In this study, the citrinin (CTN)-induced accumulation of reactive oxygen species (ROS) and the regulation of the activities of antioxidant enzymes were investigated in acute toxicity tests in Schizosaccharomyces pombe. 30% of the CTN was accumulated by the cells in 1000 μM CTN solution. In comparison with the control, exposure of 10(7) cells ml(-1) to 1000 μM CTN for 60 min at pH = 4.5 induced significantly (p < 1%) elevated levels of peroxides and total ROS, but not of superoxide or hydroxyl radicals, while there was a 3-fold increase in the concentration of glutathione. ROS-induced adaptation processes at cell and molecular levels via activation of the redox-sensitive transcription factors Pap1 and (in part) Atf1 resulted in significantly increased specific activities of glutathione peroxidases, glucose-6-phosphate dehydrogenase and glutathione S-transferase and in decreased levels of catalase and glutathione reductase, but no changes were detected in the activities of superoxide dismutases. This treatment caused a G2/M cell cycle arrest and elevated the number of fragmented nuclei, which is one of the markers of apoptosis. Comparison of these results with those for the positive control, 200 μM H2O2, suggested that CTN induced a medium level of oxidative stress.

A major challenge in applying genomics to oocyte physiology is that many RNAs are present but will not be translated into proteins, making it difficult to draw conclusions from RNAseq and array data. Oocyte maturation and early embryo development rely on maternal storage of specific RNAs with a short poly(A) tail, which must be elongated for translation. To resolve the role of key genes during that period, we aimed to characterize both extremes of mRNA: deadenylated RNA and long polyA tails mRNA population in immature bovine oocytes. Using magnetic beads coupled to oligodT, we isolated deadenylated (A-, 20-50 adenosines) from polyadenylated (A+, up to 200 adenosines) RNAs. After transcriptomic analysis, we observed that A+ candidates are associated with short-term processes required for immediate cell survival (translation or protein transport) or meiotic resumption, while several A- candidates are involved in processes (chromatin modification, gene transcription and post-transcriptional modifications) that will be extremely important in the development of the early embryo. In addition to a list of candidates probably translated early or late, sequence analysis revealed that cytoplasmic polyadenylation element (CPE) and U(3)GU(3) were enriched in A- sequences. Moreover, a motif associated with polyadenylation signals (MAPS, U(5)CU(2)) appeared to be enriched in 3'untranslated regions (UTR) with CPE or U(3)GU(3) sequences in bovine but also in zebrafish and Xenopus tropicalis. To further validate our methodology, we measured specific tail length of known candidates (AURKA, PTTG1, H2A1) but also determined the poly(A) tail length of other candidate RNAs (H3F3A, H1FOO, DAZAP2, ATF1, ATF2, KAT5, DAZL, ELAVL2). In conclusion, we have reported a methodology to isolate deadenylated from polyadenylated RNAs in samples with small total RNA quantities such as mammals. Moreover, we identified deadenylated RNAs in bovine oocytes that may be stored for the long-term process of early embryo development and described a conserved motif enriched in the 3'UTR of deadenylated RNAs.

Glutaredoxins are glutathione-specific thiol oxidoreductases. The regulation and the role of grx1(+) and grx2(+) genes encoding dithiol glutaredoxins were analyzed in Schizosaccharomyces pombe. When tested in the same genetic background including mating type, the grx1 null mutant became sensitive to hydrogen peroxide, whereas grx2 mutant became highly sensitive to paraquat, a superoxide generator. The grx1grx2 double mutant showed additive phenotype of each single mutant. The grx1(+) gene expression was induced by various stresses such as oxidants, salts, and heat, and increased in the stationary phase, whereas grx2(+) stayed constitutive. The induction was mediated via Spc1 MAP kinase path involving both Atf1 and Pap1 transcription factors. Sub-cellular fractionation as well as fluorescence microscopy revealed that Grx1 resides mainly in the cytosol, whereas Grx2 is in mitochondria. These results suggest distinct roles for Grx1 and Grx2 in S. pombe in mediating glutathione-dependent redox homeostasis.

Activating transcription factor-1 (ATF1) and cAMP response element binding protein (CREB) have been implicated in cAMP-, calcium-, and virus-induced transcriptional alterations. Although CREB and ATF1 share extensive homology, they appear to mediate distinct cellular functions. We investigated the effect on DNA binding and in vitro transcription of four monoclonal antibodies (mAb) that bound to domains in either the regulatory region (mAb 1 and 3) or unique regions near the DNA-binding domains (mAb 4 and 5) of ATF1.mAb 1 and 3 supershifted both ATF1 and CREB in a DNA binding assay but did not affect in vitro transcription. mAb 4 prevented ATF1-DNA binding while supershifting CREB.DNA complexes and inhibited in vitro transcription by 95% from the CRE-containing murine proliferating cell nuclear antigen promoter. mAb 5 reacted specifically with ATF1 and did not prevent DNA binding or affect in vitro transcription. The mAb 4 epitope was located within ATF1 amino acid residues 205-219, including the first 3 basic residues in the putative DNA-binding domain. Secondary structural analysis predicted that this region comprises a transition site from alpha-helix to a turn-like conformation in ATF1. The transition to turn-like motifs is predicted to occur in CREB after 5 additional residues, with a correspondingly longer alpha-helical domain. Although regulatory domains distinct from DNA binding regions are thought to account for most of the differences in activity of members of the CREB subfamily, our results suggest that small structural variations adjacent to DNA binding regions may also contribute to the distinct functional activities of ATF1 and CREB.

The human gene for member 3 of solute carrier family 8 (SLC8A3), encoding the Na+/Ca2+ exchanger isoform 3 (NCX3), was identified on chromosome 14q24.2. The minimal promoter region was predicted 250 bp upstream of exon 1. This was confirmed by luciferase reporter assays of pGL3-promoter constructs in transfected SH-SY5Y cells. The promoter activity was monitored during the differentiation of this cell line elicited by the sequential treatment with retinoic acid and brain-derived neurotrophic factor (BDNF). The activity was induced by cyclic AMP (cAMP) via the CRE (cAMP response element) and was stimulated by retinoic acid. The increase of intracellular Ca2+ induced by the partial depolarization of the plasma membrane with KCl down-regulated both the basal and the cAMP-stimulated transcription. The down-regulation of the latter may be mediated by the phosphorylation of the CRE-binding protein by a calmodulin-dependent kinase (CaMKII). The exposure of cells to BDNF after treatment with retinoic acid rapidly induced promoter activity during the initial five hours and phosphorylation of CRE-binding protein during the first two hours. The promoter activity was further enhanced by cAMP, but became insensitive to Ca2+. In BDNF-stimulated cells cAMP elevation caused the preferential phosphorylation of ATF1 instead of that of CRE-binding protein.

The function of activating transcription factor 1 (ATF1) and the mechanism about why ATF1 was over-phosphorylated in nasopharyngeal carcinoma (NPC) progression is completely undiscovered. In this study, a series of experiments both in vitro and in vivo were used to characterize a promotive function of ATF1 in NPC tumorigenesis and identify prolyl isomerase Pin1 as a novel regulator of ATF1 at post-transcription. First, we found that overexpression of ATF1 promoted colony formation in NPC. However, the high protein level of ATF1 in NPC was not resulted from high mRNA level. Then, a direct interaction between Pin1 and ATF1 at Thr184 was demonstrated using mammalian two-hybrid assay and coimmunoprecipitation. Cycloheximide (CHX) treatment indicated Pin1 stabilized the expression of ATF1 at post-transcription level. We confirmed that Pin1 upregulated ATF1 transcriptional activity of Bcl-2 using luciferase reporter assay, quantitative RT-PCR and western blot. Furthermore, the newly identified phosphorylation of ATF1 at Thr184 was suggested to have an important role in ATF1 function of transcription and tumor promotion. Finally, high expression of Pin1 in NPC tissue was found to be positively correlated with ATF1. The ATF1 promoted NPC tumorigenesis was regulated by Pin1 both in vitro and in vivo. All these findings clearly state that Pin1 is a novel regulator of ATF1 at Thr184 and thereby enhances ATF1 transcription activity and tumorigenesis promotive function in NPC.

How do cells position the Spo11 (Rec12)-dependent initiation of meiotic recombination at hotspots? The mechanisms are poorly understood and a prevailing view is that they differ substantially between phylogenetic groups. However, recent work discovered that individual species have multiple different DNA sequence-specific, protein-DNA complexes that regulate (and are essential for the activation of) recombination hotspots. The cis-acting elements function combinatorially with documented examples of synergism, antagonism and redundancy. Furthermore, we provide evidence that all currently well-defined modules of this multifactorial, cis-acting regulation are conserved functionally between taxa whose latest common ancestor occurred more than 1 billion years ago. Functionally conserved components include the ATF/CREB-family heterodimer Atf1-Pcr1 and its CRE-like DNA site M26, the CCAAT-box-binding complex Php2-Php3-Php5 and the CCAAT-box, and the zinc-finger protein Rst2 and its Oligo-C motif. The newfound multiplicity, functional redundancy and conservation of cis-acting controls constitute a paradigm shift with broad implications. They provide compelling evidence that most meiotic recombination is, like transcription, regulated by sequence-specific protein-DNA complexes. And the new findings provide important mechanistic insight, such as a solution to the conundrum that Prdm9 is a 'master regulator' of--yet is dispensable for--hotspot activity in mammals.

The expression of two members of an important family of transcription factors, cAMP response element-binding protein (CREB) and cAMP-dependent transcription factor ATF1 (ATF1), is essential for normal preimplantation development. There is a high degree of functional similarity between these two transcription factors, and they can both homodimerize and heterodimerize with each other to form active transcription factors. CREB is present in all stages of mouse preimplantation embryo, and we show here that ATF1 is localized to the nucleus in all preimplantation stages. Activation of these transcription factors requires their phosphorylation, and this was only observed to occur for both transcription factors (serine 133 phosphorylation of CREB and serine 63 phosphorylation of ATF1) at the two-cell stage. Nuclear localization and phosphorylation of ATF1 were constitutive. The nuclear localization and phosphorylation of CREB showed a constitutive component that was further induced by the autocrine embryotropin Paf (1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Activation of CREB by Paf was independent of cAMP but was dependent on calcium, calmodulin, and calmodulin-dependent kinase activity. ATF1 nuclear localization was unaffected by inhibition of the calcium/calmodulin pathway. A complex pattern of expression of calmodulin-dependent kinases was observed throughout preimplantation development. At the two-cell stage, only mRNAs coding for calmodulin-dependent protein kinase kinase beta, calmodulin-dependent protein kinase II gamma, and calmodulin-dependent protein kinase IV were detected. A selective antagonist for calmodulin-dependent protein kinase kinase (STO-609) and calmodulin-dependent protein kinases I, II, and IV (KN-62) blocked the Paf-induced phosphorylation of CREB. The study demonstrates a role for trophic signaling and constitutive activation of two essential transcription factors at the time of zygotic genome activation.

The transcription factor cAMP responsive element-binding protein (CREB) and activating transcription factors (ATFs) are downstream components of the insulin/IGF cascade, playing crucial roles in maintaining cell viability and embryo survival. One of the CREB target genes is adiponectin, which acts synergistically with insulin. We have studied the CREB-ATF-adiponectin network in rabbit preimplantation development in vivo and in vitro. From the blastocyst stage onwards, CREB and ATF1, ATF3, and ATF4 are present with increasing expression for CREB, ATF1, and ATF3 during gastrulation and with a dominant expression in the embryoblast (EB). In vitro stimulation with insulin and IGF-I reduced CREB and ATF1 transcripts by approximately 50%, whereas CREB phosphorylation was increased. Activation of CREB was accompanied by subsequent reduction in adiponectin and adiponectin receptor (adipoR)1 expression. Under in vivo conditions of diabetes type 1, maternal adiponectin levels were up-regulated in serum and endometrium. Embryonic CREB expression was altered in a cell lineage-specific pattern. Although in EB cells CREB localization did not change, it was translocated from the nucleus into the cytosol in trophoblast (TB) cells. In TB, adiponectin expression was increased (diabetic 427.8 ± 59.3 pg/mL vs normoinsulinaemic 143.9 ± 26.5 pg/mL), whereas it was no longer measureable in the EB. Analysis of embryonic adipoRs showed an increased expression of adipoR1 and no changes in adipoR2 transcription. We conclude that the transcription factors CREB and ATFs vitally participate in embryo-maternal cross talk before implantation in a cell lineage-specific manner. Embryonic CREB/ATFs act as insulin/IGF sensors. Lack of insulin is compensated by a CREB-mediated adiponectin expression, which may maintain glucose uptake in blastocysts grown in diabetic mothers.

As the most important group in the flavor profiles of Chinese liquor, ester aroma chemicals are responsible for the highly desired fruity odors. Alcohol acetyltransferase (AATase), which is mainly encoded by ATF1, is one of the most important enzymes for acetate ester synthesis in Saccharomyces cerevisiae. In this study, we overexpressed ATF1 in Chinese liquor yeast through precise and seamless insertion of PGK1 promoter (PGK1p) via a novel fusion PCR-mediated strategy. After two-step integration, PGK1p was embedded in the 5'-terminal of ATF1 exactly without introduction of any extraneous DNA sequence. In the liquid fermentation of corn hydrolysate, both mRNA level and AATase activity of ATF1 in mutant were pronounced higher than the parental strain. Meanwhile, productivity of ethyl acetate increased from 25.04 to 78.76 mg/l. The self-cloning strain without any heterologous sequences residual in its genome would contribute to further commercialization of favorable organoleptic characteristics in Chinese liquor.

Normal gravity wort and high gravity wort with different nitrogen levels were used to examine their effects on the fermentation performance of brewer's yeast and the formation of flavor volatiles. Results showed that both the wort gravity and nitrogen level had significant impacts on the growth rate, viability, flocculation, and gene expression of brewer's yeast and the levels of flavor volatiles. The sugar (glucose, maltose, and maltotriose) consumption rates and net cell growth decreased when high gravity worts were used, while these increased with increasing nitrogen level. Moreover, high gravity resulted in lower expression levels of ATF1, BAP2, BAT1, HSP12, and TDH, whereas the higher nitrogen level caused higher expression levels for these genes. Furthermore, the lower nitrogen level resulted in increases in the levels of higher alcohols and esters at high wort gravity. All these results demonstrated that yeast physiology and flavor balance during beer brewing were significantly affected by the wort gravity and nitrogen level.

In order to meet the need of consumer preferences for natural flavor compounds, microbial synthesis method has become a very attractive alternative to the chemical production. The 2-phenylethanol (2-PE) and its ester 2-phenylethylacetate (2-PEAc) are two extremely important flavor compounds with a rose-like odor. In recent years, Escherichia coli and yeast have been metabolically engineered to produce 2-PE. However, a metabolic engineering approach for 2-PEAc production is rare. Here, we designed and expressed a 2-PEAc biosynthetic pathway in E. coli. This pathway comprised four steps: aminotransferase (ARO8) for transamination of L-phenylalanine to phenylpyruvate, 2-keto acid decarboxylase KDC for the decarboxylation of the phenylpyruvate to phenylacetaldehyde, aldehyde reductase YjgB for the reduction of phenylacetaldehyde to 2-PE, alcohol acetyltransferase ATF1 for the esterification of 2-PE to 2-PEAc. Using the engineered E. coli strain for shake flasks cultivation with 1 g/L L-phenylalanine, we achieved co-production of 268 mg/L 2-PEAc and 277 mg/L 2-PE. Our results suggest that approximately 65% of L-phenylalanine was utilized toward 2-PEAc and 2-PE biosynthesis and thus demonstrate potential industrial applicability of this microbial platform.

Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl-CoA with an alcohol by alcohol-O-acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short- and medium-chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf-S.l). Atf1-S.l exhibited broad specificity towards acyl-CoAs with chain length from C4 to C10 and was specific towards 1-pentanol. The AATase screen also revealed new acyl-CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf-C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester-based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels.

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.

The most common recurrent translocation in clear cell sarcoma t(12;22)(q13;q12) results in an EWSR1/ATF1 chimeric gene. We present a molecular analysis of tumor overgrowing right proximal tibia with bone destruction metastatic to two groin lymph nodes. Fluorescent in situ hybridization analysis performed on paraffin-embedded tissue sections of primary tumor sample indicated one rearranged locus of EWSR1 gene and one additional red signal. Reverse transcription-polymerase chain reaction analysis revealed the presence of four different EWSR1/ATF1 chimerical transcripts in the tumor sample as well as in both metastatic lymph nodes. Two previously described transcripts EWSR1exon7/ATF1exon5 and EWSR1exon8/ATF1exon4, and two novel transcripts EWSR1exon7/ATF1exon4 and EWSR1exon9/ATF1exon4 were identified. Both novel transcripts were out-of-frame fusions and, therefore, most likely had limited biological impact in oncogenesis of clear cell sarcoma. Quantitative evaluation demonstrated unequal distribution of these transcripts, with EWSR1exon8/ATF1exon4 type being overexpressed.

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 atf1 itself. 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 also controlled by another transcription factor, Pap1. Furthermore, cells expressing a phospho-mimicking Atf1 mutant display enhanced stress resistance, and although 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 cells expressing the phospho-mimicking Atf1 mutant, the presence of Sty1 was 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 by promoting transcription initiation.

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.

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.

Clear cell sarcoma-like tumor of the gastrointestinal tract (CCSLTGT) is a very rare and relatively recently characterized mesenchymal neoplasm arising within the wall of the small bowel, stomach, or large bowel, predominantly in adolescents and young adults. Only few anecdotal reports or small series have been published and a consensus on treatment has not been formulated. Complete resection remains the only curative option for localized disease, but despite optimal surgery, CCSLTGT typically shows highly aggressive behavior with a high rate of local recurrence, metastases, and death from disease. The hallmark of CCSLTGT is the presence of EWSR1-CREB1 or EWSR1-ATF1 gene fusions, detectable with reverse transcription-polymerase chain reaction (PCR). The aim of this study was to assess all referred cases of CCSLTGT, and document the pathological features, treatment and outcome of these patients.

We retrospectively reviewed all cases of histologically- and molecularly-confirmed CCSLTGT with EWSR1-CREB1 or EWSR1-ATF1 fusions at our tertiary sarcoma center, between 2009 and 2016.

We assessed six patients diagnosed with CCSLTGT. In a median follow-up of 8 months, all patients received surgery, and additionally one patient was treated with chemotherapy and had progressive disease. Five of six patients experienced recurrence or progression of disease and 4 of 6 patients died of disease.

Our study confirms that CCSLTGT is a very rare aggressive sarcoma subtype with a very poor outcome. Greater international collaboration is required to obtain a better understanding of this disease.

The bottom-fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid, containing two S. cerevisiae (Sc)-type and two S. bayanus (Sb)-type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not previously been reported. Here we describe the construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p-hygro allele conferring hygromycin B resistance replaced ScURA3 and a KanMX cassette conferring G-418 resistance replaced SbURA3. The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant (ScURA3⁺/Scura3Δ::TDH3p-hygro, SbURA3⁺/Sbura3Δ::KanMX), which was plated on 5-fluoroorotic acid (5-FOA) plates to generate the desired Ura⁻ homozygous disruptant (Scura3Δ::TDH3p-hygro/Scura3Δ::TDH3p-hygro Sbura3Δ::KanMX/Sbura3Δ::KanMX) through LOH. This ura3 deletion strain was then used to construct a bottom-fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1-overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3∆ lager strain will be a useful host for constructing strains of relevance to brewing.

Numerous noncoding RNA transcripts are detected in eukaryotic cells. Noncoding RNAs transcribed across gene promoters are involved in the regulation of mRNA transcription via chromatin modulation. This function of noncoding RNA transcription was first demonstrated for the fission yeast fbp1 gene, where a cascade of noncoding RNA transcription events induces chromatin remodeling to facilitate transcription factor binding. We recently demonstrated that the noncoding RNAs from the fbp1 upstream region facilitate binding of the transcription activator Atf1 and thereby promote histone acetylation. Histone acetylation by histone acetyl transferases (HATs) and ATP-dependent chromatin remodelers (ADCRs) are implicated in chromatin remodeling, but the interplay between HATs and ADCRs in this process has not been fully elucidated. Here, we examine the roles played by two distinct ADCRs, Snf22 and Hrp3, and by the HAT Gcn5 in the transcriptional activation of fbp1. Snf22 and Hrp3 redundantly promote disassembly of chromatin in the fbp1 upstream region. Gcn5 critically contributes to nucleosome eviction in the absence of either Snf22 or Hrp3, presumably by recruiting Hrp3 in snf22∆ cells and Snf22 in hrp3∆ cells. Conversely, Gcn5-dependent histone H3 acetylation is impaired in snf22∆/hrp3∆ cells, suggesting that both redundant ADCRs induce recruitment of Gcn5 to the chromatin array in the fbp1 upstream region. These results reveal a previously unappreciated interplay between ADCRs and histone acetylation in which histone acetylation facilitates recruitment of ADCRs, while ADCRs are required for histone acetylation.

A new case of soft tissue myoepithelial carcinoma (MEC) with rhabdoid-like differentiation is presented including cytologic, histopathologic, immunohistochemical, and molecular biologic features. A 45-year-old woman was admitted to the Hospital with nodular mass involving the lower part of the abdominal wall. Fine-needle aspiration cytology showed a round cell tumor with abundant cytoplasm in the myxoid background. The nuclei were uniform, round to ovoid, with finely distributed chromatin, nucleoli, and pale, vacuolated, or eosinophilic cytoplasm with rhabdoid-like appearance resembling a soft tissue malignant rhabdoid tumor. The surgically removed tumor was poorly demarcated, yellow, soft, and myxoid. The histopathology revealed sheets of poorly differentiated round malignant cells with focal myxoid stroma and rhabdoid-like morphology. Immunohistochemistry showed positivity for CK (AE1/AE3), EMA, S100, vimentin, CD99, and SMA; however desmin, CD34, and gliofibrilar acid protein (GFAP) were negative. Tumor cells revealed loss of INI1 expression. The EWSR1 gene rearrangement was detected by fluorescence in situ hybridization (FISH), but molecular biology failed to detect EWSR1/ETS, EWSR1/NR4A3, EWSR1/DDIT3, EWSR1/ATF1, EWSR1-POU5F1, EWSR1/ZNF444, EWSR1-PBX1 gene fusions. The final diagnosis was soft tissue malignant myoepithelioma with rhabdoid changes and EWSR1 gene rearrangement. The differential diagnosis included soft tissue malignant rhabdoid tumor, cellular extraskeletal myxoid chondrosarcoma, proximal epithelioid sarcoma, and other soft tissue tumor with EWSR1 rearrangement. To our knowledge, this is the first case of MEC with rhabdoid features and description of fine-needle aspiration cytology.