Talin-1 (TLN1) is best known to activate integrin receptors and transmit mechanical stimuli to the actin cytoskeleton at focal adhesions. However, the localization of TLN1 is not restricted to focal adhesions. By utilizing both subcellular fractionations and confocal microscopy analyses, we show that TLN1 localizes to the nucleus in several human cell lines, where it is tightly associated with the chromatin. Importantly, small interfering RNA (siRNA)-mediated depletion of endogenous TLN1 triggers extensive changes in the gene expression profile of human breast epithelial cells. To determine the functional impact of nuclear TLN1, we expressed a TLN1 fusion protein containing a nuclear localization signal. Our findings revealed that the accumulation of nuclear TLN1 alters the expression of a subset of genes and impairs the formation of cell-cell clusters. This study introduces an additional perspective on the canonical view of TLN1 subcellular localization and function.

The heat shock factors (HSFs) form a family of transcription factors, which are evolutionarily conserved in eukaryotes. They are best known as transcriptional regulators of molecular chaperone genes, including those encoding heat shock proteins, in response to heat shock and other protein-damaging stresses. Since the discovery of the first HSF and its eponymous role in the heat shock response four decades ago, the currently known HSFs in vertebrates, that is, HSF1-5, HSFX, and HSFY, have been implicated in a wide array of physiological and pathological processes, including organismal development and cancer progression. To date, most studies have focused on individual HSFs, but it is becoming increasingly evident that the role of multiple HSFs and their potential crosstalk should be considered. In this review, we provide a comprehensive overview of the structures, functions, and regulation of the mammalian HSF family members and explore their interplay in biological processes. We highlight recent advancements regarding the roles of HSF family members in viral infection, cell adhesion, and spermatogenesis, and discuss the key questions to be addressed by forthcoming studies in HSF biology.

The heat shock factors (HSFs) form a family of transcription factors, which are evolutionarily conserved in eukaryotes. They are best known as transcriptional regulators of molecular chaperone genes, including those encoding heat shock proteins, in response to heat shock and other protein-damaging stresses. Since the discovery of the first HSF and its eponymous role in the heat shock response four decades ago, the currently known HSFs in vertebrates, that is, HSF1-5, HSFX, and HSFY, have been implicated in a wide array of physiological and pathological processes, including organismal development and cancer progression. To date, most studies have focused on individual HSFs, but it is becoming increasingly evident that the role of multiple HSFs and their potential crosstalk should be considered. In this review, we provide a comprehensive overview of the structures, functions, and regulation of the mammalian HSF family members and explore their interplay in biological processes. We highlight recent advancements regarding the roles of HSF family members in viral infection, cell adhesion, and spermatogenesis, and discuss the key questions to be addressed by forthcoming studies in HSF biology.

Heat shock factor 2 (HSF2) is a versatile transcription factor that regulates gene expression under stress conditions, during development, and in disease. Despite recent advances in characterizing HSF2-dependent target genes, little is known about the protein networks associated with this transcription factor. In this study, we performed co-immunoprecipitation coupled with mass spectrometry analysis to identify the HSF2 interactome in mouse testes, where HSF2 is required for normal sperm development. Endogenous HSF2 was discovered to form a complex with several adhesion-associated proteins, a finding substantiated by mass spectrometry analysis conducted in human prostate carcinoma PC-3 cells. Notably, this group of proteins included the focal adhesion adapter protein talin-1 (TLN1). Through co-immunoprecipitation and proximity ligation assays, we demonstrate the conservation of the HSF2-TLN1 interaction from mouse to human. Additionally, employing sequence alignment analyses, we uncovered a TLN1-binding motif in the HSF2 C terminus that binds directly to multiple regions of TLN1 in vitro . We provide evidence that the 25 C-terminal amino acids of HSF2, fused to EGFP, are sufficient to establish a protein complex with TLN1 and modify cell–cell adhesion in human cells. Importantly, this TLN1-binding motif is absent in the C-terminus of a closely related HSF family member, HSF1, which does not form a complex with TLN1. These results highlight the unique molecular characteristics of HSF2 in comparison to HSF1. Taken together, our data unveil the protein partners associated with HSF2 in a physiologically relevant context and identifies TLN1 as the first adhesion-related HSF2-interacting partner.

Heat shock factor 2 (HSF2) is a versatile transcription factor that regulates gene expression under stress conditions, during development, and in disease. Despite recent advances in characterizing HSF2-dependent target genes, little is known about the protein networks associated with this transcription factor. In this study, we performed co-immunoprecipitation coupled with mass spectrometry analysis to identify the HSF2 interactome in mouse testes, where HSF2 is required for normal sperm development. Endogenous HSF2 was discovered to form a complex with several adhesion-associated proteins, a finding substantiated by mass spectrometry analysis conducted in human prostate carcinoma PC-3 cells. Notably, this group of proteins included the focal adhesion adapter protein talin-1 (TLN1). Through co-immunoprecipitation and proximity ligation assays, we demonstrate the conservation of the HSF2-TLN1 interaction from mouse to human. Additionally, employing sequence alignment analyses, we uncovered a TLN1-binding motif in the HSF2 C terminus that binds directly to multiple regions of TLN1 in vitro. We provide evidence that the 25 C-terminal amino acids of HSF2, fused to EGFP, are sufficient to establish a protein complex with TLN1 and modify cell-cell adhesion in human cells. Importantly, this TLN1-binding motif is absent in the C-terminus of a closely related HSF family member, HSF1, which does not form a complex with TLN1. These results highlight the unique molecular characteristics of HSF2 in comparison to HSF1. Taken together, our data unveil the protein partners associated with HSF2 in a physiologically relevant context and identifies TLN1 as the first adhesion-related HSF2-interacting partner.

Extracellular vesicles (EVs) are important mediators of intercellular communication involved in local and long-range signalling of cancer metastasis. The onset of invasion is the key step of the metastatic cascade, but the secretion of EVs has remained unexplored at that stage due to technical challenges. In this study, we present a platform to track EVs over the course of invasive development of human prostate cancer cell (PC3) tumoroids utilizing in vivo-mimicking extracellular matrix-based 3D cultures. Using this EV production method, combined with proteomic profiling, we show that PC3 tumoroids secrete EVs with previously undefined protein cargo. Intriguingly, an increase in EV amounts and extensive changes in the EV protein composition were detected upon invasive transition of the tumoroids. The changes in EV protein cargo were counteracted by chemical inhibition of invasion. These results reveal the impact of the tumoroids' invasive status on EV secretion and cargo, and highlight the necessity of in vivo-mimicking conditions for uncovering novel cancer-derived EV components.

Talin 1 (TLN1) is best known for its role at focal adhesions, where it activates β-integrin receptors and transmits mechanical stimuli to the actin cytoskeleton. Interestingly, the localization of TLN1 is not restricted to the focal adhesions, but its function in other cellular compartments remains poorly understood. By utilizing both biochemical and confocal microscopy analyses, we show that TLN1 localizes to the nucleus and that it strongly interacts with the chromatin. Importantly, depletion of endogenous TLN1 results in extensive changes in the gene expression profile of human breast epithelial cells. To determine the impact of nuclear TLN1 on gene regulation, we expressed a TLN1 fusion protein containing a nuclear localization signal. Our results revealed that nuclear TLN1 regulates a specific subset of the TLN1-dependent genes. Taken together, we show that apart from localizing at the plasma membrane and cytoplasm, TLN1 also resides in the nucleus where it functions in the regulation of gene expression.

Maintenance of protein homeostasis, through inducible expression of molecular chaperones, is essential for cell survival under protein-damaging conditions. The expression and DNA-binding activity of heat shock factor 2 (HSF2), a member of the heat shock transcription factor family, increase upon exposure to prolonged proteotoxicity. Nevertheless, the specific roles of HSF2 and the global HSF2-dependent gene expression profile during sustained stress have remained unknown. Here, we found that HSF2 is critical for cell survival during prolonged proteotoxicity. Strikingly, our RNA sequencing (RNA-seq) analyses revealed that impaired viability of HSF2-deficient cells is not caused by inadequate induction of molecular chaperones but is due to marked downregulation of cadherin superfamily genes. We demonstrate that HSF2-dependent maintenance of cadherin-mediated cell-cell adhesion is required for protection against stress induced by proteasome inhibition. This study identifies HSF2 as a key regulator of cadherin superfamily genes and defines cell-cell adhesion as a determinant of proteotoxic stress resistance.

Extracellular vesicles (EVs) loaded with biomolecules are important in intercellular communication and mediate local and long-range signals in cancer metastasis. However, it is currently unknown how the development of the primary tumor and onset of invasion affect the secretion and characteristics of EVs. In this study, we developed an EV production method utilizing in vivo -mimicking extracellular matrix-based 3D cultures, which allows tracking of EVs over the course of invasive development of tumor organoids. Using this method, combined with proteomic profiling, we show that PC3 human prostate cancer organoids secrete EVs with previously undefined protein cargo, which substantially differs from EV cargo of 2D cultured cells. Intriguingly, an increase in EV amounts and extensive changes in EV protein composition were detected upon invasive transition of the organoids. These results reveal that EV secretion and cargo loading are highly dependent on the developmental status of the tumor organoid, emphasizing the necessity of in vivo -mimicking conditions for discovery of novel cancer-derived EV components, applicable as diagnostic markers for cancer.

Cellular ability to maintain proper protein homeostasis (proteostasis) is essential for survival upon protein-damaging conditions. Heat shock transcription factor 2 (HSF2) is one of the human HSFs activated in response to proteotoxic stress. HSF2 is dispensable for cell survival during acute heat stress, but its amount and DNA-binding activity increase under prolonged proteotoxic stress conditions, such as proteasome inhibition. Nevertheless, the specific role(s) of HSF2 and the global HSF2-dependent gene expression profile during sustained stress have remained elusive. We found that HSF2 is required for cell survival during prolonged proteotoxicity, as shown by treating wild-type and HSF2-deficient human osteosarcoma U2OS cells with the proteasome inhibitor Bortezomib. Strikingly, our RNA-seq analyses revealed that HSF2 disruption leads to marked downregulation of cadherin superfamily genes and subsequent functional impairment of cadherin-mediated cell-cell adhesion. We propose HSF2 as a key regulator of genes belonging to the cadherin superfamily. We also demonstrate that HSF2-dependent downregulation of cadherin-mediated cell-cell adhesion predisposes U2OS cells to Bortezomib-induced proteotoxic stress. In conclusion, we show that by maintaining cell-cell adhesion HSF2 is essential for cell survival and thereby we describe a novel regime in the HSF-mediated protection against stress-induced protein damage.

Heat shock factor 1 (HSF1) is an important transcription factor in cellular stress responses, cancer, aging, and developmental processes including gametogenesis. Disruption of Hsf1, together with another HSF family member, Hsf2, causes male sterility and complete lack of mature sperm in mice, but the specific role of HSF1 in spermatogenesis has remained unclear. Here, we show that HSF1 is transiently expressed in meiotic spermatocytes and haploid round spermatids in mouse testis. The Hsf1(-/-) male mice displayed regions of seminiferous tubules containing only spermatogonia and increased morphological abnormalities in sperm heads. In search for HSF1 target genes, we identified 742 putative promoters in mouse testis. Among them, the sex chromosomal multicopy genes that are expressed in postmeiotic cells were occupied by HSF1. Given that the sex chromatin mostly is repressed during and after meiosis, it is remarkable that HSF1 directly regulates the transcription of sex-linked multicopy genes during postmeiotic repression. In addition, our results show that HSF1 localizes to the sex body prior to the meiotic divisions and to the sex chromocenter after completed meiosis. To the best of our knowledge, HSF1 is the first known transcription factor found at the repressed sex chromatin during meiosis.

The mammalian Y chromosome is essential for spermatogenesis, which is characterized by sperm cell differentiation and chromatin condensation for acquisition of correct shape of the sperm. Deletions of the male-specific region of the mouse Y chromosome long arm (MSYq), harboring multiple copies of a few genes, lead to sperm head defects and impaired fertility. Using chromatin immunoprecipitation on promoter microarray (ChIP-chip) on mouse testis, we found a striking in vivo MSYq occupancy by heat shock factor 2 (HSF2), a transcription factor involved in spermatogenesis. HSF2 was also found to regulate the transcription of MSYq resident genes, whose transcriptional regulation has been unknown. Importantly, disruption of Hsf2 caused a similar phenotype as the 2/3 deletion of MSYq, i.e., altered expression of the multicopy genes and increased mild sperm head abnormalities. Consequently, aberrant levels of chromatin packing proteins and more frequent DNA fragmentation were detected, implying that HSF2 is required for correct chromatin organization in the sperm. Our findings define a physiological role for HSF2 in the regulation of MSYq resident genes and the quality of sperm.

Members of the homeodomain leucine zipper (HDZip) family of transcription factors are present in a wide range of plants, from mosses to higher plants, but not in other eukaryotes. The HDZip genes act in developmental processes, including vascular tissue and trichome development, and several of them have been suggested to be involved in the mediation of external signals to regulate plant growth. The Arabidopsis (Arabidopsis thaliana) genome contains 47 HDZip genes, which, based on sequence criteria, have been grouped into four different classes: HDZip I to IV. In this article, we present an overview of the class I HDZip genes in Arabidopsis. We describe their expression patterns, transcriptional regulation properties, duplication history, and phylogeny. The phylogeny of HDZip class I genes is supported by data on the duplication history of the genes, as well as the intron/exon patterning of the HDZip-encoding motifs. The HDZip class I genes were found to be widely expressed and partly to have overlapping expression patterns at the organ level. Further, abscisic acid or water deficit treatments and different light conditions affected the transcript levels of a majority of the HDZip I genes. Within the gene family, our data show examples of closely related HDZip genes with similarities in the function of the gene product, but a divergence in expression pattern. In addition, six HDZip class I proteins tested were found to be activators of gene expression. In conclusion, several HDZip I genes appear to regulate similar cellular processes, although in different organs or tissues and in response to different environmental signals.

ABSTRACTIn plants changes in cytosolic calcium ion concentrations ([Ca2+]cyt) have been detected after various stress treatments, including salt treatment. The involvement of a Ca2+ signal as an essential component of signalling pathways leading to downstream responses, such as gene expression, is supported only by a few studies. In this study the possible involvement of the salt stress‐induced increase in [Ca2+]cyt in the signalling pathway leading to the induction of ATHB7, a homeobox gene encoding a homeodomain leucine zipper (HDZip) transcription factor was analysed. The salt‐induced expression of ATHB7 was found to be independent of the Ca2+ signal evoked by salt. Instead, it was found that ATHB7 expression in shoots was not dependent on a direct contact with salt or osmoticum, whereas in roots, ATHB7 seemed to be induced by the direct contact, indicating that signals from roots cause systemic induction of ATHB7. Abscisic acid (ABA) or ABA‐dependent components were found to, at least partly, to function as the systemic signal.

Members of the homeodomain leucine zipper (HDZip) family of transcription factors are present in a wide range of plants, from mosses to higher plants, but not in other eukaryotes. The HDZip genes act in developmental processes, including vascular tissue and trichome development, and several of them have been suggested to be involved in the mediation of external signals to regulate plant growth. The Arabidopsis (Arabidopsis thaliana) genome contains 47 HDZip genes, which, based on sequence criteria, have been grouped into four different classes: HDZip I to IV. In this article, we present an overview of the class I HDZip genes in Arabidopsis. We describe their expression patterns, transcriptional regulation properties, duplication history, and phylogeny. The phylogeny of HDZip class I genes is supported by data on the duplication history of the genes, as well as the intron/exon patterning of the HDZip-encoding motifs. The HDZip class I genes were found to be widely expressed and partly to have overlapping expression patterns at the organ level. Further, abscisic acid or water deficit treatments and different light conditions affected the transcript levels of a majority of the HDZip I genes. Within the gene family, our data show examples of closely related HDZip genes with similarities in the function of the gene product, but a divergence in expression pattern. In addition, six HDZip class I proteins tested were found to be activators of gene expression. In conclusion, several HDZip I genes appear to regulate similar cellular processes, although in different organs or tissues and in response to different environmental signals.

The homeodomain leucine zipper (HDZip) proteins constitute a plant-specific family of transcription factors, that based on sequence criteria have been grouped into four classes, HDZip I-IV. This thesis describes the phylogeny, function, expression patterns and regulation of the HDZip class I genes in the model species Arabidopsis thaliana. The phylogenetic analyses, traced duplication history and exon/intron organisation of the 17 class I genes in Arabidopsis show that the genes form six monophyletic groups, clades, with an origin in early plant evolution. All genes are expressed in broad tissue distribution patterns and the majority are responsive to water availability and/or light conditions. The expression of the genes show different patterns and dependence on environmental stress conditions, indicating evolutionary changes within and between clades. Ectopic expression of the genes suggest that they regulate genes in part by conserved mechanisms. Therefore, different functional roles seem to have evolved by a divergence mainly in the regulatory properties of the genes. Detailed expression analyses of the paralogous HDZip I genes ATHB7 and ATHB12 show that they have essentially overlapping patterns of activity in response to abscisic acid, ABA, or water deficit in leaves, stems and roots. The water deficit response of ATHB7 and -12 is mediated by ABA and depends on the protein phosphateses ABI1 and ABI2. Transgenic plants with ectopic expression of ATHB7 and/or -12, and athb7 and athb12 mutants, reveal that the genes in roots mediate the growth inhibitory effects of ABA. In this aspect of their function they do not overlap. In leaves and stems, the genes might act as growth regulators redundantly with other factors. Taken together these data suggest that ATHB7 and -12 regulate growth in response water deficit and that other HDZip I genes have related functions in response to environmental stress conditions.

Homeodomain leucine zipper (HDZip) proteins constitute a large family of transcription factors characterised by the presence of a DNA-binding homeodomain and an adjacent leucine zipper motif, which mediates protein-dimer formation. The HDZip genes of Arabidopsis have been divided into four classes, HDZip I-IV. This thesis describes the characterisation and phylogeny of the class I HDZip genes and focuses on the expression and function of four HDZip I genes, ATHB5, -6, -7 and -16. The phylogenetic analyses of the 17 HDZip I sequences defined six subclasses, supported by the intron patterning and the traced duplication history of the genes. The members within each subclass showed diversification in expression, suggesting that the cis regulatory regions of the closely related genes have undergone evolutionary changes. However, similarities in the gene expression patterns between genes also exist and external factors like the availability of water and quality of light directs the expression of a subset of HDZip I genes. Expression analyses revealed that the plant hormone abscisic acid (ABA) is involved as a systemic signal for the salt or osmoticum induced ATHB7 expression, whereas light signals mediated through the blue light photoreceptors was found to direct the expression of ATHB6. Phenotypic analyses of plants with altered levels of ATHB6 or ATHB16 suggested that these paralogous genes encode proteins with very similar functions. ATHB16 was shown to act as a negative regulator of leaf cell expansion, as a suppressor of the flowering time sensitivity to photoperiod and as a positive regulator of blue light dependent inhibition of hypocotyl growth. A similar role for ATHB6 in the regulation of hypocotyl elongation was recorded. Further, analyses of multiple loss-of-function plants demonstrated that ATHB5, -6 and -16 function at least in part redundantly in mediating light effects on hypocotyl elongation.

Homeodomain leucine zipper (HDZip) proteins constitute a large family of transcription factors characterised by the presence of a DNA-binding homeodomain and an adjacent leucine zipper motif, which mediates protein-dimer formation. The HDZip genes of Arabidopsis have been divided into four classes, HDZip I-IV. This thesis describes the characterisation and phylogeny of the class I HDZip genes and focuses on the expression and function of four HDZip I genes, ATHB5, -6, -7 and -16. The phylogenetic analyses of the 17 HDZip I sequences defined six subclasses, supported by the intron patterning and the traced duplication history of the genes. The members within each subclass showed diversification in expression, suggesting that the cis regulatory regions of the closely related genes have undergone evolutionary changes. However, similarities in the gene expression patterns between genes also exist and external factors like the availability of water and quality of light directs the expression of a subset of HDZip I genes. Expression analyses revealed that the plant hormone abscisic acid (ABA) is involved as a systemic signal for the salt or osmoticum induced ATHB7 expression, whereas light signals mediated through the blue light photoreceptors was found to direct the expression of ATHB6. Phenotypic analyses of plants with altered levels of ATHB6 or ATHB16 suggested that these paralogous genes encode proteins with very similar functions. ATHB16 was shown to act as a negative regulator of leaf cell expansion, as a suppressor of the flowering time sensitivity to photoperiod and as a positive regulator of blue light dependent inhibition of hypocotyl growth. A similar role for ATHB6 in the regulation of hypocotyl elongation was recorded. Further, analyses of multiple loss-of-function plants demonstrated that ATHB5, -6 and -16 function at least in part redundantly in mediating light effects on hypocotyl elongation.