&alpha, agonist propyl pyrazole triol, led to HSF1 phosphorylation on S326 in ER&alpha, positive but not in ER&alpha, negative mammary breast cancer cells. Furthermore, we showed that MAPK signaling (via MEK1/2) but not mTOR signaling was involved in E2/ER&alpha, dependent activation of HSF1. E2&shy, activated HSF1 was transcriptionally potent and several genes essential for breast cancer cells growth and/or ER&alpha, action, including HSPB8, LHX4, PRKCE, WWC1, and GREB1, were activated by E2 in a HSF1-dependent manner. Our findings suggest a hypothetical positive feedback loop between E2/ER&alpha, and HSF1 signaling, which may support the growth of estrogen-dependent tumors.

Ability to adapt to temperature changes trough the Heat Shock Response (HSR) pathways is one of the most fundamental and clinically relevant cellular response systems. Here we report that Heat Shock (HS) induces a temporally-coordinated and stimulus-specific adaptation of the signalling and gene expression responses of the Nuclear Factor {kappa}B (NF-{kappa}B) transcription factor. We show that exposure of MCF7 breast adenocarcinoma cells to 43{degrees}C 1h HS inhibits the immediate signalling response to pro-inflammatory Interleukin 1{beta} (IL1{beta}) and Tumour Necrosis Factor (TNF) cytokines. Within 4h after HS treatment IL1{beta}-induced responses return to normal levels, but the recovery of the TNF-induced responses is delayed. Using siRNA knock-down of Heat Shock Factor 1 and mathematical modelling we show that the stimulus-specificity is conferred via the Inhibitory {kappa}B kinase signalosome, with HSR differentially controlling individual cytokine transduction pathways. Finally, using a novel mathematical model we predict and experimentally validate that the HSR cross-talk confers differential cytokine sensitivity of the NF-{kappa}B system to a range of physiological and clinically-relevant temperatures. This quantitative understanding of NF-{kappa}B and HSR cross-talk mechanisms is fundamentally important for the potential improvement of current hyperthermia protocols.

The NF-κB transcription factor is involved in different aspects of the cellular response to stress, including atypical NF-κB pathway activated by damage induced by ionizing radiation. Moreover, NF-κB could be involved in the regulation of genes activated by other stress-responsive factors. Here we aimed to perform the integrative genomics screening to compare subsets of NF-κB-dependent genes induced by a pro-inflammatory stimulus and a high dose of ionizing radiation and also to identify new genes potentially co-regulated by NF-κB and p53 transcription factors in irradiated cells. Methods. The RelA-containing NF-κB dimers were activated by TNFα cytokine (classical proinflammatory pathway) and a single 4 or 10 Gy dose (atypical radiation-induced pathway) in human osteosarcoma cells. NF-κB-dependent and p53-dependent genes were identified using the gene expression profiling (by RNA-Seq) in cells with downregulated RELA or TP53 combined with the global profiling of RelA and p53 binding sites (by ChIP-Seq). Candidate genes were subsequently validated by quantitative PCR. Results: There were 37 NF-κB-dependent protein-coding genes identified: in all cases RelA bound in their regulatory regions upon activation while downregulation of RELA suppressed their stimulus-induced upregulation, which apparently indicated the positive regulation mode (this set of genes included a few “novel” NF-κB-dependent species). The kinetics of the NF-κB activation was slower in cells exposed to radiation than in cytokine-stimulated ones. However, subsets of NF-κB-dependent genes upregulated by both types of stimuli were essentially the same. Moreover, we identified a subset of radiation-modulated genes whose expression was affected by silencing of both TP53 and RELA, and a subset of radiation-upregulated genes where radiation stimulated binding of both p53 and RelA. For three genes an antagonistic effect of both transcription factors was observed: IL4I1 was activated by NF-κB and inhibited by p53, while CDKN1A and SERPINE1 were activated by p53 and inhibited by NF-κB. Moreover, RRAD was putatively co-activated by both factors. Conclusions: One could expect that similar cellular processes resulting from activation of the NF-κB pathway could be induced in cells responding to pro-inflammatory cytokines and in cells where so-called “sterile inflammation” response was initiated by radiation-induced damage. Moreover, certain stress-responsive genes induced by ionizing radiation could be co-regulated by NF-κB and p53. publishedVersion

Elevated temperature induces the heat shock (HS) response, which modulates cell proliferation, apoptosis, the immune and inflammatory responses. However, specific mechanisms linking the HS response pathways to major cellular signaling systems are not fully understood. Here we used integrated computational and experimental approaches to quantitatively analyze the crosstalk mechanisms between the HS-response and a master regulator of inflammation, cell proliferation, and apoptosis the Nuclear Factor κB (NF-κB) system. We found that populations of human osteosarcoma cells, exposed to a clinically relevant 43°C HS had an attenuated NF-κB p65 response to Tumor Necrosis Factor α (TNFα) treatment. The degree of inhibition of the NF-κB response depended on the HS exposure time. Mathematical modeling of single cells indicated that individual crosstalk mechanisms differentially encode HS-mediated NF-κB responses while being consistent with the observed population-level responses. In particular “all-or-nothing” encoding mechanisms were involved in the HS-dependent regulation of the IKK activity and IκBα phosphorylation, while others involving transport were “analogue”. In order to discriminate between these mechanisms, we used live-cell imaging of nuclear translocations of the NF-κB p65 subunit. The single cell responses exhibited “all-or-nothing” encoding. While most cells did not respond to TNFα stimulation after a 60 min HS, 27% showed responses similar to those not receiving HS. We further demonstrated experimentally and theoretically that the predicted inhibition of IKK activity was consistent with the observed HS-dependent depletion of the IKKα and IKKβ subunits in whole cell lysates. However, a combination of “all-or-nothing” crosstalk mechanisms was required to completely recapitulate the single cell data. We postulate therefore that the heterogeneity of the single cell responses might be explained by the cell-intrinsic variability of HS-modulated IKK signaling. In summary, we show that high temperature modulates NF-κB responses in single cells in a complex and unintuitive manner, which needs to be considered in hyperthermia-based treatment strategies., Author summary Hyperthermia has been considered a promising strategy to sensitize cancer cells to treatment. As such, it might potentially increase treatment efficacy while reducing negative side effects. So far, this potential has not been fully realized. One of the major obstacles is a lack of quantitative understanding of crosstalk mechanisms involved in the heat shock and other cellular stress-factor systems. In this work, we used experimental population-level and live-cell imaging approaches to characterize the NF-κB signaling system response to physiologically high temperature. We then iteratively applied mathematical modeling of single cells to infer the underlying regulatory mechanisms. We showed experimentally that elevated temperature attenuated cytokine-induced NF-κB system responses, resulting in complex and unintuitive single cell behavior. Our analyses suggested that these responses are consistent with stochastic cell-intrinsic heat-shock-mediated regulation of the NF-κB system via the IKK signaling module. Considering the oncogenic associations of the NF-κB pathway, the crosstalk mechanisms described in this work are important for the understanding of chemo- and radio-resistance to treatment. However, more but similar, quantitative studies are required to ultimately change currently used therapy protocols.

SPEN (spen family transcription repressor) is a nucleic acid-binding protein putatively involved in repression of gene expression. We hypothesized that SPEN could be involved in general downregulation of the transcription during the heat shock response in mouse spermatogenic cells through its interactions with chromatin. We documented predominant nuclear localization of the SPEN protein in spermatocytes and round spermatids, which was retained after heat shock. Moreover, the protein was excluded from the highly condensed chromatin. Chromatin immunoprecipitation experiments clearly indicated interactions of SPEN with chromatinin vivo. However, ChIP-Seq analyses did not reveal any strong specific peaks both in untreated and heat shocked cells, which might suggest dispersed localization of SPEN and/or its indirect binding to DNA. Usingin situproximity ligation assay we found closein vivoassociations of SPEN with MTA1 (metastasis-associated 1), a member of the nucleosome remodeling complex with histone deacetylase activity, which might contribute to interactions of SPEN with chromatin.

Heat shock inhibits NF-κB signaling, yet the knowledge about its influence on the regulation of NF-κB-dependent genes is limited. Using genomic approaches, i.e., expression microarrays and ChIP-Seq, we aimed to establish a global picture for heat shock-mediated impact on the expression of genes regulated by TNFα cytokine. We found that 193 genes changed expression in human U-2 osteosarcoma cells stimulated with cytokine (including 77 genes with the κB motif in the proximal promoters). A large overlap between sets of genes modulated by cytokine or by heat shock was revealed (86 genes were similarly affected by both stimuli). Binding sites for heat shock-induced HSF1 were detected in regulatory regions of 1/3 of these genes. Furthermore, pre-treatment with heat shock affected the expression of 2/3 of cytokine-modulated genes. In the largest subset of co-affected genes, heat shock suppressed the cytokine-mediated activation (antagonistic effect, 83 genes), which genes were associated with the canonical functions of NF-κB signaling. However, subsets of co-activated and co-repressed genes were also revealed. Importantly, pre-treatment with heat shock resulted in the suppression of NF-κB binding in the promoters of the cytokine-upregulated genes, either antagonized or co-activated by both stimuli. In conclusion, we confirmed that heat shock inhibited activation of genes involved in the classical cytokine-mediated functions of NF-κB. On the other hand, genes involved in transcription regulation were over-represented in the subset of genes upregulated by both stimuli. This suggests the replacement of NF-κB-mediated regulation by heat shock-mediated regulation in the latter subset of genes, which may contribute to the robust response of cells to both stress conditions. Electronic supplementary material The online version of this article (doi:10.1007/s00438-015-1055-1) contains supplementary material, which is available to authorized users.

Heat shock transcription factors (HSFs), as regulators of heat shock proteins (HSPs) expression, are well known for their cytoprotective functions during cellular stress. They also play important yet less recognized roles in gametogenesis. All HSF family members are expressed during mammalian spermatogenesis, mainly in spermatocytes and round spermatids which are characterized by extensive chromatin remodeling. Different HSFs could cooperate to maintain proper spermatogenesis. Cooperation of HSF1 and HSF2 is especially well established since their double knockout results in meiosis arrest, spermatocyte apoptosis, and male infertility. Both factors are also involved in the repackaging of the DNA during spermatid differentiation. They can form heterotrimers regulating the basal level of transcription of target genes. Moreover, HSF1/HSF2 interactions are lost in elevated temperatures which can impair the transcription of genes essential for spermatogenesis. In most mammals, spermatogenesis occurs a few degrees below the body temperature and spermatogenic cells are extremely heat-sensitive. Pro-survival pathways are not induced by heat stress (e.g., cryptorchidism) in meiotic and postmeiotic cells. Instead, male germ cells are actively eliminated by apoptosis, which prevents transition of the potentially damaged genetic material to the next generation. Such a response depends on the transcriptional activity of HSF1 which in contrary to most somatic cells, acts as a proapoptotic factor in spermatogenic cells. HSF1 activation could be the main trigger of impaired spermatogenesis related not only to elevated temperature but also to other stress conditions; therefore, HSF1 has been proposed to be the quality control factor in male germ cells.

Heat shock transcription factors (HSFs), as regulators of heat shock proteins (HSPs) expression, are well known for their cytoprotective functions during cellular stress. They also play important yet less recognized roles in gametogenesis. All HSF family members are expressed during mammalian spermatogenesis, mainly in spermatocytes and round spermatids which are characterized by extensive chromatin remodeling. Different HSFs could cooperate to maintain proper spermatogenesis. Cooperation of HSF1 and HSF2 is especially well established since their double knockout results in meiosis arrest, spermatocyte apoptosis, and male infertility. Both factors are also involved in the repackaging of the DNA during spermatid differentiation. They can form heterotrimers regulating the basal level of transcription of target genes. Moreover, HSF1/HSF2 interactions are lost in elevated temperatures which can impair the transcription of genes essential for spermatogenesis. In most mammals, spermatogenesis occurs a few degrees below the body temperature and spermatogenic cells are extremely heat-sensitive. Pro-survival pathways are not induced by heat stress (e.g., cryptorchidism) in meiotic and postmeiotic cells. Instead, male germ cells are actively eliminated by apoptosis, which prevents transition of the potentially damaged genetic material to the next generation. Such a response depends on the transcriptional activity of HSF1 which in contrary to most somatic cells, acts as a proapoptotic factor in spermatogenic cells. HSF1 activation could be the main trigger of impaired spermatogenesis related not only to elevated temperature but also to other stress conditions; therefore, HSF1 has been proposed to be the quality control factor in male germ cells.

HSF1 (Heat Shock transcription Factor 1) is the main transcription factor activated in response to proteotoxic stress. Once activated, it induces an expression of heat shock proteins (HSPs) which enables cells to survive in suboptimal conditions. HSF1 could be also activated by altered kinase signaling characteristic for cancer cells, which is a probable reason for its high activity found in a broad range of tumors. There is rapidly growing evidence that HSF1 supports tumor initiation and growth, as well as metastasis and angiogenesis. It also modulates the sensitivity of cancer cells to therapy. Functions of HSF1 in cancer are connected with HSPs' activity, which generally protects cells from apoptosis, but also are independent of its classical targets. HSF1-dependent regulation of non-HSPs genes plays a role in cell cycle progression, glucose metabolism, autophagy and drug efflux. HSF1 affects the key cell-survival and regulatory pathways, including p53, RAS/MAPK, cAMP/PKA, mTOR and insulin signaling. Although the exact mechanism of HSF1 action is still somewhat obscure, HSF1 is becoming an attractive target in anticancer therapies, whose inhibition could enhance the effects of other treatments.

The binding of capacitated spermatozoa to the egg's extracellular coat and induction of acrosome reaction are necessary for successful fertilization in mammals. Biogenesis of acrosome is complicated, and not all proteins involved in this process are known. In this study, we have cloned a novel mouse gene,Spaca7, that is expressed exclusively in the testes. During the postnatal development, transcripts of the gene could be detected at a very low level in 18-day-old mouse testes and at a higher level in 21-day-old mouse testes and later, which corresponds to an expansion of round spermatids. In the stably transfected PT67 cells, SPACA7 fused with EGFP was predominantly localized in the Golgi apparatus. In transgenic mouse testes, the fusion protein was found in acrosome (starting from the first stages of acrosome formation in late pachytene spermatocytes and finally in spermatozoa isolated from caput and cauda of epididymis). Confocal microscopy studies revealed an intra-acrosomal not membrane-bound localization of SPACA7/EGFP, which suggests that the protein can be released during acrosome reaction and involved in fertilization. Acrosomal localization of endogenous SPACA7 protein was also found in human spermatozoa.

NF-κB transcription factor regulates numerous genes important for inflammation, immune responses and cell survival. HSF1 is the primary transcription factor activated under stress conditions that is responsible for induction of genes encoding heat shock proteins. Previous studies have shown that the NF-κB activation pathway is blocked by heat shock possibly involving heat shock proteins. Here, we investigate whether active HSF1 inhibited this pathway in the absence of stress conditions. Activation of the NF-κB pathway and expression of NF-κB-dependent genes were analyzed in TNFα-stimulated U-2 OS human osteosarcoma cells that were either heat-shocked or engineered to express a constitutively active form of HSF1 in the absence of heat shock. As expected, heat shock resulted in a general blockade in the degradation of the IκBα inhibitor, nuclear translocation of NF-κB and expression of NF-κB-dependent target genes. In marked contrast, the presence of constitutively active HSF1 did not block TNFα-induced activation of the NF-κB pathway or expression of a set of the NF-κB-dependent genes. We conclude that in the absence of heat shock, the NF-κB activation pathway is inhibited by neither active HSF1 transcription factor nor by increased levels of HSF1-induced heat shock proteins.

Expression of constitutively active heat shock transcription factor 1 (HSF1) in mouse spermatocytes induces apoptosis and leads to male infertility. We report here that prior to the onset of massive apoptosis caused by expression of active HSF1 in spermatocytes a marked reduction in spermatocyte-specific Hsp70.2 mRNA and protein levels occurs. In addition, HSP70.2 protein relocalizes from a predominant cytoplasmic to a nuclear position in developing spermatocytes that express active HSF1. Later in the developmental stages, cells undergoing HSF1-induced apoptosis essentially lack the HSP70.2 protein. The down-regulation of Hsp70.2 gene expression by HSF1 is paradoxical because HSF1 is the prototypical activator of HSP genes. Furthermore, HSF1-mediated repression neither involved a heat shock element (HSE)-like sequence adjacent to the Hsp70.2 gene nor were Hsp70.2 promoter sequences associated directly with HSF1. Interestingly, other spermatocyte- and spermatid-specific transcripts are also down-regulated in testes of transgenic mice expressing active HSF1, suggesting involvement of a putative HSF1-dependent block of development of spermatogenic cells. Importantly however, transcription of the Hsp70.2 gene is down-regulated in testes of wild-type mice subjected to a hyperthermia that induces transient activation of HSF1, indicating that the spermatocyte-specific activity of HSF1 might misdirect a network of transcription factors required for proper regulation of Hsp70.2.

Accumulation of inducible heat shock proteins (e.g. Hsp70i) during cellular stress confers thermotolerance, reduces the consequences of damage and facilitates cellular recovery, while abrogation of Hsp70i expression renders sensitivity to apoptosis. Testis translocation into abdominal cavity, which results in temperature elevation, does not induce expression of the Hsp70i proteins. Despite constitutive expression of testis-specific Hsp70 proteins, spermatocytes are very sensitive to damage at elevated temperatures. To test whether Hsp70i protein could protect testes from heat-induced damage, we have engineered transgenic mice that over-express this protein selectively in spermatocytes and spermatids. We demonstrate that the testes of cryptorchid transgenic mice, like those of wild-type mice, exhibit reduced weight and smaller sizes of their seminiferous tubules, disorganization of their germinal epithelium structures, appearance of multinucleated giant cells, and reduced populations of germ cells. The data show that constitutive expression of Hsp70i does not protect the seminiferous epithelium against cryptorchidism-induced damage.

The hst70 gene which belongs to rat HSP70 multigene family is highly expressed in pachytene spermatocytes. Using a transgenic mice model it was found that the promoter of the rat hst70 gene directs the expression of the chloramphenicol acetyl transferase (CAT) reporter gene not only to testis but also to multiple somatic tissues. In wild-type rats the expression of the hst70 gene in tissues other than testis was confirmed by non-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Beside the testis, the CAT expression in transgenic mice and the hst70 gene transcripts in wild-type rats were found in brain, pituitary, epididymis, vas deferens, adrenals, spleen, lung, ovary, oviduct and uterus. The only tissue in which both the CAT expression and the hst70 gene activity has not been found was the liver. These observations suggest possibly more universal, not confined to spermatogenesis, function of the hst70 gene.

Background Heat Shock Transcription Factor 1 (HSF1) is activated under stress conditions. In turn, it induces expression of Heat Shock Proteins (HSPs), which are well-known regulators of protein homeostasis. Elevated levels of HSF1 and HSPs were observed in many types of tumors. The aim of the present study was to determine whether HSF1 could have an effect on the survival of cancer cells treated with chemotherapeutic cytotoxic agents. Methods We constructed mouse (B16F10) and human (1205Lu, WM793B) melanoma cells overexpressing full or mutant form of human HSF1: a constitutively active one with a deletion in regulatory domain or a dominant negative one with a deletion in the activation domain. The impact of different forms of HSF1 on the expression of HSP and ABC genes was studied by RT-PCR and Western blotting. Cell cultures were treated with increasing amounts of doxorubicin, paclitaxel, cisplatin, vinblastine or bortezomib. Cell viability was determined by MTT, and IC50 was calculated. Cellular accumulation of fluorescent dyes and side population cells were studied using flow cytometry. Results Cells overexpressing HSF1 and characterized by increased HSPs accumulation were more resistant to doxorubicin or paclitaxel, but not to cisplatin, vinblastine or bortezomib. This resistance correlated with the enhanced efflux of fluorescent dyes and the increased number of side population cells. The expression of constitutively active mutant HSF1, also resulting in HSPs overproduction, did not reduce the sensitivity of melanoma cells to drugs, unlike in the case of dominant negative form expression. Cells overexpressing a full or dominant negative form of HSF1, but not a constitutively active one, had higher transcription levels of ABC genes when compared to control cells. Conclusions HSF1 overexpression facilitates the survival of melanoma cells treated with doxorubicin or paclitaxel. However, HSF1-mediated chemoresistance is not dependent on HSPs accumulation but on an increased potential for drug efflux by ABC transporters. Direct transcriptional activity of HSF1 is not necessary for increased expression of ABC genes, which is probably mediated by HSF1 regulatory domain.

NF-κB transcription factor regulates numerous genes important for inflammation, immune responses and cell survival. HSF1 is the primary transcription factor activated under stress conditions that is responsible for induction of genes encoding heat shock proteins. Previous studies have shown that the NF-κB activation pathway is blocked by heat shock possibly involving heat shock proteins. Here, we investigate whether active HSF1 inhibited this pathway in the absence of stress conditions. Activation of the NF-κB pathway and expression of NF-κB-dependent genes were analyzed in TNFα-stimulated U-2 OS human osteosarcoma cells that were either heat-shocked or engineered to express a constitutively active form of HSF1 in the absence of heat shock. As expected, heat shock resulted in a general blockade in the degradation of the IκBα inhibitor, nuclear translocation of NF-κB and expression of NF-κB-dependent target genes. In marked contrast, the presence of constitutively active HSF1 did not block TNFα-induced activation of the NF-κB pathway or expression of a set of the NF-κB-dependent genes. We conclude that in the absence of heat shock, the NF-κB activation pathway is inhibited by neither active HSF1 transcription factor nor by increased levels of HSF1-induced heat shock proteins.

Expression of constitutively active heat shock transcription factor 1 (HSF1) in mouse spermatocytes induces apoptosis and leads to male infertility. We report here that prior to the onset of massive apoptosis caused by expression of active HSF1 in spermatocytes a marked reduction in spermatocyte-specific Hsp70.2 mRNA and protein levels occurs. In addition, HSP70.2 protein relocalizes from a predominant cytoplasmic to a nuclear position in developing spermatocytes that express active HSF1. Later in the developmental stages, cells undergoing HSF1-induced apoptosis essentially lack the HSP70.2 protein. The down-regulation of Hsp70.2 gene expression by HSF1 is paradoxical because HSF1 is the prototypical activator of HSP genes. Furthermore, HSF1-mediated repression neither involved a heat shock element (HSE)-like sequence adjacent to the Hsp70.2 gene nor were Hsp70.2 promoter sequences associated directly with HSF1. Interestingly, other spermatocyte- and spermatid-specific transcripts are also down-regulated in testes of transgenic mice expressing active HSF1, suggesting involvement of a putative HSF1-dependent block of development of spermatogenic cells. Importantly however, transcription of the Hsp70.2 gene is down-regulated in testes of wild-type mice subjected to a hyperthermia that induces transient activation of HSF1, indicating that the spermatocyte-specific activity of HSF1 might misdirect a network of transcription factors required for proper regulation of Hsp70.2.

Accumulation of inducible heat shock proteins (e.g. Hsp70i) during cellular stress confers thermotolerance, reduces the consequences of damage and facilitates cellular recovery, while abrogation of Hsp70i expression renders sensitivity to apoptosis. Testis translocation into abdominal cavity, which results in temperature elevation, does not induce expression of the Hsp70i proteins. Despite constitutive expression of testis-specific Hsp70 proteins, spermatocytes are very sensitive to damage at elevated temperatures. To test whether Hsp70i protein could protect testes from heat-induced damage, we have engineered transgenic mice that over-express this protein selectively in spermatocytes and spermatids. We demonstrate that the testes of cryptorchid transgenic mice, like those of wild-type mice, exhibit reduced weight and smaller sizes of their seminiferous tubules, disorganization of their germinal epithelium structures, appearance of multinucleated giant cells, and reduced populations of germ cells. The data show that constitutive expression of Hsp70i does not protect the seminiferous epithelium against cryptorchidism-induced damage.

The rat Hst70 gene and its mouse counterpart Hsp70.2 are expressed specifically in pachytene primary spermatocytes and spermatids. Here we demonstrate that a 165 bp fragment of the Hst70 gene promoter, containing the T1 transcription start site region, entire exon 1 and 42 bp 5´ region of the intron, is sufficient to drive testis-specific expression of the chloramphenicol acetyltransferase reporter gene in transgenic mice with the same developmentally regulated pattern as the endogenous Hsp70.2 gene. We show further that high-level tissue-specific gene expression requires additional sequences localized upstream of the T2 transcription start site. Electrophoretic mobility-shift assay analysis revealed that only testes of juvenile rats, when Hst70 gene expression is repressed, contain proteins that specifically bind to the Oct (octamer) sequence localized directly downstream of the T1 site.

The influence of partial hepatectomy on the level of 2-aminofluorene (2-AF) induced DNA adducts in rat liver was studied. We found that partial hepatectomy performed either 3 weeks before or simultaneously with the injection of 2-AF affected the amounts of adducts in rat hepatic DNA compared to controls. The level of DNA adducts in rats that were treated with 2-AF and simultaneously hepatectomized was higher (19.9 fmol/microgram DNA) than in non-hepatectomized ones (14.4 fmol/microgram DNA) when measured 48 h after 2-AF administration. In rats treated with the carcinogen 3 weeks after hepatectomy the level of DNA adducts was significantly higher than in nonhepatectomized rats when measured 15 days after the injection of 2-AF (10.9 fmol/microgram DNA and 5.9 fmol/microgram DNA respectively). The high level of DNA adducts in that group of hepatectomized animals was correlated with a relatively lower rate of 2-AF-induced radioactive thymidine incorporation into hepatic DNA (in comparison to non-hepatectomized rats).

The rat hst70 gene belongs to a heat shock hsp70 multigene family and its expression has been detected so far solely in spermatocytes. To investigate the cis-elements responsible for testis-specific expression of the hst70 gene we produced several lines of transgenic mice carrying fragments of the 5'-flanking regions of the hst70 gene fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Hybrid genes of series B were constructed such that, besides the 780 bp, 343 bp and 163 bp 5'-flanking region these plasmids contained no other sequences of the hst70 gene. In hybrid genes of series D the CAT gene was ligated to 343 bp and 252 bp 5'-flanking regions together with the 57 bp of the 5'-end nontranslated (leader) sequences of the hst70 gene. We found that in 780/B, 343/B, 343/D and 252/D adult mice the transgene was specifically and highly expressed in testes. In developing testes the high CAT activity appeared in transgenic mice aged 3 weeks and older. None of the three 163/B transgenic lines exhibited CAT activity in any tissue analyzed. In all CAT expressing lines a weak but significant CAT activity (up to 5% of that in testis) was detected also in the brain. RNase protection assay confirmed that the endogenous hst70 gene transcripts are present in testis as well as in brain of nontransgenic rats and mice. Our data show that the cis-regulatory sequences responsible for testis-specific and developmentally regulated expression of the hst70 gene are localized within the 252 bp region 5' to the gene and neither the 5'-end nor 3'-end nontranslated sequences of the gene are important for this specificity.

Heat shock transcription factor 1 (HSF1), the major regulator of stress response, is frequently activated in cancer and has an apparent role in malignant transformation. Here we analyzed the influence of the over-expression of a constitutively active transcriptionally-competent HSF1 mutant form on phenotypes of mouse and human melanoma cells. We observed that the expression of active HSF1 supported anchorage-independent growth in vitro, and metastatic spread in the animal model in vivo, although the proliferation rate of cancer cells was not affected. Furthermore, active HSF1 enhanced cell motility, reduced the adherence of cells to a fibronectin-coated surface, and affected the actin cytoskeleton. We found that although the expression of active HSF1 did not affect levels of epithelial-to-mesenchymal transition markers, it caused transcriptional down-regulation of vinculin, protein involved in cell motility, and adherence. Functional HSF1-binding sites were found in mouse and human Vcl/VCL genes, indicating a direct role of HSF1 in the regulation of this gene. An apparent association between HSF1-induced down-regulation of vinculin, increased motility, and a reduced adherence of cells suggests a possible mechanism of HSF1-mediated enhancement of the metastatic potential of cancer cells.

Heat Shock Factor 1 (HSF1) is the primary transcription factor responsible for the response to cellularstress, while HSF2 becomes activated during development and differentiation, including spermatogen-esis. Although both factors are indispensable for proper spermatogenesis, activation of HSF1 by heatshock initiates apoptosis of spermatogenic cells leading to infertility of males. To characterize mecha-nisms assisting such heat induced apoptosis we studied how HSF1 and HSF2 cooperate during the heatshock response. For this purpose we used chromatin immunoprecipitation and the proximity ligationapproaches. We looked for co-occupation of binding sites by HSF1 and HSF2 in untreated (32◦C) or heatshocked (at 38◦C or 43◦C) spermatocytes, which are cells the most sensitive to hyperthermia. At thephysiological temperature or after mild hyperthermia at 38◦C, the sharing of binding sites for both HSFswas observed mainly in promoters of Hsp genes and other stress-related genes. Strong hyperthermiaat 43◦C resulted in an increased binding of HSF1 and releasing of HSF2, hence co-occupation of pro-moter regions was not detected any more. The close proximity of HSF1 and HSF2 (and/or existence ofHSF1/HSF2 complexes) was frequent at the physiological temperature. Temperature elevation resultedin a decreased number of such complexes and they were barely detected after strong hyperthermia at43◦C. We have concluded that at the physiological temperature HSF1 and HSF2 cooperate in spermato-genic cells. However, temperature elevation causes remodeling of chromatin binding and interactionsbetween HSFs are disrupted. This potentially affects the regulation of stress response and contributes tothe heat sensitivity of these cells. publishedVersion

The Hsp70.2/Hst70 gene, which belongs to the 70 kDa heat-shock protein (HSP) family, is expressed specifically in primary spermatocytes and spermatids. The regulatory elements required for a high level of testis-specific expression of the gene are placed between the two major transcription start sites T1 and T2 (approximately 350 and 115 bp upstream of the starting ATG codon). Here we have shown that sequences proximal to the exon1/intron splicing site in the 5' untranslated region of the Hsp70.2/Hst70 gene, which include a highly conserved element called box B, are required for efficient expression of the chloramphenicol acetyltransferase reporter gene in testes of transgenic mice. However, in spite of the drastically reduced overall activity, the stage-specific expression pattern of the transgene was preserved after removal of these sequences. We have also shown that GC-box located downstream of the box B (approximately 210 bp upstream of the starting ATG codon) is indispensable for efficient expression of the Hsp70.2/Hst70 gene promoter in spermatogenic cells. The GC-box specifically binds proteins present in nuclear extracts from testes (putatively Sp1-like factors). A change in the pattern of such GC-box-interacting factors corresponds to activation of the Hsp70.2/Hst70 gene, confirming the importance of this regulatory element.

Background Elevated temperatures induce activation of the heat shock transcription factor 1 (HSF1) which in somatic cells leads to heat shock proteins synthesis and cytoprotection. However, in the male germ cells (spermatocytes) caspase-3 dependent apoptosis is induced upon HSF1 activation and spermatogenic cells are actively eliminated. Results To elucidate a mechanism of such diverse HSF1 activity we carried out genome-wide transcriptional analysis in control and heat-shocked cells, either spermatocytes or hepatocytes. Additionally, to identify direct molecular targets of active HSF1 we used chromatin immunoprecipitation assay (ChIP) combined with promoter microarrays (ChIP on chip). Genes that are differently regulated after HSF1 binding during hyperthermia in both types of cells have been identified. Despite HSF1 binding to promoter sequences in both types of cells, strong up-regulation of Hsps and other genes typically activated by the heat shock was observed only in hepatocytes. In spermatocytes HSF1 binding correlates with transcriptional repression on a large scale. HSF1-bound and negatively regulated genes encode mainly for proteins required for cell division, involved in RNA processing and piRNA biogenesis. Conclusions Observed suppression of the transcription could lead to genomic instability caused by meiotic recombination disturbances, which in turn might induce apoptosis of spermatogenic cells. We propose that HSF1-dependent induction of cell death is caused by the simultaneous repression of many genes required for spermatogenesis, which guarantees the elimination of cells damaged during heat shock. Such activity of HSF1 prevents transmission of damaged genetic material to the next generation.