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The nucleotide sequences of alcohol acetyltransferase genes isolated from lager brewing yeast, Saccharomyces carlsbergensis have been determined. S. carlsbergensis has one ATF1 gene and another homologous gene, the Lg-ATF1 gene. There was a high degree of homology between the amino acid sequences deduced for the ATF1 protein and the Lg-ATF1 protein (75.7%), but the N-terminal region has a relatively low degree of homology. Southern analysis and contour-clamped homogeneous electric field analysis of Saccharomyces strains suggest that the ATF1 gene is located on chromosome XV in S. cerevisiae and that the Lg-ATF1 gene might originate from the "non-S. cerevisiae' genome of S. carlsbergensis, which is similar to that of S. bayanus and S. pastorianus.

Rationale Monocytes entering tissues, including advanced atherosclerotic plaques, rapidly mature and adapt to their new microenvironment. Intraplaque haemorrhage promotes human atherosclerosis progression and destabilisation via a dual metabolic challenge: cholesterol-enriched erythrocyte membranes and haem-iron. In human coronary culprit lesions, we recently described a novel macrophage subset (M-haem) with atheroprotective properties (IL-10high HO1high CD163high HLADRlow MPOlow 8keto-Guanosinelow). Methodology We dissected the mechanism of M-haem differentiation by microarray, computational biology and gene manipulation in human primary macrophages and human coronary lesions. Results Microarray analysis showed that M-haem cells were distinct from conventional M1 or M2 subsets. Using computational biology, we identified a simple transcriptional network motif with activating transcription factor 1 (ATF1) as a hub co-inducing HO1, SOCS1 and NR1H2 (LXR-α). Silencing RNA experiments showed that ATF1 induction was required for haem to upregulate HO-1 SOCS1 and NR1H2. Luciferase analysis confirmed that ATF1 transcriptionally activated both HO-1 and LXR. Luciferase deletion mutants indicated that the key ATF1-site in the HO-1 enhancer was distal (−2.2 kb to −4.9 kb). Binding experiments showed that ATF1 bound to this sequence. ATF1 overexpression in human macrophages conferred the characteristics of M-haem cells, including co-induction of HO-1, NR1H2, resistance to foam cell formation, increased survival and antioxidant protection. Conclusions Our data define the molecular basis of the differentiation of M-haem, a novel atheroprotective macrophage subset. Our data indicate that redirection of macrophage phenotype in atherosclerosis progression is a modality appropriate for therapeutic development.

Lkh1, the LAMMER kinase homolog in Schizosaccharomyces pombe, has dual-specific kinase activity. The lkh1+ deletion mutant exhibits flocculation upon reaching stationary phase in liquid media and filamentous adhesion growth on solid media [1]. Furthermore, the lkh1+ null mutant is sensitive to oxidative stresses, such as increased H2O2. This oxidant sensitivity is due to reduced levels of atf1+ mRNA, which reduces expression of the antioxidant enzymes catalase (Ctt1) and superoxide dismutase (SOD1) [2]. Recently, the Csx1 protein, which contains three RNA-recognition motifs, has been reported to bind directly to atf1+ mRNA in response to oxidative stress but not other stresses, and also to maintain normal levels of Atf1 by stabilizing atf1+ mRNA under oxidative-stress conditions [3]. Csx1 is phosphorylated by Lkh1 in response to oxidative stress, and the stress-activated binding of Csx1 to atf1+ mRNA is also affected by Lkh1 and Spc1 [4]. Previously, a parallel proteomic and metabolomic analysis of the Sty1p-dependent hydrogen peroxide stress response has been reported [5], but there has been no report focused on a proteome analysis of Lkh1-related proteins. The present report compares the cellular and extracellular proteomes of the wild type and lkh1+ null mutant to estimate possible roles of the Lkh1 kinase in cellular responses.

Background Stress-activated protein kinases regulate multiple cellular responses to a wide variety of intracellular and extracellular conditions. The conserved, multifunctional, ATF/CREB protein Atf1 (Mts1, Gad7) of fission yeast binds to CRE-like (M26) DNA sites. Atf1 is phosphorylated by the conserved, p38-family kinase Spc1 (Sty1, Phh1) and is required for many Spc1-dependent stress responses, efficient sexual differentiation, and activation of Rec12 (Spo11)-dependent meiotic recombination hotspots like ade6-M26. Methodology/Principal Findings We sought to define mechanisms by which Spc1 regulates Atf1 function at the ade6-M26 hotspot. The Spc1 kinase was essential for hotspot activity, but dispensable for basal recombination. Unexpectedly, a protein lacking all eleven MAPK phospho-acceptor sites and detectable phosphorylation (Atf1-11M) was fully proficient for hotspot recombination. Furthermore, tethering of Atf1 to ade6 in the chromosome by a heterologous DNA binding domain bypassed the requirement for Spc1 in promoting recombination. Conclusions/Significance The Spc1 protein kinase regulates the pathway of Atf1-promoted recombination at or before the point where Atf1 binds to chromosomes, and this pathway regulation is independent of the phosphorylation status of Atf1. Since basal recombination is Spc1-independent, the principal function of the Spc1 kinase in meiotic recombination is to correctly position Atf1-promoted recombination at hotspots along chromosomes. We also propose new hypotheses on regulatory mechanisms for shared (e.g., DNA binding) and distinct (e.g., osmoregulatory vs. recombinogenic) activities of multifunctional, stress-activated protein Atf1.

Recent studies have shown that global gene expression during oxidative stress in Schizosaccharomyces pombe is regulated by stress-induced activation and binding of Csx1 to atf1+ mRNA. However, the kinase responsible for the activation of Csx1 has not been identified. Here, we describe, for the first time, that Csx1 is phosphorylated by S. pombe LAMMER kinase, Lkh1, under oxidative conditions and that the stress-activated binding of the Csx1 to the atf1+ mRNA was also affected by Lkh1 and Spc1. These data indicate that concerted actions of Spc1 and Lkh1 are required for the activation of Csx1 during oxidative condition in the fission yeast S. pombe.

In Schizosaccharomyces pombe, the Wis1-Sty1 MAP (mitogen-activated protein) kinase signaling cascade is known to play a major role in cellular adaptation to adverse external stimuli, including osmotic stress, oxidative stress, nutrient deprivation, DNA-damaging agents, and heat stress. Nonetheless, it is not known whether or not this particular MAPK cascade is also involved in response to the most common stress, salinity. In this study, we provide evidence that the Wis1-Sty1 MAP cascade is implicated in salt stress response through regulating expression of a salinity-inducible gene. The downstream target gene thus identified is the cta3+ gene, which encodes a cation-transporting P-type ATPase. The salt stress-responsive nature of cta3+ expression was characterized extensively. It was found that not only the Sty1 MAP kinase but also the Atf1 transcription factor is crucial for the inducible expression of cta3+. As far as we know, this is the first instance that the stress-activated Wis1-Sty1 MAPK cascade plays a role in salt stress response in S. pombe.

The mechanism underlying stringently controlled sequence of events in the eukaryotic cell cycle involves periodic transcription of a number of genes encoding important regulators of cell cycle, growth, proliferation and apoptosis. Deregulated activities of transcription factors that contribute to this programmed gene expression, are associated with many diseases including cancer. A detailed mechanistic understanding of the transcriptional control associated with cell division is, therefore, important. We have reported earlier that the transcription factor Atf1 in Schizosaccharomyces pombe can regulate G2–M transition by directly controlling the expression of the mitotic cyclin Cdc13 (1).To gain a better understanding of the role of Atf1 in cell cycle, we performed a microarray based identification of cell cycle related targets of Atf1. The microarray data are available at NCBI's Gene Expression Omnibus (GEO) Series (accession number GSE71820). Here we report the annotation of the genes whose expression get altered by Atf1 overexpression and also provide details related to sample processing and statistical analysis of our microarray data.

Clear cell sarcoma of soft tissue (CCSST) represents a highly malignant tumor of the musculoskeletal system that is characterized by the chromosomal translocation t(12;22)(g13;q12) of the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1). In a former microarray expression study, we identified ERBB3, a member of the epidermal growth factor receptor (EGFR) family, as a promising new diagnostic marker in the differential diagnosis of CCSST. Here we show that, besides ErbB3, all CCSST cell lines (n = 8) also express the ErbB2 receptor or the ErbB4 receptor, representing an adequate coreceptor of ErbB3. The phosphorylation status of ErbB3 revealed these receptor pairs to be either constitutively activated in CCSST cells with high neuregulin-1 (NRG1) expression (n= 4) or activatable by exogenic NRG1 in cells showing low amounts of NRG1 mRNA (n = 4). Exogenous NRG1 stimulated the growth of a subset of CCSST cells but did not affect the kinetics of another subset. This difference was not strictly dependent on endogenous NRG1 expression; however, the growth-inhibiting effect of the pan-ErbB tyrosine kinase inhibitor Cl-1033 or PD158780 clearly correlated with NRG1 expression indicating an autocrine growth stimulation loop, which may constitute an interesting target of new therapeutic strategies in this tumor entity.

Adaptation to stress triggers the most dramatic shift in gene expression in fission yeast (Schizosaccharomyces pombe), and this response is driven by signaling via the MAPK Sty1. Upon activation, Sty1 accumulates in the nucleus, and stimulates expression of hundreds of genes via the nuclear transcription factor Atf1, including expression of atf1itself. However, the role of stress-induced, Sty1-mediated Atf1 phosphorylation in transcriptional activation is unclear. To this end, we expressed Atf1 phosphorylation mutants from a constitutive promoter, to uncouple Atf1 activity from endogenous, stress-activated Atf1 expression. We found that cells expressing a nonphosphorylatable Atf1 variant are sensitive to oxidative stress because of impaired transcription of a subset of stress genes whose expression is controlled also by another transcription factor, Pap1. Furthermore, cells expressing a phosphomimicking Atf1 mutant display enhanced stress resistance, and while expression of the Pap1-dependent genes still relied on stress induction, another subset of stress-responsive genes was constitutively expressed in these cells. We also observed that in the cells expressing the phosphomimicking Atf1 mutant, the presence of Sty1 is completely dispensable, with all stress defects of Sty1-deficient cells being suppressed by expression of the Atf1 mutant. We further demonstrated that Sty1-mediated Atf1 phosphorylation does not stimulate binding of Atf1 to DNA, but rather establishes a platform of interactions with the basal transcriptional machinery to facilitate transcription initiation. In summary, our results provide evidence that Atf1 phosphorylation by the MAPK Sty1 is required for oxidative stress responses in fission yeast cells and by promoting transcription initiation. 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 by the Ministerio de Economía y Competitividad (Spain). EH is recipient of an ICREA Academia Award (Generalitat de Catalunya, Spain).

The cooperation of transcriptional and post-transcriptional levels of control to shape gene regulation is only partially understood. Here we show that a combination of two simple and non-invasive genomic techniques, coupled with kinetic mathematical modeling, affords insight into the intricate dynamics of RNA regulation in response to oxidative stress in the fission yeast Schizosaccharomyces pombe. This study reveals a dominant role of transcriptional regulation in response to stress, but also points to the first minutes after stress induction as a critical time when the coordinated control of mRNA turnover can support the control of transcription for rapid gene regulation. In addition, we uncover specialized gene expression strategies associated with distinct functional gene groups, such as simultaneous transcriptional repression and mRNA destabilization for genes encoding ribosomal proteins, delayed mRNA destabilization with varying contribution of transcription for ribosome biogenesis genes, dominant roles of mRNA stabilization for genes functioning in protein degradation, and adjustment of both transcription and mRNA turnover during the adaptation to stress. We also show that genes regulated independently of the bZIP transcription factor Atf1p are predominantly controlled by mRNA turnover, and identify putative cis-regulatory sequences that are associated with different gene expression strategies during the stress response. This study highlights the intricate and multi-faceted interplay between transcription and RNA turnover during the dynamic regulatory response to stress.

Abstract Regulation of signal transduction pathways is crucial for the maintenance of cellular homeostasis and organismal development in fungi. Transcription factors are key elements of this regulatory network. The basic-region leucine zipper (bZIP) domain of the bZIP-type transcription factors is responsible for DNA binding while their leucine zipper structural motifs are suitable for dimerization with each other facilitiating the formation of homodimeric or heterodimeric bZIP proteins. This review highlights recent knowledge on the function of fungal orthologs of the Schizosaccharomyces pombe Atf1, Aspergillus nidulans AtfA, and Fusarium verticillioides FvAtfA, bZIP-type transcription factors with a special focus on pathogenic species. We demonstrate that fungal Atf1-AtfA-FvAtfA orthologs play an important role in vegetative growth, sexual and asexual development, stress response, secondary metabolite production, and virulence both in human pathogens, including Aspergillus fumigatus, Mucor circinelloides, Penicillium marneffei, and Cryptococcus neoformans and plant pathogens, like Fusarium ssp., Magnaporthe oryzae, Claviceps purpurea, Botrytis cinerea, and Verticillium dahliae. Key points • Atf1 orthologs play crucial role in the growth and development of fungi. • Atf1 orthologs orchestrate environmental stress response of fungi. • Secondary metabolite production and virulence are coordinated by Atf1 orthologs.

The CREB/ATF family of dimeric basic/leucine zipper (bZIP) transcription factors have been implicated in signal transduction by several agents including intracellular cAMP. The ubiquity of the cAMP-signaling pathway in mammalian cells has made it difficult to assay individual family members in their native form and dissect their roles in signal transduction. In light of observations that ATF1 exhibits several cell-specific characteristics and is allosterically regulated, the above problem has represented a particular limitation for studying ATF1. To address this we have exploited the modular nature of the bZIP domain to design fusion proteins that closely resemble native ATF1 but have altered DNA binding and dimerization specificity. The activity of these novel proteins can be monitored following their introduction into a cAMP-responsive cell type. Thus we describe an assay for members of the CREB family that should be generally applicable in a range of cell types and to other bZIP protein families.