The nuclear effector of Wnt-signaling, Tcf1, functions as a T-cell-specific tumor suppressor for development of lymphomas.

The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1(-/-) mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1(-/-) mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1(-/-) mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell-specific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus.

Expression of the Aspergillus terreus itaconic acid biosynthesis cluster in Aspergillus niger.

BACKGROUND: Aspergillus terreus is a natural producer of itaconic acid and is currently used to produce itaconic acid on an industrial scale. The metabolic process for itaconic acid biosynthesis is very similar to the production of citric acid in Aspergillus niger. However, a key enzyme in A. niger, cis-aconitate decarboxylase, is missing. The introduction of the A. terreus cadA gene in A. niger exploits the high level of citric acid production (over 200 g per liter) and theoretically can lead to production levels of over 135 g per liter of itaconic acid in A. niger. Given the potential for higher production levels in A. niger, production of itaconic acid in this host was investigated. RESULTS: Expression of Aspergillus terreus cis-aconitate decarboxylase in Aspergillus niger resulted in the production of a low concentration (0.05 g/L) of itaconic acid. Overexpression of codon-optimized genes for cis-aconitate decarboxylase, a mitochondrial transporter and a plasma membrane transporter in an oxaloacetate hydrolase and glucose oxidase deficient A. niger strain led to highly increased yields and itaconic acid production titers. At these higher production titers, the effect of the mitochondrial and plasma membrane transporters was much more pronounced, with levels being 5-8 times higher than previously described. CONCLUSIONS: Itaconic acid can be produced in A. niger by the introduction of the A. terreus cis-aconitate decarboxylase encoding cadA gene. This results in a low itaconic acid production level, which can be increased by codon-optimization of the cadA gene for A. niger. A second crucial requirement for efficient production of itaconic acid is the expression of the A. terreus mttA gene, encoding a putative mitochondrial transporter. Expression of this transporter results in a twenty-fold increase in the secretion of itaconic acid. Expression of the A. terreus itaconic acid cluster consisting of the cadA gene, the mttA gene and the mfsA gene results in A. niger strains that produce over twenty five-fold higher levels of itaconic acid and show a twenty-fold increase in yield compared to a strain expressing only CadA.

Global Transcriptional Response of Aspergillus niger to Blocked Active Citrate Export through Deletion of the Exporter Gene.

Aspergillus niger is the major industrial citrate producer worldwide. Export as well as uptake of citric acid are believed to occur by active, proton-dependent, symport systems. Both are major bottlenecks for industrial citrate production. Therefore, we assessed the consequences of deleting the citT gene encoding the A. niger citrate exporter, effectively blocking active citrate export. We followed the consumption of glucose and citrate as carbon sources, monitored the secretion of organic acids and carried out a thorough transcriptome pathway enrichment analysis. Under controlled cultivation conditions that normally promote citrate secretion, the knock-out strain secreted negligible amounts of citrate. Blocking active citrate export in this way led to a reduced glucose uptake and a reduced expression of high-affinity glucose transporter genes, mstG and mstH. The glyoxylate shunt was strongly activated and an increased expression of the OAH gene was observed, resulting in a more than two-fold higher concentration of oxalate in the medium. Deletion of citT did not affect citrate uptake suggesting that citrate export and citrate uptake are uncoupled from the system.

DNA microarray studies of hematopoietic subpopulations.

In recent years, one of the most quickly incorporated methods in biomedical research has been microarray technology. Microarrays have been designed for the purpose of genotyping (e.g., SNP analysis), expression analysis (mRNA, miRNA, exon arrays), chromatin immunoprecipitations (ChIP-on-chip technology), and DNA sequencing. In this chapter we will focus on the application of DNA microarrays in gene expression analysis of mRNA. This technology allows for the simultaneous analysis of transcription patterns of literally thousands of genes, making it a very powerful approach. Hematopoietic sub-populations are relatively easy to obtain by using fluorescence-activated cell sorting or magnetic bead cell separation. Over the last decade, the combination of these purification techniques with microarray analysis has resulted in an enormous gain of knowledge of blood lineages and their development.

Aspergillus niger citrate exporter revealed by comparison of two alternative citrate producing conditions.

Currently, there is no consensus regarding the mechanism underlying Aspergillus niger citrate biosynthesis and secretion. We hypothesise that depending on the experimental setup, extracellular citrate accumulation can have fundamentally different underlying transcriptomic landscapes. We show that varying the amount and type of supplement of an arginine auxotrophic A. niger strain results in transcriptional down-regulation of citrate metabolising enzymes in the condition in which more citrate is accumulated extracellularly. This contrasts with the transcriptional adaptations when increased citrate production is triggered by iron limitation. By combining gene expression data obtained from these two very distinct experimental setups with hidden Markov models and transporter homology approaches, we were able to compile a shortlist of the most likely citrate transporter candidates. Two candidates (An17g01710 and An09g06720m.01) were heterologously expressed in the yeast Saccharomyces cerevisiae, and one of the resultant mutants showed the ability to secrete citrate. Our findings provide steps in untangling the complex interplay of different mechanisms underlying A. niger citrate accumulation, and we demonstrate how a comparative transcriptomics approach complemented with further bioinformatics analyses can be used to pinpoint a fungal citrate exporter.

Aspergillus niger Secretes Citrate to Increase Iron Bioavailability.

Aspergillus niger has an innate ability to secrete various organic acids, including citrate. The conditions required for A. niger citrate overproduction are well described, but the physiological reasons underlying extracellular citrate accumulation are not yet fully understood. One of the less understood culture conditions is the requirement of growth-limiting iron concentrations. While this has been attributed to iron-dependent citrate metabolizing enzymes, this straightforward relationship does not always hold true. Here, we show that an increase in citrate secretion under iron limited conditions is a physiological response consistent with a role of citrate as A. niger iron siderophore. We found that A. niger citrate secretion increases with decreasing amounts of iron added to the culture medium and, in contrast to previous findings, this response is independent of the nitrogen source. Differential transcriptomics analyses of the two A. niger mutants NW305 (gluconate non-producer) and NW186 (gluconate and oxalate non-producer) revealed up-regulation of the citrate biosynthesis gene citA under iron limited conditions compared to iron replete conditions. In addition, we show that A. niger can utilize Fe(III) citrate as iron source. Finally, we discuss our findings in the general context of the pH-dependency of A. niger organic acid production, offering an explanation, besides competition, for why A. niger organic acid production is a sequential process influenced by the external pH of the culture medium.

Toolkit for visualization of the cellular structure and organelles in Aspergillus niger.

Aspergillus niger is a filamentous fungus that is extensively used in industrial fermentations for protein expression and the production of organic acids. Inherent biosynthetic capabilities, such as the capacity to secrete these biomolecules in high amounts, make A. niger an attractive production host. Although A. niger is renowned for this ability, the knowledge of the molecular components that underlie its production capacity, intercellular trafficking processes and secretion mechanisms is far from complete. Here, we introduce a standardized set of tools, consisting of an N-terminal GFP-actin fusion and codon optimized eforRed chromoprotein. Expression of the GFP-actin construct facilitates visualization of the actin filaments of the cytoskeleton, whereas expression of the chromoprotein construct results in a clearly distinguishable red phenotype. These experimentally validated constructs constitute the first set of standardized A. niger biomarkers, which can be used to study morphology, intercellular trafficking, and secretion phenomena.

Overexpression of a modified 6-phosphofructo-1-kinase results in an increased itaconic acid productivity in Aspergillus niger.

A modified 6-phosphofructo-1-kinase was expressed in a citrate producing Aspergillus niger strain in combination with cis-aconitate decarboxylase from Aspergillus terreus to study the effect on the production of itaconic acid. The modified pfkA gene was also expressed in combination with the itaconic acid biosynthetic cluster from A. terreus, which consists of cis-aconitate decarboxylase cadA, a putative mitochondrial transporter mttA and a putative plasmamembrane transporter mfsA. The combined expression of pfkA and cadA resulted in increased citrate levels, but did not show increased itaconic acid levels. The combined expression of pfkA with the itaconic acid biosynthetic cluster resulted in significantly increased itaconic acid production at earlier time points. Also the itaconic acid productivity increased significantly. The maximum itaconic acid productivity that was reached under these conditions was 0.15 g/L/h, which is only a factor 17 lower than the 2.5 g/L/h that according to the US Department of Energy should be achieved to have an economically feasible production process.