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Activating transcription factors, ATFs, are a group of bZIP transcription factors that act as homodimers or heterodimers with a range of other bZIP factors. In general, ATFs respond to extracellular signals, indicating their important roles in maintaining homeostasis. The ATF family includes ATF1, ATF2, ATF3, ATF4, ATF5, ATF6, and ATF7. Consistent with the diversity of cellular processes reported to be regulated by ATFs, the functions of ATF are also diverse. ATFs play an important role in cell proliferation, apoptosis, differentiation and inflammation-related pathological processes. The expression and phosphorylation status of ATFs are also related to neurodegenerative diseases and polycystic kidney disease. Various miRNAs target ATF to regulate cancer proliferation, apoptosis, autophagy, sensitivity and resistance to radiotherapy and chemotherapy. Moreover, ATFs are necessary to maintain cell redox homeostasis. Therefore, deepening our understanding of the regulation and function of ATFs will provide insights into the basic regulatory mechanisms that influence how cells integrate extracellular and intracellular signals into genomic responses through transcription factors. Under pathological conditions, especially in cancer biology and response to treatment, the characterization of ATF dysfunction is important for understanding how to therapeutically utilize ATF2 or other pathways controlled by transcription factors. In this review, we will demonstrate how ATF1, ATF2, ATF3, ATF4, ATF5, ATF6, and ATF7 function in promoting or suppressing cancer development and identify their roles in tumour immunotherapy.

Homologous recombination (HR)-deficient cells are sensitive to PARP inhibitors through a synthetic lethal effect. We previously developed an HR activity assay named Assay of Site-Specific HR Activity (ASHRA). Here, we evaluated the HR activity of 30 missense variants of BRCA1 by ASHRA and found that several BRCA1 variants showed intermediate HR activity, which was not clearly discerned by our previous analyses using a conventional method. HR activity measured by ASHRA was significantly correlated with sensitivity to olaparib. However, cells expressing the severely HR-deficient BRCA1-C61G variant were resistant to olaparib, and resistance was dependent on high expression of activating transcription factor 1 (ATF1), which binds to BRCA1 and activates the transcription of target genes to regulate cell proliferation. The BRCA1-C61G variant bound to ATF1 and stimulated ATF1-mediated transactivation similar to wild-type BRCA1. High expression of ATF1 conferred resistance to olaparib and cisplatin activating BRCA1/ATF1-mediated transcription without affecting HR activity in BRCA2-knockdown or RAD51-knockdown cells, but not in BRCA1-knockdown cells. These results suggest that ASHRA is a useful method to evaluate HR activity in cells and to predict the sensitivity to PARP inhibitors. The expression level of ATF1 might be an important biomarker of the effect of PARP inhibitors and platinum agents on HR-deficient tumors with the BRCA1-C61G variant or alteration of non-BRCA1 HR factors such as BRCA2 and RAD51. Significance: ASHRA could evaluate HR activity in cells and predict the sensitivity to PARP inhibitors. High expression level of ATF1 may predict the resistance of BRCAness tumors with alterations of non-BRCA1 HR factors to PARP inhibitors and platinum agents.

The activating transcription factor 1 (ATF1) has been identified as a vital pathogenic factor in the progression of colorectal cancer (CRC), whiles, the precise regulatory mechanisms remain elusive. Here, we comprehensively characterized the ATF1 cistrome by RNA-seq and ChIP-seq assays in CRC cell lines. As the results, we identified 358 genes differentially regulated and 15,029 ATF1 binding sites and demonstrated that ATF1 was widely involved in major signaling pathways in CRC, such as Wnt, TNF, Jak-STAT. Subsequently, by the expression quantitative trait loci (eQTL) analyses, we found that rs7017386 was associated with the expression of CCAT1 and PVT1 in the Wnt pathway. By a two-stage population study with 6,131 CRC cases and 10,022 healthy controls, we identified the variant was associated with CRC risk. Mechanistically, we found rs7017386 allele-specifically enhanced the binding affinity of ATF1 and promoted the expressions of PVT1 and CCAT1, via forming a long-range chromatin loop. Moreover, those two lncRNAs could synergistically facilitate c-Myc expression to activate the Wnt pathway in CRC progression. Our findings not only demonstrated the transcriptomic profiling of ATF1 in CRC, but also provided important clues for the etiology of CRC.

Transcriptional regulation, a pivotal biological process by which cells adapt to environmental fluctuations, is achieved by the binding of transcription factors to target sequences in a sequence-specific manner. However, how transcription factors recognize the correct target from amongst the numerous candidates in a genome has not been fully elucidated. We here show that, in the fission-yeast fbp1 gene, when transcription factors bind to target sequences in close proximity, their binding is reciprocally stabilized, thereby integrating distinct signal transduction pathways. The fbp1 gene is massively induced upon glucose starvation by the activation of two transcription factors, Atf1 and Rst2, mediated via distinct signal transduction pathways. Atf1 and Rst2 bind to the upstream-activating sequence 1 region, carrying two binding sites located 45 bp apart. Their binding is reciprocally stabilized due to the close proximity of the two target sites, which destabilizes the independent binding of Atf1 or Rst2. Tup11/12 (Tup-family co-repressors) suppress independent binding. These data demonstrate a previously unappreciated mechanism by which two transcription-factor binding sites, in close proximity, integrate two independent-signal pathways, thereby behaving as a hub for signal integration.

Chromosomal translocations constitute driver mutations in solid tumors and leukemias. The mechanisms of how related or even identical gene fusions drive the pathogenesis of various tumor types remain elusive. One remarkable example is the presence of EWSR1 fusions with CREB1 and ATF1, members of the CREB family of transcription factors, in a variety of sarcomas, carcinomas and mesotheliomas. To address this, we have developed in vitro models of oncogenic fusions, in particular, EWSR1-CREB1 and EWSR1-ATF1, in human embryonic stem (hES) cells, which are capable of multipotent differentiation, using CRISPR-Cas9 technology and HDR together with conditional fusion gene expression that allows investigation into the early steps of cellular transformation. We show that expression of EWSR1-CREB1/ATF1 fusion in hES cells recapitulates the core gene signatures, respectively, of angiomatoid fibrous histiocytoma (AFH) and gastrointestinal clear cell sarcoma (GI-CCS), although both fusions lead to cell lethality. Conversely, expression of the fusions in hES cells differentiated to mesenchymal progenitors is compatible with prolonged viability while maintaining the core gene signatures. Moreover, in the context of a mesenchymal lineage, the proliferation of cells expressing the EWSR1-CREB1 fusion is further extended by deletion of the tumor suppressor TP53. We expect the generation of isogenic lines carrying oncogenic fusions in various cell lineages to expand our general understanding of how those single genetic events drive tumorigenesis while providing valuable resources for drug discovery.

Activating transcription factor 1 (ATF1), belonging to the CREB/ATF family of transcription factors, is highly expressed in the testes. However, its role in spermatogenesis has not yet been established. Here, we aimed to elucidate the impact of ATF1 in spermatogenesis by examining the expression pattern of ATF1 in mice and the effect of ATF1 knockdown in the mouse testes. We found that ATF1 is expressed in various organs, with very high levels in the testes. Immunohistochemical staining showed that ATF1 was localized in the nuclei of spermatogonia and co-localized with proliferating cell nuclear antigen. In ATF1-deficient mice, the seminiferous tubules of the testis contained cells at all developmental stages; however, the number of spermatocytes was decreased. Proliferating cell nuclear antigen expression was decreased and apoptotic cells were rare in the seminiferous tubules. These results indicate that ATF1 plays a role in male germ cell proliferation and sperm production.

Myoepithelial tumors of soft tissue are rare mesenchymal neoplasms that overlap with their salivary gland and skin counterparts at both the histopathologic and molecular levels. EWSR1 gene rearrangements with various fusion partners represent a common genetic event in myoepithelial tumors of soft tissue, whether benign or malignant, and may prove useful as a diagnostic tool in difficult cases. However, the number of diagnostic entities with EWSR1 gene rearrangements has grown considerably in recent years, and there is significant morphologic and immunophenotypic overlap amongst this group, underscoring the importance of fusion testing to detect fusion partners that are characteristic of discrete diagnostic entities. Herein, we report a malignant myoepithelial tumor of soft tissue/myoepithelial carcinoma with an undifferentiated round cell morphology arising in a pediatric patient with a EWSR1-ATF1 gene fusion.

Gene fusions constitute pivotal driver mutations often encoding aberrant chimeric transcription factors. However, an increasing number of gene fusion events have been shown not to be histotype specific and shared among different tumor types, otherwise completely unrelated clinically or phenotypically. One such remarkable example of chromosomal translocation promiscuity is represented by fusions between EWSR1 or FUS with genes encoding for CREB-transcription factors family (ATF1, CREB1, and CREM), driving the pathogenesis of various tumor types spanning mesenchymal, neuroectodermal, and epithelial lineages. In this study, we investigate a group of 13 previously unclassified malignant epithelioid neoplasms, frequently showing an epithelial immunophenotype and marked predilection for the peritoneal cavity, defined by EWSR1/FUS-CREB fusions. There were seven females and six males, with a mean age of 36 (range 9-63). All except three cases occurred intra-abdominally, including one each involving the pleural cavity, upper, and lower limb soft tissue. All tumors showed a predominantly epithelioid morphology associated with cystic or microcystic changes and variable lymphoid cuffing either intermixed or at the periphery. All except one case expressed EMA and/or CK, five were positive for WT1, while being negative for melanocytic and other mesothelioma markers. Nine cases were confirmed by various RNA-sequencing platforms, while in the remaining four cases the gene rearrangements were detected by FISH. Eleven cases showed the presence of CREM-related fusions (EWSR1-CREM, 7; FUS-CREM, 4), while the remaining two harbored EWSR1-ATF1 fusion. Clinically, seven patients presented with and/or developed metastases, confirming a malignant biologic potential. Our findings expand the spectrum of tumors associated with CREB-related fusions, defining a novel malignant epithelioid neoplasm with an immunophenotype suggesting epithelial differentiation. This entity appears to display hybrid features between angiomatoid fibrous histiocytoma (cystic growth and lymphoid cuffing) and mesothelioma (peritoneal/pleural involvement, epithelioid phenotype, and cytokeratin and WT1 co-expression).

Transcription factors are often the downstream effectors of signaling cascades. In fission yeast, the transcription factor Atf1 is phosphorylated by the MAP kinase Sty1 under several environmental stressors to promote transcription initiation of stress genes. However, Sty1 and Atf1 have also been involved in other cellular processes such as homologous recombination at hotspots, ste11 gene expression during mating and meiosis, or regulation of fbp1 gene transcription under glucose starvation conditions. Using different phospho-mutants of Atf1, we have investigated the role of Atf1 phosphorylation by Sty1 in those biological processes. An Atf1 mutant lacking the canonical MAP kinase phosphorylation sites cannot activate fbp1 transcription when glucose is depleted, but it is still able to induce recombination at ade6.M26 and to induce ste11 after nitrogen depletion; in these last cases, Sty1 is still required, suggesting that additional non-canonical sites are activating the transcription factor. In all cases, an Atf1 phosphomimetic mutant bypasses the requirement of the Sty1 kinase in these diverse biological processes, highlighting the essential role of the DNA binding factor Atf1 on chromatin remodeling and cell adaptation to nutritional changes. We propose that post-translational modifications of Atf1 by Sty1, either at canonical or non-canonical sites, are sufficient to activate some of the functions of Atf1, those involving chromatin remodeling and transcription initiation. However, in the case of fbp1 where Atf1 acts synergistically with other transcription factors, elimination of the canonical sites is sufficient to hamper some of the interactions required in this complex scenario and to impair transcription initiation. We are thankful to Dr. Masayuki Yamamoto for providing strain JX233. This work is supported by the Ministerio de Ciencia, Innovación y Universidades (Spain), PLAN E and FEDER (PGC2018-093920-B-I00 to E.H.). The Oxidative Stress and Cell Cycle group is also supported by Generalitat de Catalunya (Spain) (2017-SGR-539) and by Unidad de Excelencia María de Maeztu from the Ministerio de Ciencia, Innovación y Universidades (CEX2018-000792-M) (Spain).

Background Chondroclasts and osteoclasts have been previously identified as the cells capable of resorbing mineralized cartilage and bone matrices, respectively. While both cell types appear morphologically similar, contain comparable ultrastructural features, and express tartrate-resistant acid phosphatase (TRAP), however, no information is available about the genomic similarities and differences between osteoclasts and chondroclasts. Methods To address this question, we laser captured homogeneous populations of TRAP-positive cells that interact with bone (osteoclasts) and TRAP-positive cells that interact with mineralized cartilage (chondroclasts) on the same plane from murine femoral fracture callus sections. We then performed a global transcriptome profiling of chondroclasts and osteoclasts by utilizing a mouse genome Agilent GE 4X44K V2 microarray platform. Multiple computational approaches and interaction networks were used to analyze the transcriptomic landscape of osteoclasts and chondroclasts. Results Our systematic and comprehensive analyses using hierarchical clustering and principal component analysis (PCA) demonstrate that chondroclasts and osteoclasts are transcriptionally distinct cell populations and exhibit discrete transcriptomic signatures as revealed by multivariate analysis involving scatter plot, volcano plot, and heatmap analysis. TaqMan qPCR was used to validate the microarray results. Intriguingly, the functional enrichment and integrated network analyses revealed distinct Gene Ontology terms and molecular pathways specific to chondroclasts and osteoclasts and further suggest that subsets of metabolic genes were specific to chondroclasts. Protein-protein interaction (PPI) network analysis showed an abundance of structured networks of metabolic pathways, ATP synthesis, and proteasome pathways in chondroclasts. The regulatory network analysis using transcription factor-target gene network predicted a pool of genes including ETV6, SIRT1, and ATF1 as chondroclast-specific gene signature. Conclusions Our study provides an important genetic resource for further exploration of chondroclast function in vivo. To our knowledge, this is the first demonstration of genetic landscape of osteoclasts from chondroclasts identifying unique molecular signatures, functional clustering, and interaction network.

The fungus Verticillium dahliae causes vascular wilt disease on hundreds of plant species. Homologs of the bZIP transcription factor Atf1 are required for virulence in most pathogenic fungi, but the molecular basis for their involvement is largely unknown. We performed targeted gene deletion, expression analysis, biochemistry and pathogenicity assays to demonstrate that VdAtf1 governs pathogenesis via the regulation of nitrosative resistance and nitrogen metabolism in V. dahliae. VdAtf1 controls pathogenesis via the regulation of nitric oxide (NO) resistance and inorganic nitrogen metabolism rather than oxidative resistance and is important for penetration peg formation in V. dahliae. VdAtf1 affects ammonium and nitrate assimilation in response to various nitrogen sources. VdAtf1 may be involved in regulating the expression of VdNut1. VdAtf1 responds to NO stress by strengthening the fungal cell wall, and by causing over-accumulation of methylglyoxal and glycerol, which in turn impacts NO detoxification. We also verified that the VdAtf1 ortholog in Fusarium graminearum mediates nitrogen metabolism, suggesting conservation of this function in related plant pathogenic fungi. Our findings revealed new functions of VdAtf1 in pathogenesis, response to nitrosative stress and nitrogen metabolism in V. dahliae. The results provide novel insights into the regulatory mechanisms of the transcription factor VdAtf1 in virulence.

ATF1, CREB1, and CREM constitute the CREB family of transcription factors. The genes encoding these factors are involved in gene fusion events in human tumors. EWSR1-ATF1 and EWSR1-CREB1 are the 2 most characterized fusions, whereas EWSR1-CREM has been less studied. To better understand the phenotypic spectrum of mesenchymal tumors associated with the EWSR1-CREM fusion, we investigated archival cases using fluorescence in situ hybridization and/or RNA sequencing. Among 33 clear cell sarcomas of soft tissue tested, we found 1 specimen, a hand tumor bearing the rearrangements of EWSR1 and CREM, with classic histology and immunophenotype. None of 6 clear cell sarcoma-like tumors of the gastrointestinal tract tested harbored the EWSR1-CREM fusion. Among 11 angiomatoid fibrous histiocytomas, we found that 3 tumors of myxoid variant harbored the rearrangements of EWSR1 and CREM. All 3 tumors occurred in middle-aged men and involved the distal extremities (N=2) and the lung (N=1). Prominent lymphoid cuff, fibrous pseudocapsule, and amianthoid fiber were present in 3, 2, and 2 tumors, respectively, whereas none showed pseudoangiomatoid spaces. All 3 tumors were immunohistochemically positive for epithelial membrane antigen and desmin. These cases suggested a closer relationship between angiomatoid fibrous histiocytoma and a recently proposed novel group of myxoid tumors with CREB family fusions. Our cohort also included 2 unclassifiable sarcomas positive for EWSR1-CREM. One of these was an aggressive pediatric tumor of the abdominal cavity characterized by proliferation of swirling spindle cells immunopositive for cytokeratin and CD34. The other tumor derived from the chest wall of an adult and exhibited a MUC4-positive sclerosing epithelioid fibrosarcoma-like histology. Our study demonstrates that a wider phenotypic spectrum is associated with the EWSR1-CREM fusion than previously reported.

Clear cell carcinoma (CCC) is a rare epithelial malignant tumor of the salivary glands. It is characterized by tumor cells with clear cytoplasm, hyalinized stroma, and most importantly the fusion genes EWSR1-ATF1, EWSR1-CREM, and EWSR1-PLAG1. Break-apart FISH has been performed for multiple CCC cases, but direct sequencing analysis has been performed in relatively few. Herein, we report an interesting case of CCC harboring three EWSR1-ATF1 translocations: EWSR1 exon 8-ATF1 exon 4, EWSR1 exon 7-ATF1 exon 4, and EWSR1 exon 7-ATF1 exon 5. This case indicates the possibility of independent EWSR1-ATF1 gene translocations, and could provide insight into CCC tumorgenesis.

Dogs have been considered as an excellent immunocompetent model for human melanoma due to the same tumor location and the common clinical and pathological features with human melanoma. However, the differences in the melanoma transcriptome between the two species have not been yet fully determined. Considering the role of oncogenes in melanoma development, in this study, we first characterized the transcriptome in canine oral melanoma and then compared the transcriptome with that of human melanoma. The global transcriptome from 8 canine oral melanoma samples and 3 healthy oral tissues were compared by RNA‑Seq followed by RT‑qPCR validation. The results revealed 2,555 annotated differentially expressed genes, as well as 364 novel differentially expressed genes. Dog chromosomes 1 and 9 were enriched with downregulated and upregulated genes, respectively. Along with 10 significant transcription site binding motifs; the NF‑κB and ATF1 binding motifs were the most significant and 4 significant unknown motifs were indentified among the upregulated differentially expressed genes. Moreover, it was found that canine oral melanoma shared >80% significant oncogenes (upregulated genes) with human melanoma, and JAK‑STAT was the most common significant pathway between the species. The results identified a 429 gene signature in melanoma, which was up‑regulated in both species; these genes may be good candidates for therapeutic development. Furthermore, this study demonstrates that as regards oncogene expression, human melanoma contains an oncogene group that bears similarities with dog oral melanoma, which supports the use of dogs as a model for the development of novel therapeutics and experimental trials before human application.

Recent studies showed that long non-coding RNAs (lncRNAs) could play critical roles in tumors progression. However, the performance of LINC01354 is still limited in non-small cell lung cancer (NSCLC). In the current study, our results showed that LINC01354 was significantly increased in NSCLC tissues and cell lines. High LINC01354 expression was associated with advanced TNM stage and poor prognosis in NSCLC patients. Loss-of-function assays revealed that knockdown of LINC01354 reduced lung cancer cells proliferation and invasive ability in vitro. Subsequently, mechanism studies showed that LINC01354 positively regulated the ATF1 expression via competitive binding to miR-340-5p. Therefore, our results illustrated that LINC01354 might act as an oncogenic role by modulating the miR-340-5p/ATF1 axis, providing a novel therapeutic therapy for NSCLC treatment.

AIMS: In thymic carcinomas, focal clear cell change is a frequent finding. In addition to a prominent, diffuse clear cell morphology, some of these carcinomas show an exuberant hyalinised extracellular matrix, and therefore probably represent a separate entity. However, a characteristic genomic alteration remains elusive. We hypothesised that, analogous to hyalinising clear cell carcinomas of the salivary gland, hyalinising clear cell carcinomas of the thymus might also harbour EWSR1 translocations. METHODS AND RESULTS: We identified nine archived cases of thymic carcinoma with focal clear cell features and two cases that showed remarkable hyalinised stroma and prominent, diffuse clear cell morphology. These two cases expressed p40 and were negative for Pax8, CD5, and CD117. Programmed death-ligand 1 was highly positive in one case (70%), and negative in the other one. EWSR1 translocation was identified in both cases of hyalinising clear cell carcinoma, and was absent in all nine carcinomas that showed clear cell features without substantial hyalinisation. In one of the EWSR1-translocated cases, a fusion between exon 13 and exon 6 of EWSR1 and ATF1, respectively was identified by next-generation sequencing. CONCLUSIONS: These findings suggest that the EWSR1 translocation and possibly the EWSR1-ATF1 fusion might be unifying genomic alterations for thymic clear cell carcinomas with prominent hyalinised stroma, for which we propose the term 'hyalinising clear cell carcinoma of the thymus'. Because the immunophenotype is unspecific, testing for the EWSR1 translocation might be helpful in discriminating this entity from other thymic neoplasms or metastases, in particular those with clear cell change. ispartof: HISTOPATHOLOGY vol:75 issue:3 pages:431-436 ispartof: location:England status: published

Clear cell odontogenic carcinoma (CCOC) is a rare, low-grade malignant epithelial neoplasm, occurring in the jawbones, mainly affecting the mandible of elderly patients. In addition to hyalinizing clear cell carcinoma of the salivary gland, it is one of the epithelial neoplasms known to harbor an EWSR1-ATF1 fusion. Therefore, a link between these tumors seems plausible. We describe six cases of CCOC showing EWSR1 rearrangements, with two cases being positive for the ATF1 partner gene using FISH analysis. In one case, an EWSR1-CREB1 fusion was identified using RT-PCR, which we report for the first time in this tumor type. The other three cases investigated by FISH were negative for ATF1, CREB1 and CREB3L2. In conclusion, our data show that EWSR1-CREB1 is an alternative fusion gene to EWSR1-ATF1 in CCOC.

Our previous work reported activating transcription factor 1 (ATF1) is a promotive factor of nasopharyngeal carcinoma (NPC) tumorigenesis. This study is to further explore the association between the human ATF1 rs11169571 polymorphism and the risk of NPC occurrence. The association between ATF1 rs11169571 and risk of NPC occurrence was investigated in clinical samples of 560 patients and 661 controls obtained from southern China with high incidence of NPC. The genotypes were detected by PCR‐RFLP. The differential expression activity of alleles ‐T and ‐C was analyzed with CNE‐2 and C666‐1 cells by luciferase reporter assay. Our data suggested that the allelic frequency and genotypes were significantly different between patients and controls. Compared to the TT homozygote, the TC and CC genotypes have been shown to be significantly decreased in NPC patients (OR = 0.494, 95% CI = 0.387‐0.629, P

Geranylgeranyl acetate have been identified as potent and selective inhibitors against Mycobacterium tuberculosis. In this study, the MVA pathway was engineered in Escherichia coli to biosynthesis of the antimycobacterial agent geranylgeranyl acetate from glucose for the first time. Three modules were constructed in E. coli to achieve this objective. AtoB, ERG13 and tHMG1 were overexpressed in the upstream module to accumulate MVA. Then MVA were catalyzed to IPP and DMAPP in the midstream module, which overexpressed Idi, ERG8, MVD1 and ERG12. Finally, in the downstream module, geranylgeranyl acetate were biosynthesized from IPP and DMAPP by overexpressing GGPPS, PgpB and ATF1. The engineered strain DG108 accumulated 57 ± 3.8 mg/L of geranylgeranyl acetate. Further overexpression the Idi, the yield was increased to 119 ± 6.1 mg/L in DG109. The approach described here could be expanded to biosynthesize many antimycobacterial agents and expand the design strategy of metabolic engineering for bioproducts.

Translocations between EWSR1 and members of the CREB family of transcription factors (CREB1, ATF1, and CREM) are rare genetic findings occurring in various sarcomas. Of these, the EWSR1-CREM translocation is the most rarely reported. We present the case of a 9-year-old boy who presented with a year of fatigue, weight loss, and abulia. A brain MRI revealed a frontal interhemispheric tumor arising from the falx. After resection, pathology demonstrated a myxoid mesenchymal tumor with an EWSR1-CREM translocation. A series of recent reports of similar tumors has generated ongoing debate in the literature over the classification of these tumors either as intracranial angiomatoid fibrous histiocytomas, which also harbor EWSR1-CREB family translocations, or as a novel diagnostic entity. The present case provides another example of the rare EWSR1-CREM fusion in an intracranial myxoid mesenchymal tumor that recurred in just 6 months despite gross total resection. The findings are discussed in the context of the existing literature and the ongoing effort to appropriately classify this type of tumor.

The ability to cope with environmental abiotic stress and biotic stress is crucial for the survival of plants and microorganisms, which enable them to occupy multiple niches in the environment. Previous studies have shown that transcription factors play crucial roles in regulating various biological processes including multiple stress tolerance and response in eukaryotes. This work identified multiple critical transcription factor genes, metabolic pathways and gene ontology (GO) terms related to abiotic stress response were broadly activated by analyzing the transcriptome of phytopathogenic fungus Alternaria alternata under metal ions stresses, oxidative stress, salt stresses, and host-pathogen interaction. We investigated the biological functions and regulatory roles of the bZIP transcriptional factor (TF) genes in the phytopathogenic fungus A.alternata by analyzing targeted gene disrupted mutants. Morphological analysis provides evidence that the bZIP transcription factors (Gcn4, MeaB, Atf1, the ER stress regulator Hac1, and the all development altered-1 gene Ada1) are required for morphogenesis as the colony morphology of these gene deletion mutants was significantly different from that of the wild-type. In addition, bZIPs are involved in the resistance to multiple stresses such as oxidative stress (Ada1, Yap1, MetR) and virulence (Hac1, MetR, Yap1, Ada1) at varying degrees. Transcriptome data demonstrated that the inactivation of bZIPs (Hac1, Atf1, Ada1 and Yap1) significantly affected many genes in multiple critical metabolism pathways and gene ontology (GO) terms. Moreover,the ΔHac1 mutants displayed reduced aerial hypha and are hypersensitivity to endoplasmic reticulum disruptors such as tunicamycin and dithiothreitol. Transcriptome analysis showed that inactivation of Hac1 significantly affected the proteasome process and its downstream unfolded protein binding, indicating that Hac1 participates in the endoplasmic reticulum stress response through the conserved unfolded protein response. Taken together, our findings reveal that bZIP transcription factors function as key regulators of fungal morphogenesis, abiotic stress response and pathogenesis, and expand our understanding of how microbial pathogens utilize these genes to deal with environmental stresses and achieve successful infection in the host plant.

OBJECTIVE To determine the functional effects of ATF1, WNT10B and GREM2 gene variants identified in individuals with tooth agenesis (TA). SETTINGS AND SAMPLE POPULATION Stem cells from human exfoliated deciduous teeth (SHED) were used as an in vitro model system to test the effect of TA-associated variants. MATERIALS AND METHODS Plasmid constructs containing reference and mutant alleles for ATF1 rs11169552, WNT10B rs833843 and GREM2 rs1414655 variants were transfected into SHED for functional characterization of variants. Allele-specific changes in gene transcription activity, protein expression, cell migration and proliferation, and expression of additional tooth development genes (MSX1, PAX9 and AXIN2) were evaluated. Data analyses were performed using Student's t-test. P-values ≤ .05 were considered statistically significant. RESULTS Mutant variants resulted in significantly decreased transcriptional activity of respective genes (P

The presence of melanocytic differentiation in tumors of non-melanocyte origin is uncommon and is typically associated with the overexpression of MITF, the master regulator of melanin synthesis, or another member of the MIT/TFE3 family. In clear cell sarcoma, the presence of either an EWSR1-ATF1 or EWSR1-CREB1 translocation-derived fusion protein is thought to drive melanocytic differentiation by directly stimulating the expression of MITF. Here, we describe a clear cell neoplasm with melanocytic differentiation that is characterized by a novel MITF-CREM gene fusion. CREM is the third member of the ATF1/CREB1/CREM family, and the nature of the MITF-CREM fusion appears analogous to the EWSR1-ATF1 and EWSR1-CREB1 fusions. Thus, this MITF-CREM-rearranged clear cell tumor represents a novel entity with morphologic, immunohistochemical, and molecular similarity to clear cell sarcoma.

Calcium/calmodulin‐dependent protein kinase (CAMKs) can control a wide range of cancer‐related functions in multiple tumour types. Herein, we explore the expressions and clinical significances of calcium/calmodulin‐dependent protein kinase 1 (CAMK1) in pancreatic cancer (PC). The expression of CAMK1 in PC was analysed by Gene Expression Profiling Interactive Analysis 2 (GEPIA 2) database and the Oncomine database. For further validation, the protein level of CAMK1 in PC tissues was also detected in the Human Protein Atlas (HPA) database and the tissue microarray (TMA)‐based immunohistochemistry (IHC). GEPIA 2 and Kaplan‐Meier Plotter (KM Plotter) databases were used to explore the prognostic significances of CAMK1 in overall survival (OS) and disease‐free survival (DFS) of PC at mRNA level. The relationship between CAMK1 expression and the clinicopathological characteristics of PC was further explored. Additionally, the Search Tool for the Retrieval of Interacting Genes (STRING) database was used to analyse protein‐protein interactions (PPI). We found CAMK1 was highly expressed in PC both in bioinformatics analyses and TMA‐IHC results. The prognostic analyses from the public databases also showed consistent results with follow‐up data. The PPI network suggested that CALM1, CALM3, CREB1, CALM2, SYN1, NOS3, ATF1, GAPDH, PPM1F and FBXL12 were important significant genes associated with CAMK1. Our finding revealed CAMK1 has prognostic value in PC patients, suggesting that CAMK1 may has a distinct role in PC patients and can be used as a candidate marker for investigating clinical prognosis of PC.

Mitogen-activated protein kinase (MAPK) cascades are broadly conserved and play essential roles in multiple cellular processes, including fungal development, pathogenicity, and secondary metabolism. Their function, however, also exhibits species and strain specificity. Penicillium oxalicum secretes plant-biomass-degrading enzymes (PBDEs) that contribute to the carbon cycle in the natural environment and to utilization of lignocellulose in industrial processes. However, knowledge of the MAPK pathway in P. oxalicum has been relatively limited. In this study, comparative transcriptomic analysis of P. oxalicum, cultured on different carbon sources, found ten putative kinase genes with significantly modified transcriptional levels. Six of these putative kinase genes were knocked out in the parental strain ∆PoxKu70, and deletion of the gene, Fus3/Kss1-like PoxMK1 (POX00158), resulted in the largest reduction (91.1%) in filter paper cellulase production. Further tests revealed that the mutant ∆PoxMK1 lost 37.1 to 92.2% of PBDE production, under both submerged- and solid-state fermentation conditions, compared with ∆PoxKu70. In addition, the mutant ∆PoxMK1 had reduced vegetative growth and increased pigment biosynthesis. Comparative transcriptomic analysis showed that PoxMK1 deletion from P. oxalicum downregulated the expression of major PBDE genes and known regulatory genes such as PoxClrB and PoxCxrB, whereas the transcription of pigment biosynthesis-related genes was upregulated. Comparative phosphoproteomic analysis revealed that PoxMK1 deletion considerably modified phosphorylation of key transcription- and signal transduction-associated proteins, including transcription factors Mcm1 and Atf1, RNA polymerase II subunits Rpb1 and Rpb9, MAPK-associated Hog1 and Ste7, and cyclin-dependent kinase Kin28. These findings provide novel insights into understanding signal transduction and regulation of PBDE gene expression in fungi. Key points • PoxMK1 is involved in expression of PBDE- and pigment synthesis-related genes. • PoxMK1 is required for vegetative growth of P. oxalicum. • PoxMK1 is involved in phosphorylation of key TFs, kinases, and RNA polymerase II.

Clear cell carcinoma (CCC) is a rare low-grade malignant salivary gland carcinoma. EWSR1-ATF1 fusion has been characterized as a consistent finding in CCC, with breakpoints described between EWSR1 exon 11 and ATF1 exon 3. So far, over 100 cases of CCC harboring EWSR1 rearrangement arising from salivary gland of the oral cavity have been reported. Although EWSR1 involvement in these cases was confirmed by EWSR1 break-apart FISH indicating the translocation, sequence analysis for EWSR1-ATF1 fusion type has been reported only in three cases of CCC so far. Herein, we report a CCC case with novel EWSR1-ATF1 fusion (EWSR1 exon 15 and ATF1 exon 5) arising in minor salivary gland and review the role of the chimeric variants in some malignancies with EWSR1-ATF1 rearrangement. Current tumor was composed of the small nests of clear tumor cells and hyalized fibrous stroma. Immunohistochemically, the tumor was positive for AE1/AE3, CK5/6 and p63, negative for S100, Melan-A, SMA and CD10. After 8 months of follow-up, there are no evidence of recurrence.

Aluminum (Al) toxicity is a major abiotic stress that restricts crop production in acid soils. Plants have evolved internal and external mechanisms of tolerance, and among them it is well known that AtSTOP1 and OsART1 are key transcription factors involved in tolerance through regulation of multiple downstream genes. Here, we identified the closest homolog of these two proteins in barley, namely HvATF1, Al-tolerance Transcription Factor 1, and determined its potential function in Al stress. HvATF1 is expressed in the nucleus, and functions in transcriptional activation. The transcription of HvATF1 was found to be constitutive in different tissues, and was little affected by Al stress. Knockdown of HvATF1 by RNAi resulted in increased Al sensitivity. Transcriptomics analysis identified 64 differently expressed genes in the RNAi lines compared to the wild-type, and these were considered as candidate downstream genes regulated by HvATF1. This study provides insights into the different molecular mechanisms of Al tolerance in barley and other plants.

Appropriate concentrations and proportion of acetate esters and higher alcohols improve the quality of Chinese Baijiu. To regulate the concentrations of acetate esters in Chinese Baijiu, we constructed a PGK1 promoter library through error-prone PCR. Then, we used an enhanced green fluorescent protein as a reporter to characterize the activities of PGK1p mutants. The PGK1p library contained 28 PGK1p mutants and spanned an activity that ranged between 0.1% and 141% of wild-type PGK1p. Seven PGK1p mutants were characterized by an additional reporter β-galactosidase and then used for the overexpression of ATF1 with BAT2 deletion in Saccharomyces cerevisiae a45. The production of ethyl acetate in strains A8, A17, A18, A27, A22, A25, A28, and AWT were 1.66-, 3.09-, 10.59-, 13.07-, 15.99-, 22.67-, 24.06-, and 27.22-fold higher than that of the parental strain. The results on alcohol acetyltransferase (AATase) activity showed that the PGK1p library precisely controlled ATF1 expression and regulated the acetate esters production.

Mezcal, a traditional beverage that originated in Mexico, is produced from species of the Agavaceae family. The esters associated with the yeasts utilized during fermentation are important for improving the organoleptic properties of the beverage. We improved the ester contents in a mezcal beverage by using the yeast Kluyveromyces marxianus, which was engineered with the ATF1 gene. ATF1 expression in the recombinant yeast significantly increased compared with that in the parental yeast, but its fermentative parameters were unchanged. Volatile-organic-compound-content analysis showed that esters had significantly increased in the mezcal produced with the engineered yeast. In a sensory-panel test, 48% of the panelists preferred the mezcal produced from the engineered yeast, 30% preferred the mezcal produced from the wild type, and 15 and 7% preferred the two mezcal types produced following the routine procedure. Correlation analysis showed that the fruitiness/sweetness description of the mezcal produced using the ATF1-engineered K. marxianus yeast correlated with the content of the esters, whose presence improved the organoleptic properties of the craft mezcal beverage.

About a third of soft tissue tumors are recognized by gene fusions. Some of these fusions are not histotype specific and occur in entities with totally different clinicopathological features. A good example is the fusion between EWSR1 and genes of the CREB transcription factor gene family (CREB1 or ATF1). This article is protected by copyright. All rights reserved. ispartof: Histopathology vol:72 issue:5 pages:880-883 ispartof: location:England status: published

Nitric Oxide (NO) and its associated reactive nitrogen species (RNS) produce nitrosative stress under various pathophysiological conditions in eukaryotes. The fission yeast Schizosaccharomyces pombe regulates stress response mainly through the Sty1-Atf1 MAP Kinase pathway. The present study deals with the role of transcription factor Atf1 and Sty1 in S. pombe under nitrosative stress. In this study, exposure to an NO donor resulted in S-phase slowdown with associated mitotic block in S. pombe. Deletion of sty1 and atf1 in S. pombe had differential growth sensitivity towards NO donor. Both Sty1 and Atf1 were involved in regulating mitotic slowdown in S. pombe under nitrosative stress. Experimental data obtained in this study reveals a novel role of Atf1 in initiating the replication slowdown in S. pombe under nitrosative stress. Both Sty1 and Atf1 were accumulated in the nucleus in S. pombe under nitrosative stress in a concentration and time dependent manner. Atf1 is also found to be nuclear delocalized under longer nitrosative stress.

Introduction: Ewing sarcoma breakpoint region 1- activating transcription factor 1 (EWSR1-ATF1) fusion is mentioned as a diagnostic marker in favor of clear cell carcinoma in the differential diagnosis of clear cell neoplasms of salivary glands. However, in literature, its positivity is reported between 82% and 93%. In salivary glands, various benign and malignant primary tumors, as well as odontogenic neoplasms and metastatic cases, present with a varied number of clear cells. Clear cell carcinoma of the salivary gland is a rare entity with distinct histochemistry and immunohistochemistry features.Case Presentation: We present a clear cell carcinoma of the salivary gland showing cytoplasmic Periodic acid–Schiff (PAS) (+), mucin (-) staining, cytokeratin (CK), CK7, and P63 immunoreactivity without EWSR1-ATF1 fusion. Conclusions: We conclude that clinical, morphological, and immunohistochemical analyses should be the guide for diagnosis even though molecular analysis does not support it.

NAC (NAM, ATF1/2 and CUC2) is a transcription factor that can regulate many physiological and biochemical reactions in plants. Two NAC genes, SNAC4 (NM_001279348.2) and SNAC9 (NM_001365397.1), with similar secondary structures have been isolated from tomato (Solanum lycopersicum L.). The pattern of SNAC4/9 co-regulation of tomato ripening with abscisic acid (ABA) and ethylene through yeast two-hybrid and bimolecular fluorescence complementation assays has revealed that SNAC4/9 can interact with genes related to ABA and ethylene (SAPK3, SlPYL9, SlAREB1, SlACS2 and SlACO1) at the protein level. Electrophoretic mobility shift assay and yeast one-hybrid showed that SNAC4, but not SNAC9, acts directly on the promoter regions of SAPK3, SlCYP707A1, SlACS8 and SlACO6 and can activates them. In addition, quantitative real-time PCR analyses of SNAC4/9-silenced fruit obtained by virus-induced gene silencing, and phytohormone-treated fruit, confirmed that the interaction genes are regulated by SNAC4/9 and thereby affect fruit ripening. In summary, we found that SNAC4/9 can regulate fruit ripening by positively acting on key genes in the synthesis and signal transduction of ABA and ethylene. SNAC4/9 cooperates with these phytohormones as part of the tomato fruit ripening regulatory network.

Objectives Salivary gland tumors are comprised of a diverse group of malignancies with widely varying prognoses. These cancers can be difficult to differentiate, especially in cases with limited potential for immunohistochemistry (IHC)-based characterization. Here, we sought to define the molecular profile of a rare salivary gland cancer called hyalinizing clear cell carcinoma (HCCC), and identify a molecular gene signature capable of distinguishing between HCCC and the histopathologically similar disease, mucoepidermoid carcinoma (MEC). Materials and methods We performed the first integrated full characterization of five independent HCCC cases. Results We discovered insulin-like growth factor alterations and aberrant IGF2 and/or IGF1R expression in HCCC tumors, suggesting a potential dependence on this pathway. Further, we identified a 354 gene signature that differentiated HCCC from MEC, and was significantly enriched for genes with an ATF1 binding motif in their promoters, supporting a transcriptional pathogenic mechanism of the characteristic EWSR1-ATF1 fusion found in these tumors. Of the differentially expressed genes, IGF1R, SGK1 and SGK3 were found to be elevated in the HCCCs relative to MECs. Finally, analysis of immune checkpoints and subsequent IHC demonstrated that CXCR4 protein was elevated in several of the HCCC cases. Conclusion Collectively, our data identify an ATF1-motif enriched gene signature that may have clinical utility for molecular differentiation of HCCCs from other salivary gland tumors and discover potential actionable alterations that may benefit the clinical care of recurrent HCCC patients.

Acetate esters and higher alcohols significantly determine the flavor profiles of Chinese Baijiu (Chinese liquor). The aminotransferase encoded by BAT2 and alcohol acetyltransferases encoded by ATF1, ATF2, and Lg-ATF1 are involved in the production of branched-chain alcohols and synthesis of acetate esters, respectively. In this study, the effects of ATF1, ATF2, and Lg-ATF1 overexpressions with BAT2 deletion were explored in Chinese Baijiu yeast. The differences among these effects were also investigated. Results showed that the productions of acetate esters by mutant overexpressing ATF1 with BAT2 deletion and that overexpressing ATF2 with BAT2 deletion were 1353.45 and 73.40 mg/L, respectively, which were 43.16- and 2.34-fold higher than that by the original strain. Compared with mutant overexpressing ATF2 with BAT2 deletion, mutant overexpressing ATF1 with BAT2 deletion exhibited 48.17% decreased higher alcohol productivity. The production of higher alcohols in mutant overexpressing ATF2 with BAT2 deletion was similar to that in the BAT2 deletion mutant. Furthermore, no significant difference was observed between the BAT2 deletion mutant and the mutant overexpressing Lg-ATF1 with BAT2 deletion in terms of acetate ester and higher alcohol production. The mutants that have varying capacities for acetate ester and higher alcohol production can be potentially developed and applied.

Aged mice consumed water supplemented with Pedro Ximenez must (PXM). After 20 weeks, histological analyses showed that PXM consumption highly alleviated the age-related hepatic damage observed in control mice, restoring the cytoplasmic appearance and normal arrangement of hepatic cords of young mice. Bidimensional electrophoresis of 5-IAF labeled hepatic proteins showed changes in the reversibly oxidized thiol status of 42 protein spots. Fourteen proteins were identified. Seven (FAM210B, RPE, SRSF1, KRT1, ATF1, ZEB1, IgM) resulted reduced and other seven (RPL29, GABPB1, ACTB, DDC, INA, PRKAR1B, ZFP738) oxidized. For 6 proteins (ATF1, RPE, ZEB1, IGM, PRKAR1B and ZFP738) the susceptibility of a particular Cys to reversible oxidation was analyze. The histological changes, the variations in the oxidation status of protein thiolic groups and the identification of specific Cys residues affected, indicated that the PXM must is acting as an antioxidant but also a pro-oxidant that might help the elderly liver to regenerate.

Sin1 is a substrate-binding subunit of target of rapamycin complex 2 (TORC2), an evolutionarily conserved protein kinase complex. In fission yeast, Sin1 has also been identified as a protein that interacts with Spc1 (also known as Sty1) in the stress-activated protein kinase (SAPK) pathway. Therefore, this study examined the relationship between TORC2 and Spc1 signaling. We found that the common docking (CD) domain of Spc1 interacts with a cluster of basic amino acid residues in Sin1. Although diminished TORC2 activity in the absence of the functional Spc1 cascade suggests positive regulation of TORC2 by Spc1, such regulation appears to be independent of the Sin1-Spc1 interaction. Hyperosmotic stress transiently inhibits TORC2, and its swift recovery is dependent on Spc1, the transcription factor Atf1, and the glycelrol-3-phosphate dehydrogenase Gpd1, whose expression is induced upon osmostress by the Spc1-Atf1 pathway. Thus, cellular adaptation to osmostress seems important for TORC2 reactivation, though Spc1 and Atf1 contribute to TORC2 activation also in the absence of osmostress. These results indicate coordinated actions of the SAPK and TORC2 pathways, both of which are essential for fission yeast cells to survive environmental stress.

Lung cancer is among the most frequently occurring cancers and the leading cause of cancer-related deaths worldwide. Nonsmall cell lung cancer is accountable for 85% to 90% of all lung cancer cases and develops distant metastases with high mortality. In this work, we elucidated the role of activating transcription factor 1 (ATF1) in migration and invasion of lung cancer cells. We found that the migration and invasion were inhibited with ATF1 silencing in lung cancer cells. By contrast, ATF1 overexpression led to promotion in migration and invasion. The alteration in ATF1 expression induced a change in the epidermal growth factor receptor (EGFR) and matrix metalloproteinases (MMP)-2 expression level in the same tendency. Thus, we provided a potential new candidate for therapies against lung cancer, showing the possible mechanism underlying the invasion and migration of lung cancer cells.

The security of engineering Saccharomyces cerevisiae is becoming more focused on industrial production in consideration of the public concern regarding genetically modified organisms. In this work, a rapid and highly efficient system for seamless gene deletion in S. cerevisiae was developed through two-step integration protocol combined with endonuclease I-SCEI expression. The factors affecting the frequency of the second homologous recombination were optimized, and studies indicated that the mutant strains with 500 bp direct repeats and that have been incubating in galactose (0.5 g/100 mL) medium at 30°C and 180 r/min for 24 h permit high frequency (6.86 × 10−4) of the second homologous recombination. Furthermore, DNA sequence assays showed only self-DNA in native location without any foreign genes after deletion using this method. The seamless gene deletion method was applied to the construction of the engineering strains with BAT2 (encoding aminotransferase) deletion and ATF1 (alcohol acetyltransferases) overexpression. The mutants exhibited significant effects on higher alcohol reduction and ester improvement after Baijiu fermentation. The engineered strains can be used in industrial production in security, thereby meeting the requirements of modern science and technology.

As one of the most aggressive malignancies, cervical cancer (CC) which mainly affects females has high risks of relapse and death. Long non-coding RNA (lncRNA) RP11-552M11.4 is verified to promote the progression of ovarian cancer; nevertheless, its role and the probable molecular mechanisms in CC remain unclear until the present study. Herein, we unveiled that the expression of lncRNA RP11-552M11.4 tested by qRT-PCR was enhanced in tumor tissues compared with the para-carcinoma tissues and related to FIGO Stage, lymph node metastasis, vascular invasion and distant metastasis in CC. Additionally, CC patients with high lncRNA RP11-552M11.4 level suffered from poor clinical outcomes. Moreover, silenced lncRNA RP11-552M11.4 restrained cell proliferation, migration and invasion in CC cells. Subsequently, the mechanistic studies revealed that lncRNA RP11-552M11.4 functioned as a ceRNA of ATF1 in CC by acting as the endogenous sponge for miR-3941, which was identified as a tumor suppressor in CC. Moreover, both miR-3941 inhibition and ATF1 overexpression restored the impacts of inhibited lncRNA RP11-552M11.4 on cellular processes in CC cells. Our observations elucidated the carcinogenic role of lncRNA RP11-552M11.4 in CC was mediated through miR-3941/ATF1 axis, giving a new insight into the effective target for the treatment and prognosis of cervical cancer.

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.

Summary Upon heat shock, the fission yeast Hsp40 chaperone Mas5 drives temperature-sensitive proteins toward protein aggregate centers (PACs) to avoid their degradation until lower temperatures favor their refolding. We show here that cells lacking Mas5 are resistant to oxidative stress. Components of the general stress pathways, the MAP kinase Sty1 and the transcription factor Atf1, are suppressors of this phenotype. Strain Δmas5 expresses higher levels of Sty1- and Atf1-dependent stress genes than wild-type cells. Pyp1, the main tyrosine phosphatase maintaining Sty1 inactive in the absence of stress, is a temperature-sensitive protein that aggregates upon temperature up-shifts in a Mas5-dependent manner. In strain Δmas5, Pyp1 is sent to proteasomal degradation even in the absence of stress. We propose that Pyp1 is a thermo-sensitive phosphatase, which during heat stress coalescences into PACs in a Mas5-dependent manner, to promote full activation of the anti-stress Sty1-Atf1 cascade., Graphical Abstract, Highlights • Pyp1 phosphatase requires Mas5 to escape from degradation in unstressed conditions • Pyp1 assembles into protein aggregate centers (PACs) upon temperature up-shifts • Temporal sequestration of Pyp1 into PACs upon heat shock allows Sty1 activation • Pyp1 incorporation into PACs depends on Mas5, Biological Sciences; Molecular Biology; Cell Biology

Background Clear cell sarcoma of soft tissue (CCSST) and clear cell sarcoma-like gastrointestinal tumor (CCSLGT) are malignant mesenchymal tumors that share some pathological features, but they also have several different characteristics. They are well known to express chimeric fusions of Ewing sarcoma breakpoint region 1 (EWSR1) and cAMP response element-binding protein (CREB) family members; namely, EWSR1-activating transcription factor 1 (ATF1) and EWSR1-CREB1. In addition, recent studies have suggested the presence of other fusions. Methods We used fluorescence in situ hybridization to detect specific rearrangements including EWSR1, ATF1, CREB1, and cAMP response element modulator (CREM) in 16 CCSST and 6 CCSLGT cases. We also used reverse transcription polymerase chain reaction (RT-PCR) to detect specific chimeric fusions of EWSR1-ATF1 and EWSR1-CREB1 using fresh tumor samples in available cases. Results A total of 15 of 16 CCSST cases (93.8%) had EWSR1 rearrangement, of which 11 (68.8%) also had ATF1 rearrangement, suggestive of the presence of EWSR1-ATF1 fusions. One CCSST case (6.3%) was found to have EWSR1 and CREM rearrangements, and 4 of 6 CCSLGT cases (66.7%) had EWSR1 rearrangement, of which 2 (33.3%) showed ATF1 rearrangement and the other 2 cases (33.3%) showed CREB1 rearrangement. These cases most likely had EWSR1-ATF1 and EWSR1-CREB1 fusions, respectively. RT-PCR was performed in 8 available cases, including 6 CCSSTs and 2 CCSLGTs. All CCSSTs showed EWSR1-ATF1 fusions. Among the 2 CCSLGT cases, one had EWSR1-ATF1 fusion and the other had EWSR1-CREB1 fusion. Conclusions Rearrangements of EWSR1 and ATF1 or EWSR1-ATF1 fusion were predominantly found in CCSST, whereas those of EWSR1 and CREB1 or EWSR1-CREB1 tended to be detected in CCSLGT. A novel CREM fusion was also detected in a few cases of CCSST and CCSLGT. The cases in which EWSR1 rearrangement was detected without definitive partner genes should be considered for the presence of CREM rearrangement.

We describe a novel gene fusion, EWSR1-CREM, identified in 3 cases of clear cell carcinoma (CCC) using anchored multiplex polymerase chain reaction, a next-generation sequencing-based technique. CCC is a low-grade salivary tumor recently characterized to have EWSR1-ATF1 fusions in the majority of cases. Three cases of malignant tumor presenting in the base of tongue, lung, and nasopharynx were studied. All cases shared a clear cell morphology with hyalinized stroma, presence of mucin and p63 positivity and were initially diagnosed as mucoepidermoid carcinoma but were negative for evidence of any of the expected gene fusions. Anchored multiplex polymerase chain reaction demonstrated a EWSR1-CREM fusion in all 3 cases to confirm a diagnosis of CCC. This finding is biologically justified as CREM and ATF1 both belong to the CREB family of transcription factors. EWSR1-CREM fusions have not been previously reported in CCC and have only rarely been reported in other tumors. We show that the ability to discover novel gene variants with next-generation sequencing-based assays has clinical utility in the pathologic classification of fusion gene-associated tumors.

Azole antifungals directly inhibit enzymes for ergosterol biosynthesis, and this direct action is thought to underlie antifungal actions of these drugs. Recent studies showed that azoles alter expression of genes for various cellular functions. However, transcription factors regulated by azoles and their roles in antifungal actions remain poorly characterized. Using luciferase assay, we found that miconazole increased luciferase activity under the promoter containing the cAMP response element (CRE) motif. This azole-induced activation of CRE reporter was abolished in Atf1-deficient cells, suggesting that azoles induce Atf1 activation. As Atf1 is activated by stress-activated MAP kinase Sty1 upon various stressors, we examined its involvement. Azoles increased phosphorylation of Sty1 for its activation, and Sty1 deletion impaired azole-induced CRE reporter activation. In contrast, deletion of Pyp1, a tyrosine phosphatase which negatively regulates Sty1, increased CRE reporter activation. In addition, cells deficient in Atf1 and stress-activated MAP kinase pathway showed resistance to azoles, whereas cells lacking Pyp1 increased azole susceptibility, suggesting a critical role for azole-induced activation of MAP kinase-Atf1 pathway in antifungal actions of azoles. Collectively, these results suggest that azoles activate stress-activated MAP kinase pathway, thereby facilitating Atf1-mediated transcription for antifungal effects.

Morphological, ultrastructural and immunohistochemical characteristics of clear cell sarcoma (CCS) of the soft tissue frequently overlap with those of malignant melanoma. Thus, the differential diagnosis between the two lesions represents an important diagnostic dilemma. However, a number of genetic factors can be used to differentiate the two tumors; in particular, the t(12;22)(q13;q12) chromosomal translocation is typical of CCS, resulting in fusion of the EWSR1 gene on chromosome 22q12 and the ATF1 gene on chromosome 12q13. The detection of this molecular alteration has proved useful in the differential diagnosis of the two lesions. The present study reports the case of a 71-year-old male patient with a suspicious lymph node mass. Immunohistochemical analysis of the lesion indicated a diagnosis of metastatic melanoma, however, cytogenetic analysis using fluorescence in situ hybridization was additionally performed to investigate the chromosomal rearrangements of the 22q12 region and completely exclude the possibility of CCS. The current case did not demonstrate the presence of the translocation, supporting the diagnosis of melanoma. However, a clear orange amplification signal was observed relative to an ~500-kb region adjacent to the EWSR1 gene in the centromeric direction of chromosome 22q12. To the best of our knowledge, this is the first description of a 22q12 chromosomal alteration in melanoma. Furthermore, despite the presence of numerous genes in this region, their amplification has not previously been associated with the pathogenesis of melanoma.

Vitamin D deficiency is common in the Caucasian population and is associated with increased incidence and unfavorable outcome of many diseases, including various types of cancer, infectious, cardiovascular, and autoimmune diseases. Individual factors that predispose for a person's vitamin D status, such as skin type, have been identified, but limited data exist on genetic determinants of serum 25-hydroxyvitamin D (25[OH]D) concentration. We have tested the hypothesis that variants of genes (single nucleotide polymorphisms [SNPs]) involved in skin pigmentation are predictive of serum 25(OH)D levels. Serum 25(OH)D and SNPs (n = 960) related to genes with relevance for skin pigmentation (tyrosinase [TYR], TYR-related protein 1 [TYRP1], dopachrome tautomerase [DCT], oculocutaneous albinism II [OCA2], two pore segment channel 2 [TPCN2], solute carrier family 24 A4 [SLC24A4], solute carrier family 45 A2 [SLC45A2], agouti signalling peptide [ASIP], cyclic AMP-dependent transcription factor [ATF1], microphthalmia-associated transcription factor [MITF], proopiomelanocortin [POMC], cAMP-dependent protein kinase catalytic subunit beta [PRKACB], cAMP-dependent protein kinase catalytic subunit gamma [PRKACG], cAMP-dependent protein kinase type I-alpha regulatory subunit [PRKAR1A], cAMP-dependent protein kinase type II-alpha regulatory subunit [PRKAR2A], cAMP-dependent protein kinase type II-beta regulatory subunit [PRKAR2B], tubulin beta-3 chain/melanocortin receptor 1 [TUBB3/MC1R], Cadherin-1 [CDH1], catenin beta 1 [CTNNB1], Endothelin 1 [EDN1], endothelin 3 [EDN3], endothelin receptor type B [EDNRB], fibroblast growth factor 2 [FGF2], KIT, KIT ligand [KITLG], nerve growth factor [NGF], interferon regulatory factor 4 [IRF4], exocyst complex component 2 [EXOC2], and tumor protein 53 [TP53]) were analyzed in a cohort of participants of the Ludwigshafen Risk and Cardiovascular Health Study (n = 2970). A total of 46 SNPs were associated (P

Previously reported co-occurrence of colorectal cancer (CRC) and tooth agenesis (TA) and the overlap in disease-associated gene variants suggest involvement of similar molecular pathways. In this study, we took an unbiased approach and tested genome-wide significant CRC-associated variants for association with isolated TA. Thirty single nucleotide variants (SNVs) in CRC-predisposing genes/loci were genotyped in a discovery dataset composed of 440 individuals with and without isolated TA. Genome-wide significant associations were found between TA and DUSP10 rs6687758 (P=1.25 × 10−9) and ATF1 rs11169552 (P=4.36 × 10−10), with strong association found with CASC8 rs10505477 (P=8.2 × 10−5). Additional CRC marker haplotypes were also significantly associated with TA (PCASC8.Atf1 and Dusp10 expression was detected in the mouse developing teeth from early bud stages to the formation of the complete tooth, suggesting a potential role for these genes and their encoded proteins in tooth development. Our findings suggest Atf1 and Dusp10 as new tooth development genes, while having a role in colorectal cancer. While their individual contributions in tooth development remain to be elucidated, these genes may be considered additional candidates to be tested in future human genetic studies.

The focus of modern biomedical research concentrates on molecular level regulatory mechanisms and how the normal and abnormal phenotypes of tissue functional are affected by regulatory mechanisms. Most of the research on regulatory mechanism starts from the reconstruction of gene regulation network. At present, a large number of reconstruction methods construct the network using a single data set. These methods of inferring and predicting the relationship between the target gene and the transcription factor (TF) can be used to identify individual interactions between genes, while there is not much research on the interaction of many functional-related genes. In this paper, an integrated approach based on multi-data fusion is used to reconstruct the network on Alzheimer's disease (AD) which is the most common form of dementia. It not only considers the interaction between many functional-related genes and the TFs that have important implications for regulatory mechanisms, but also detects new genes associated with specific gene function expression. Protein interaction data, motif data and gene expression data of AD were integrated to gain insight into the underlying biological processes of AD. This method takes into account the TF on the target gene regulation, at the same time also considers co-expression mechanism of the TF and co-regulatory mechanism of the target gene. Eventually, not only a number of genes such as E2F4 and ATF1 related to the pathogenesis of AD have been identified, but also several significant biological processes, such as immunoregulation and neurogenesis, have been found to be associated with AD.

Stress-dependent activation of signaling cascades is often mediated by phosphorylation events, but the exact nature and role of these phosphorelays are frequently poorly understood. Here, we review which are the consequences of the stress-dependent phosphorylation of a transcription factor on gene activation. In fission yeast, the MAP kinase Sty1 is activated upon several environmental hazards and promotes cell adaptation and survival, greatly through activation of a gene program mediated by the transcription factor Atf1. Although described decades ago, the role of the phosphorylation of Atf1 by Sty1 is still a matter of debate. We present here a brief review of recent data, obtained through the characterization of several phosphorylation mutant derivatives of Atf1, demonstrating that Atf1 phosphorylation does not stabilize the factor nor stimulates its binding to DNA. Rather, it provides a structural platform of interaction with the transcriptional machinery. Based on these findings, future work will establish how this phosphorylated trans-activation domain promotes the massive gene expression shift allowing cellular adaptation to stress. This work was supported by the Ministerio de Economía y Competitividad (Spain), PLAN E and FEDER (BFU2015-68350-P to EH), and by 2014-SGR-154 from Generalitat de Catalunya (Spain) to EH. LSM is recipient of a Juan de la Cierva post-doctoral contract, and CSC is recipient of a María de Maeztu pre-doctoral fellowship, both from the Ministerio de Economía y Competitividad (Spain). EH is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain).