The mat2,3-region of Schizosaccharomyces pombe is flanked by two inverted repeat elements, IRL and IRR, which define the boundaries of the silent domain resulting from heterochromatin assembly in the region. We employed a genetic screen to isolate factors whose mutations allowed spreading of heterochromatin across boundary elements. Surprisingly, this screen revealed that mutations in the genes required for deoxyribonucleotide biosynthesis, cdc22 (encoding the large subunit of ribonucleotide reductase) and tds1 (putative thymidylate synthase), cause silencing of marker genes inserted outside of the silent domain. Chromatin-immunoprecipitation analysis showed that histone H3 lysine 9 methylation modification, an epigenetic mark associated with gene silencing, is enriched by two- to three-fold in the cdc22 mutant as compared to the level found in the wild-type strain in regions outside the silent domain. The spreading of heterochromatin across barriers required functional Atf1/Pcr1, ATF-CREB family proteins, but not the RNA-interference Dcr1, Ago1, or Rdp1 factors, previously implicated in silencing. These results implicate the deoxyribonucleotide biosynthesis pathway in limiting epigenetic controls at barrier elements at the mating-type region, but the mechanism remains unknown.

A universal response to elevated temperature and other forms of physiological stress is the induction of heat shock proteins (HSPs). Hsp16 in Schizosaccharomyces pombe encodes a polypeptide of predicted molecular weight 16 kDa that belongs to the HSP20/alpha-crystallin family whose members range in size from 12 to 43 kDa. Heat shock treatment increases expression of the hsp16 gene by 64-fold in wild-type cells and 141-fold in cdc22-M45 (ribonucleotide reductase) mutant cells. Hsp16 expression is mediated by the spc1 MAPK signaling pathway through the transcription factor atf1 and in addition through the HSF pathway. Nucleotide depletion or DNA damage as occurs in cdc22-M45 mutant cells, or during hydroxyurea or camptothecin treatment, is sufficient to activate hsp16 expression through atf1. Our findings suggest a novel role for small HSPs in the stress response following nucleotide depletion and DNA damage. This extends the types of damage that are sensed by the spc1 MAPK pathway via atf1.

We have demonstrated previously that the EGFR (epidermal growth factor receptor) is a calmodulin (CaM)-binding protein. To establish whether or not the related receptor ErbB2/Neu/HER2 also binds CaM, we used human breast adenocarcinoma SK-BR-3 cells, because these cells overexpress this receptor thus facilitating the detection of this interaction. In the present paper, we show that ErbB2 could be pulled-down using CaM-agarose beads in a Ca2+-dependent manner, as detected by Western blot analysis using an anti-ErbB2 antibody. ErbB2 was also isolated by Ca2+-dependent CaM-affinity chromatography. We also demonstrate using an overlay technique with biotinylated CaM that CaM binds directly to the immunoprecipitated ErbB2. The binding of biotinylated CaM to ErbB2 depends strictly on the presence of Ca2+, since it was prevented by the presence of EGTA. Moreover, the addition of an excess of free CaM prevents the binding of its biotinylated form, demonstrating that this was a specific process. We excluded any interference with the EGFR, as SK-BR-3 cells express considerably lower levels of this receptor, and no detectable EGFR signal was observed by Western blot analysis in the immunoprecipitated ErbB2 preparations used to perform the overlay assays with biotinylated CaM. We also demonstrate that treating living cells with W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a cell-permeant CaM antagonist, down-regulates ErbB2 phosphorylation, and show that W7 does not interfere non-specifically with the activity of ErbB tyrosine kinases. We also show that W7 inhibits the phosphorylation (activation) of both ERK1/2 (extracellular-signal-regulated kinases 1 and 2) and Akt/PKB (protein kinase B), in accordance with the inhibition observed in ErbB2 phosphorylation. In contrast, W7 treatment increased the phosphorylation (activation) of CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor-1), two Ca2+-sensitive transcription factors that operate downstream of these ErbB2 signalling pathways, most likely because of the absence of calcineurin activity. We conclude that ErbB2 is a new CaM-binding protein, and that CaM plays a role in the regulation of this receptor and its downstream signalling pathways in vivo.

The conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine by tyrosine hydroxylase (TH) is the first and rate-limiting step in biosynthesis of catecholamine neurotransmitters. TH gene expression is regulated in a cell type-specific and cAMP-dependent manner. Evidence from this laboratory and others indicates that the cAMP response element (CRE), residing at -45 to -38 bp upstream of the transcription initiation site, is essential for both basal and cAMP-inducible transcription of the TH gene. To understand the control mechanisms of TH gene transcription in greater detail, we sought to identify and characterize the transcription factors involved in recognition and activation of the CRE of the TH gene. Remarkably, electrophoretic mobility shift assay and antibody supershift experiments indicated that all three major CRE-binding protein factors, i.e. CREB, ATF1, and CREM, may participate in forming specific DNA/protein complexes with the CRE of the TH gene. To address the transcriptional activation function of individual factors, we replaced the TH CRE with a GAL4-binding site and cotransfected this modified TH promoter-reporter gene with an effector plasmid that encodes GAL4-fused transcription factor. Our results indicate that CREB but not ATF1 can support basal promoter activity while both can robustly induce the promoter activity in response to co-expression of the catalytic subunit of cAMP-dependent protein kinase (PKA). We further show that the coactivator CBP up-regulates PKA-mediated activation of the TH promoter and, if tethered to the TH promoter by a GAL4-fusion, can robustly transactivate the TH promoter even in the absence of PKA. Collectively, our results suggest that multiple CRE-binding factors interact with the CRE and regulate, in conjunction with the coactivator CBP, the transcriptional activity of the TH gene.

Fission yeast Atf1 is a member of the ATF/CREB basic leucine zipper (bZIP) family of transcription factors with strong homology to mammalian ATF2. Atf1 regulates transcription in response to stress stimuli and also plays a role in controlling heterochromatin formation and recombination. However, its DNA binding independent role is poorly studied. Here, we report that Atf1 has a distinct role in regulating the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. We have identified atf1(+) as a dose-dependent suppressor of apc5-1, a mutation causing mitotic arrest. Remarkably, the suppression is not dependent upon the bZIP domain and is therefore independent of the ability of Atf1 to bind DNA. Interestingly, Atf1 physically binds the APC/C in vivo. Furthermore, we show that addition of purified Atf1 proteins into a cell-free system stimulates ubiquitylation of cyclin B and securin by the APC/C. These results reveal a novel role for Atf1 in cell cycle control through protein-protein interaction.

Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.

The next WHO classification should abandon "salivary duct carcinoma"; conventional salivary duct carcinoma should be classified as "high-grade salivary duct carcinoma". Low-grade salivary duct carcinoma should replace the current nosology of "low-grade cribriform cystadenocarcinoma". Cystadenocarcinoma should be classified with the descriptor "Not Otherwise Specified" and should be considered an exclusionary diagnostic category. On the other hand, "Not Otherwise Specified" does not fit for hyalinizing clear cell carcinoma (HCCC). The EWSR1-ATF1 fusion is specific for HCCC within the context of salivary neoplasia. We recommend adding "hyalinizing" even though this feature is not present in all cases; the benefit of which is the mental association with a salivary clear cell malignancy. Sinonasal Renal Cell-like Adenocarcinoma (SNRCLA) is a distinct clear cell neoplasm and should be added to the next WHO classification. Future studies will bear out whether SNRCLA is even a low-grade carcinoma, or may be reclassified as "adenoma". Lastly, the next WHO monograph should include the Risk Model in the general introductory statements on oral squamous cell carcinoma, under a subheading of "Histological Prognosticators". The positive predictive value for developing locoregional recurrence in patients with low-stage oral cavity squamous carcinoma (OSCC) and "worst pattern of invasion type-5" (WPOI-5) is 42 %. Low-stage high-risk OSCC with a combination of features other than WPOI-5 is associated with 32 % likelihood for locoregional progression. WPOI-5 also predicts occult metastatic disease (p = 0.0001, Chi squared, 2 DF). Thus the Risk Model can also be used to make decisions regarding staged elective neck dissections.

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of an aggressive malignancy of CD4+ T lymphocytes. Since the viral transactivator Tax-1 is a major player in T-cell transformation, targeting Tax-1 protein is regarded as a possible strategy to arrest viral replication and to counteract neoplastic transformation. We demonstrate that CIITA, the master regulator of major histocompatibility complex class II gene transcription, inhibits HTLV-1 replication by blocking the transactivating function of Tax-1 both when exogenously transfected in 293T cells and when endogenously expressed by a subset of U937 promonocytic cells. Tax-1 and CIITA physically interact in vivo via the first 108 amino acids of Tax-1 and two CIITA adjacent regions (amino acids 1 to 252 and 253 to 410). Interestingly, only CIITA 1-252 mediated Tax-1 inhibition, in agreement with the fact that CIITA residues from positions 64 to 124 were required to block Tax-1 transactivation. CIITA inhibitory action on Tax-1 correlated with the nuclear localization of CIITA and was independent of the transcription factor NF-YB, previously involved in CIITA-mediated inhibition of Tax-2 of HTLV-2. Instead, CIITA severely impaired the physical and functional interaction of Tax-1 with the cellular coactivators p300/CBP-associated factor (PCAF), cyclic AMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1), which are required for the optimal activation of HTLV-1 promoter. Accordingly, the overexpression of PCAF, CREB, and ATF1 restored Tax-1-dependent transactivation of the viral long-terminal-repeat promoter inhibited by CIITA. These findings strongly support our original observation that CIITA, beside increasing the antigen-presenting function for pathogen antigens, acts as an endogenous restriction factor against human retroviruses by blocking virus replication and spreading.

Oxidative stress constitutes the basis of physio-pathological situations such as neurodegenerative diseases and aging. However, sublethal exposure to toxic molecules such as reactive oxygen species can induce cellular responses that result in stress fitness. Studies in Schizosaccharomyces pombe have recently showed that the Sty1 MAP kinase, known to be activated by hydrogen peroxide and other cellular stressors, plays a pivotal role in promoting fitness and longevity when it becomes activated by calorie restriction, a situation which induces oxidative metabolism and reactive oxygen species production. Activation of the MAP kinase by calorie restriction during logarithmic growth induces a transcriptional anti-stress response including genes essential to promote lifespan extension. Importantly enough, the lifespan promotion exerted by deletion of the pka1 or sck2 genes, inactivating the two main nutrient-responsive pathways, is dependent on the presence of a functional Sty1 stress pathway, since double mutants also lacking Sty1 or its main substrate Atf1 do not display extended viability. In this Research Perspective, we review these findings in relation to previous reports and extend important aspects of the original study. We propose that moderate stress levels that are not harmful for cells can make them stronger.

Fusarium graminearum is a necrotrophic plant pathogen of cereals that produces mycotoxins such as deoxynivalenol (DON) and zearalenone (ZEA) in grains. The stress-activated mitogen-activated protein kinase FgOS-2 is a central regulator in F. graminearum and controls, among others, virulence and DON and ZEA production. Here, we characterized the ATF/CREB-activating transcription factor FgAtf1, a regulator that functions downstream of FgOS-2. We created deletion and overexpression mutants of Fgatf1, the latter being also in an FgOS-2 deletion mutant. FgAtf1 localizes to the nucleus and appears to interact with FgOS-2 under osmotic stress conditions. Deletion mutants in Fgatf1 (ΔFgatf1) are more sensitive to osmotic stress and less sensitive to oxidative stress compared with the wild type. Furthermore, sexual reproduction is delayed. ΔFgatf1 strains produced higher amounts of DON under in vitro induction conditions than that of the wild type. However, during wheat infection, DON production by ΔFgatf1 is strongly reduced. The ΔFgatf1 strains displayed strongly reduced virulence to wheat and maize. Interestingly, constitutive expression of Fgatf1 in the wild type led to hypervirulence on wheat, maize, and Brachypodium distachyon. Moreover, constitutive expression of Fgatf1 in the ΔFgOS-2 mutant background almost complements ΔFgOS-2-phenotypes. These data suggest that FgAtf1 may be the most important transcription factor regulated by FgOS-2.

DNMT3L i.e. DNA (cytosine-5)-methyltransferase 3-like protein, is devoid of cytosine methyltransferase activity, despite clear homology to DNMT3A and DNMT3B, due to the mutation of key catalytic residues. However, DNMT3L participates in de novo methylation reactions through its direct interaction with DNMT3A and DNMT3B. In the present study, we investigated if DNMT3L interacts also directly with transcription factors (TFs). Using TF arrays, we identified 73 TFs that interacted with DNMT3L, 13 of which (ASH2L, ATF1, ATF3, BLZF1, CDX2, CERM, E2F3, E2F4, GCNF, GTF2I, GTF3C5, NFkB-p65 and RXRα) interacted only with DNMT3L, but not with DNMT3A/B. By focusing on the interaction with NFkB-p65, we demonstrate that DNMT3L forms a complex with DNMT3B and NFkB-p65 and that this complex is required for the control of DNA methylation at the TRAF1 promoter in the T98G glioma cell line. In addition, our experiments describe the DNA methylation at TRAF1 as being dynamic with a demethylation phase involving TET3. Thus, our data suggests that DNMT3L can address DNMT3A/B to specific sites by directly interacting with TFs that do not directly interact with DNMT3A/B. In summary, our data provide a new avenue for the direction of site-specific de novo DNA methylation catalyzed by DNMT3A/B.

We report a case of a large, deep-seated, diagnostically difficult angiomatoid fibrous histiocytoma. The neoplastic cells were positive for vimentin, calponin, CD99, and, focally, for desmin and contained intertwining cytoplasmic processes joined by desmosomelike junctions. Fusion of codon 175 of the gene to codon 110 of the gene was detected by reverse transcription-polymerase chain reaction. Because identical fusion of the and genes has been recently reported in another case of angiomatoid fibrous histiocytoma, fusion of these genes may be characteristic for at least a subset of these tumors.

The Atf1 transcription factor is critical for directing stress-induced gene expression in fission yeast [1]. Upon exposure to stress, Atf1 is hyperphosphorylated by the mitogen-activated protein kinase (MAPK) Sty1 [2, 3], which results in its stabilization [4]. The resulting increase in Atf1 is vital for a robust response to certain stresses [4]. Here we investigated the mechanism by which phosphorylation stabilizes Atf1. We show that Atf1 is a target for the ubiquitin-proteasome system and that its degradation is dependent upon an SCF E3 ligase containing the F box protein Fbh1. Turnover of Atf1 requires an intact F box, but not DNA helicase activity of Fbh1. Accordingly, disruption of Fbh1 F box function suppresses phenotypes associated with loss of Atf1 phosphorylation. Atf1 and Fbh1 interact under basal conditions, but this binding is lost upon stress. In contrast, a version of Atf1 lacking all intact MAPK sites still interacts with Fbh1 upon stress, indicating that the association between the F box protein and substrate is disrupted by stress-induced phosphorylation. Most F box protein-substrate interactions described to date are mediated positively by phosphorylation [5]. Thus, our findings represent a novel means of regulating the interaction between an F box protein and its substrate. Moreover, Atf1 is the first target described in any organism for the Fbh1 F box protein.

While studying the effect of steroids on the growth of the yeast Saccharomyces cerevisiae, we found that pregnenolone was converted into the acetate ester. This reaction was identified as a transfer of the acetyl group from acetyl-CoA to the 3beta-hydroxyl group of pregnenolone. The corresponding enzyme, acetyl-CoA:pregnenolone acetyltransferase (APAT) is specific for Delta5- or Delta4-3beta-hydroxysteroids and short-chain acyl-CoAs. The apparent Km for pregnenolone is approximately 0.5 microm. The protein associated with APAT activity was partially purified and finally isolated from an SDS/polyacrylamide gel. Tryptic peptides were generated and N-terminally sequenced. Two peptide sequences allowed the identification of an open reading frame (YGR177c, in the S. cerevisiae genome database) translating into a 62-kDa protein of hitherto unknown function. This protein encoded by a gene known as ATF2 displays 37% identity with an alcohol acetyltransferase encoded by the yeast gene ATF1. Disruption of ATF2 led to the complete elimination of APAT activity and consequently abolished the esterification of pregnenolone. In addition, a toxic effect of pregnenolone linked to the disruption of ATF2 was observed. Pregnenolone toxicity is more pronounced when the atf2-Delta mutation is introduced in a yeast strain devoid of the ATP-binding cassette transporters, PDR5 and SNQ2. Our results suggest that Atf2p (APAT) plays an active role in the detoxification of 3beta-hydroxysteroids in association with the efflux pumps Pdr5p and Snq2p.

A clear cell sarcoma, arising primarily in the ileum of a 35-year-old man, is reported. Histologically, the neoplasm infiltrated the full thickness of the intestinal wall. It consisted of strands and sheets of round to spindle-shaped cells with clear to eosinophilic cytoplasm, vesicular nuclei and prominent nucleoli. Vascular invasion was present at diagnosis. Tumour cells expressed S-100 protein, melan-A and tyrosinase. They were negative for HMB45, CD117, cytokeratins, epithelial membrane antigen, smooth muscle actin, desmin, CD31, CD34, chromogranin and synaptophysin. Reverse transcription-polymerase chain reaction analysis performed on paraffin-embedded tissue showed EWS-ATF1 fusion transcripts representative of the t(12;22) (q13;q12) clear cell sarcoma reciprocal translocation. The patient, who developed liver metastases 2 months after diagnosis, died of disease at 15 months. This case demonstrates that the gastrointestinal tract is a potential site for primary clear cell sarcoma of soft tissues, and, furthermore, that cytogenetics and/or molecular techniques play a central role in the diagnosis.

Among the several effectors that mediate TNF-alpha action is AP-1, which consists of transcription factors belonging to the JUN and FOS families. Although the effects of TNF-alpha in immune cells, such as the induction of NF-kappaBeta, are well known, the mechanisms by which it induces transcriptional activation of AP-1 in pulmonary epithelial cells are not well defined. In this study, we report that TNF-alpha stimulates the expression of the FRA-1 protooncogene in human pulmonary epithelial cells using c-Jun, acting via a 12-O-tetradecanoylphorbol-13 acetate response element located at -318. Although TNF-alpha stimulates phosphorylation of c-Jun, the inhibition of JNK activity had no significant effect on FRA-1 induction. Consistent with this result, ectopic expression of a c-Jun mutant lacking JNK phosphorylation sites had no effect on the TNF-alpha-induced expression of the promoter. In contrast, inhibition of the ERK pathway or ectopic expression of an ERK1 mutant strikingly reduced FRA-1 transcription. ERK inhibition not only blocked phosphorylation of Elk1, CREB, and ATF1, which constitutively bind to the FRA-1 promoter, but also suppressed the recruitment of c-Jun to the promoter. We found that short interfering RNA-mediated silencing of FRA-1 enhances TNF-alpha-induced IL-8 expression, whereas overexpression causes an opposite effect. Our findings collectively indicate that ERK signaling plays key roles in both Elk1, CREB, and ATF-1 activation and the subsequent recruitment of c-Jun to the FRA-1 promoter in response to TNF-alpha in pulmonary epithelial cells.

Casein kinase II (CKII) is thought to regulate a broad range of transcription factors, but its mode of action is not well characterized. We previously showed that CKII is co-purified with the ATF family of transcription factors using DNA-affinity latex beads. Here we report a functional and physical interaction between CKII and transcription factors. We demonstrate that CKII binds through its catalytic alpha and alpha' subunits to the basic leucine zipper (bZIP) DNA-binding domains of many transcription factors, including ATF1. Kinetic analysis using a surface plasmon resonance sensor suggests that CKII loosely associates with ATF1 in vivo . Deletion of the bZIP domain of ATF1 markedly reduces its phosphorylation by CKII, suggesting that the bZIP recruits CKII to the vicinity of the target site. ATF1-CKII complex is also formed on DNA. Using CKIIalpha fusedto a heterologous DNA-binding domain, we also demonstrate that CKII, when bound to DNA, efficiently phosphorylates its substrate, which is bound to the same DNA molecule. Taken together, CKII may regulate transcription (and possibly other events) by phosphorylating proteins on DNA.

The ATF1 gene encodes an alcohol acetyl transferase (AATase), that catalyses the synthesis of acetate esters from acetyl CoA and several kinds of alcohols. ATF1 transcription is negatively regulated by unsaturated fatty acids and oxygen. A series of analyses of the ATF1 promoter identified an 18 bp element essential for transcriptional activation. Ligation of the 18 bp element into a plasmid carrying the CYC1 promoter deleted UAS-activated transcription and conferred transcriptional repression by unsaturated fatty acids. The 18 bp element contains a binding sequence for Rap1p, which is a transcriptional repressor and activator. In vitro binding studies showed that Rap1p binds to the 18 bp element essential for transcriptional activation. The results of internal deletion studies of the promoter region suggested that there was also a region responsible for ATF1 oxygen regulation. This region contained the consensus binding sequence for the hypoxic repressor Rox1p. In vitro binding studies showed that Rox1p binds to the region responsible for oxygen regulation. To investigate the effect of the hypoxic repressor complex on transcription, ATF1 expression was measured in rox1, tup1 and ssn6 disruptant strains. It was found that rox1, tup1 and ssn6 disruption caused elevated expression of ATF1 under aerobic conditions. Thus, the activation of ATF1 transcription is dependent on Rap1p, and the Rox1p-Tup1p-Ssn6p hypoxic repressor complex is responsible for repression by oxygen. Furthermore, a study of ATF1 expression in a sch9 null mutant suggested that the Sch9p protein kinase is involved in ATF1 trancriptional activation.

BACKGROUND

Clear cell sarcoma (CCS) is a rare tumor with a very poor prognosis that occurs predominantly in the distal extremities of young adults. Most patients bear the t(12;22) reciprocal translocation, which involves the EWS and ATF1 genes. The diagnosis of CCS usually is easy but may be challenging in unusual sites, and the detection of EWS-ATF1 fusion transcripts is helpful to rule out a metastatic melanoma.

METHODS

Forty-four patients with CCS and 14 conventional melanomas were examined for the presence of EWS-ATF1 transcripts by using real-time polymerase chain reaction (PCR) analysis on paraffin embedded tissues, including frozen samples for 9 CCS samples and 9 melanoma samples. Prior to molecular analysis, the diagnosis of CCS was considered certain in 35 patients and as probable in 9 patients on the basis of location, histologic features, and immunohistochemical profile. Treatment modalities and follow-up were available for 41 patients with CCS.

RESULTS

EWS-ATF1 fusion transcripts were detected in 38 paraffin embedded CCS tissues (86% of all samples; 93% of interpretable samples), 3 samples (7%) were negative, and 3 samples (7%) were considered uninterpretable. Fusion transcripts were detected in 7 of 9 samples for which the diagnosis of CCS was considered probable. EWS-ATF1 transcripts were not detected in the 14 samples of melanoma. Results from frozen tissues were concordant with those from all corresponding paraffin embedded samples. Twenty-eight of 41 patients (68%) experienced lymph node and/or distant metastasis, and the 5 year-survival rate was 44%. Mitotic index and histologic grade were predictive of survival and distant metastasis.

CONCLUSIONS

The results of this study showed that the molecular detection of EWS-ATF1 fusion transcript by real-time PCR on paraffin embedded tissues is a sensitive and specific method for the diagnosis of CCS. It is an efficient tool for the diagnosis of unusual tumors, especially with regard to its distinction from melanoma. The current results also confirmed the poor prognosis for patients with this tumor type. Mitotic index and grade were predictive factors for survival and distant metastasis.

The commercial application of genetically modified industrial microorganisms has been problematic due to public concerns. We constructed a "self-cloning" sake yeast strain that overexpresses the ATF1 gene encoding alcohol acetyltransferase, to improve the flavor profile of Japanese sake. A constitutive yeast overexpression promoter, TDH3p, derived from the glyceraldehyde-3-phosphate dehydrogenase gene from sake yeast was fused to ATF1; and the 5' upstream non-coding sequence of ATF1 was further fused to TDH3p-ATF1. The fragment was placed on a binary vector, pGG119, containing a drug-resistance marker for transformation and a counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the ATF1 locus of a sake yeast strain. This integration constructed tandem repeats of ATF1 and TDH3p-ATF1 sequences, between which the plasmid was inserted. Loss of the plasmid, which occurs through homologous recombination between either the TDH3p downstream ATF1 repeats or the TDH3p upstream repeat sequences, was selected by growing transformants on counter-selective medium. Recombination between the downstream repeats led to reversion to a wild type strain, but that between the upstream repeats resulted in a strain that possessed TDH3p-ATF1 without the extraneous DNA sequences. The self-cloning TDH3p-ATF1 yeast strain produced a higher amount of isoamyl acetate. This is the first expression-controlled self-cloning industrial yeast.

Angiomatoid fibrous histiocytoma (AFH) is a rare soft tissue tumor of low malignant potential and uncertain differentiation. Only three genetically investigated cases of AFH have been reported. Two of them displayed a FUS-ATF1 fusion gene and one showed an EWSR1-ATF1 chimera. Using RT-PCR analysis, we have identified the EWSR1-ATF1 fusion transcript, and delineated the genomic breakpoints, in two new cases of AFH. Previously, the EWSR1-ATF1 fusion protein has been suggested to activate expression of the MITF-M transcript, and therefore the expression pattern of the MITF gene was studied. The MITF-M transcript was not detected in either AFH, in line with the finding that the co-activator SOX10 was not expressed. Thus, of the five AFH that have been molecularly analyzed to date, two have displayed a FUS-ATF1 fusion gene and three have shown an EWSR1-ATF1 chimera. There is no apparent correlation between the type of fusion gene and clinicopathologic features. Nonetheless, RT-PCR for these fusion transcripts remains a valuable diagnostic adjunct in the distinction between AFH and other soft tissue tumors or metastases that may simulate it.

Clear cell sarcoma is a rare and malignant soft tissue tumor that shows phenotypic and immunohistochemical overlap with cutaneous malignant melanoma; identification of biomarkers that differentiate clear cell sarcoma from malignant melanoma is therefore needed. In this study, we performed mutation analysis of BRAF and NRAS, investigated the EWSR1 gene rearrangement and evaluated the protein expression of insulin-like growth factor 2 and insulin-like growth factor 1R in 31 cases of malignant melanoma and 16 cases of clear cell sarcoma. By direct sequencing and high-resolution melting analysis, we identified BRAF and NRAS mutations in 51.6% and 12.9% of malignant melanoma cases, respectively, while none of clear cell sarcoma harbored BRAF or NRAS mutations. Fluorescence in situ hybridization showed that 78.6% of clear cell sarcoma exhibited the t(12;22)(q13;q12) translocation. The presence of type 1, 2, and 3 EWSR1/ATF1 fusion gene transcripts was confirmed by reverse transcriptase polymerase chain reaction analysis, but type 4 and EWSR1/CREB1 fusion gene transcripts were not found. No fusion transcript could be detected in any of the malignant melanoma cases. Additionally, immunohistochemistry showed that the majority of clear cell sarcoma and malignant melanoma had insulin-like growth factor 2 and insulin-like growth factor receptor 1 expression; however the expression of insulin-like growth factor 1R was significantly higher in clear cell sarcoma compared to melanoma (p = .006). Our results suggest that the combination of BRAF and NRAS mutation analysis with fusion gene detection contributes to diagnosis of malignant melanoma and clear cell sarcoma, and that insulin-like growth factor 1R might be a novel target for the treatment of these two malignancies.

Branched-chain alcohols, such as isoamyl alcohol and isobutanol, and isoamyl acetate are important flavor components of yeast-fermented alcoholic beverages. Analysis of a null mutant of the BAT2 gene encoding cytosolic branched-chain amino acid aminotransferase, and a transformant with multi-copy plasmids containing the BAT2 gene showed that the BAT2 gene product plays an important role in the production of branched-chain alcohols and isoamyl acetate. Fermentation tests using the bat2 null mutant transformed with multi-copy plasmids carrying the ATF1 gene, which encodes alcohol acetyltransferase, indicated that modified expression of BAT2 and ATF1 genes could significantly alter the proportion of branched-chain alcohols and isoamyl acetate synthesized. Furthermore, fermentation tests using different ratios of nitrogen source and RNA blot analyses demonstrated that transcription of L-leucine biosynthetic ( LEU) and BAT genes is co-regulated by nitrogen source, that production of isoamyl alcohol depends on this transcription, and that ATF transcription increased with increased concentrations of nitrogen source. Our data suggest that changes in isoamyl alcohol production by nitrogen source are due to transcriptional co-regulation of LEU and BAT genes, and that production of isoamyl acetate is dependent on isoamyl alcohol production and ATF transcription.

MicroRNAs are central players in the control of insulin secretion, but their transcriptional regulation is poorly understood. Our aim was to investigate cAMP-mediated transcriptional regulation of the miR-212/miR-132 cluster and involvement of further upstream proteins in insulin secreting β-cells. cAMP induced by forskolin+IBMX or GLP-1 caused increased expression of miR-212/miR-132, and elevated phosphorylation of cAMP-response-element-binding-protein (CREB)/Activating-transcription-factor-1 (ATF1) and Salt-Inducible-Kinases (SIKs). CyclicAMP-Regulated Transcriptional Co-activator-1 (CRTC1) was concomitantly dephosphorylated and translocated to the nucleus. Silencing of miR-212/miR-132 reduced, and overexpression of miR-212 increased, glucose-stimulated insulin secretion. Silencing of CRTC1 expression resulted in decreased insulin secretion and miR-212/miR-132 expression, while silencing or inhibition of SIKs was associated with increased expression of the microRNAs and dephosphorylation of CRTC1. CRTC1 protein levels were reduced after silencing of miR-132, suggesting feed-back regulation. Our data propose cAMP-dependent co-regulation of miR-212/miR-132, in part mediated through SIK-regulated CRTC1, as an important factor for fine-tuned regulation of insulin secretion.