Transcription enhancers as major determinants of SV40 polyomavirus growth efficiency and host cell tropism.

The non-coding control region (NCCR) of polyomaviruses includes the promoters for early and late genes, a transcription enhancer and the origin of DNA replication. Particularly virulent variants of the human pathogens BKPyV and JCPyV, as well as of simian virus 40 (SV40), occur in vitro and in vivo. These strains often harbour rearrangements in their NCCR, typically deletions of some DNA segment(s) and/or duplications of others. Using an SV40-based model system we provide evidence that duplications of enhancer elements, whether from SV40 itself or from the related BKPyV and JCPyV, increase early gene transcription and replicative capacity. SV40 harbouring subsegments of the strong cytomegalovirus (HCMV) enhancer replicated better than the common 'wild-type' SV40 in the human cell lines HEK293 and U2OS. In conclusion, replacing the SV40 enhancer with heterologous enhancers can profoundly influence SV40's infective capacity, underscoring the potential of small DNA viruses to overcome cell type and species barriers.

Head to head evaluation of the analytical performance of two commercial methotrexate immunoassays and comparison with liquid chromatography-mass spectrometry and the former fluorescence polarization immunoassay.

BACKGROUND: Monitoring of plasma drug levels is mandatory in patients receiving high-dose methotrexate. This study evaluated the analytical performance of the novel Architect and the established ARK™ methotrexate immunoassay (running on the Roche Cobas© c502 analyzer) in comparison with liquid chromatography-mass spectrometry (LC-MS) and the TDx/TDxFLx Methotrexate II assay. METHODS: Imprecision and linearity were verified for the Architect and ARK assay according to CLSI EP15-A3 and EP6-A guidelines, respectively. The reported limit of quantitation (0.04 µmol/L) was tested for both assays according to the CLSI EP17-A2 guideline. Correlation and agreement between the different assays were evaluated using residual plasma samples (n=153). RESULTS: Total imprecision was <6.3% and <9.5% for the Architect and ARK assay, respectively. The claimed linearity and limit of quantitation were confirmed for the Architect assay. For the ARK assay, imprecision at the limit of quantitation was <18% with a positive bias resulting in a high total error up to 58%, and hence the linearity could not be confirmed. Both assays showed strong correlations with the TDX assay and LC-MS but a positive bias of 12.2% and 20.5% in comparison to LC-MS for the Architect and ARK assay, respectively. For the ARK assay this bias increased dramatically for samples with concentrations towards the limit of quantitation. CONCLUSIONS: The Architect assay is suitable for monitoring plasma methotrexate, but the ARK assay showed unsatisfactory performance in the analysis of low concentrated samples. Unlike the TDX assay, both assays require manual dilution of samples at higher concentrations, which delays sample processing in clinical routine.

A recent evolutionary change affects a regulatory element in the human FOXP2 gene.

The FOXP2 gene is required for normal development of speech and language. By isolating and sequencing FOXP2 genomic DNA fragments from a 49,000-year-old Iberian Neandertal and 50 present-day humans, we have identified substitutions in the gene shared by all or nearly all present-day humans but absent or polymorphic in Neandertals. One such substitution is localized in intron 8 and affects a binding site for the transcription factor POU3F2, which is highly conserved among vertebrates. We find that the derived allele of this site is less efficient than the ancestral allele in activating transcription from a reporter construct. The derived allele also binds less POU3F2 dimers than POU3F2 monomers compared with the ancestral allele. Because the substitution in the POU3F2 binding site is likely to alter the regulation of FOXP2 expression, and because it is localized in a region of the gene associated with a previously described signal of positive selection, it is a plausible candidate for having caused a recent selective sweep in the FOXP2 gene.

The legless lizard Anguis fragilis (slow worm) has a potent metal-responsive transcription factor 1 (MTF-1).

The metal-responsive transcription factor-1 (MTF-1) is a key regulator of heavy metal homeostasis and detoxification. Here we characterize the first MTF-1 from a reptile, the slow worm Anguis fragilis. The slow worm, or blind worm, is a legless lizard also known for its long lifespan of up to several decades. Anguis MTF-1 performs well and matches the strong zinc and cadmium response of its human ortholog, clearly surpassing the activity of rodent MTF-1s. Some amino acid positions critical for metal response are the same in humans and slow worm but not in rodent MTF-1. This points to a divergent evolution of rodent MTF-1, and we speculate that rodents can afford a less sophisticated metal handling than humans and (some) reptiles.

Characterization of the promoter and the transcriptional regulation of the lipoxin A4 receptor (FPR2/ALX) gene in human monocytes and macrophages.

The lipoxin A4 receptor FPR2/ALX plays an important part in host defense and inflammation. The receptor binds structurally diverse agonistic ligands, which mainly regulate chemotaxis and activation of leukocytes. However, little is known about the promoter region of the FPR2/ALX gene and its transcriptional regulation in leukocytes. We identified two TATA-less promoter regions, separated by 224 bp, that drive the expression of FPR2/ALX in macrophages. Both promoter regions increased transcription in a reporter assay, and the basal transcription factors OCT1 and SP1 were shown to bind the first and the second promoter, respectively, and to transactivate transcription. Although monocytes expressed high levels of FPR2/ALX mRNA from the second promoter region, differentiation into macrophages abrogated FPR2/ALX expression. Stimulation of macrophages with a set of cytokines revealed that only IFN-γ and LPS increased FPR2/ALX expression from the first promoter to levels similar to those detected in monocytes. The upregulation by IFN-γ is in part mediated by the interaction of IFN regulatory factor 1 with an IFN-responsive sequence element transcription factor binding site located in the first promoter region of the FPR2/ALX gene. However, this upregulation on the mRNA level did not translate into FPR2/ALX protein expression in macrophages owing to reduced translation of the longer mRNA from the first promoter. In contrast, FPR2/ALX mRNA transcribed from the second promoter was translated into surface expression of FPR2/ALX in monocytes. These data support a model in which FPR2/ALX plays a role in chemotaxis and activation of monocytes; however, they also suggest that its function in resident tissue macrophages is limited.

The taste of heavy metals: gene regulation by MTF-1.

The metal-responsive transcription factor-1 (MTF-1, also termed MRE-binding transcription factor-1 or metal regulatory transcription factor-1) is a pluripotent transcriptional regulator involved in cellular adaptation to various stress conditions, primarily exposure to heavy metals but also to hypoxia or oxidative stress. MTF-1 is evolutionarily conserved from insects to humans and is the main activator of metallothionein genes, which encode small cysteine-rich proteins that can scavenge toxic heavy metals and free radicals. MTF-1 has been suggested to act as an intracellular metal sensor but evidence for direct metal sensing was scarce. Here we review recent advances in our understanding of MTF-1 regulation with a focus on the mechanism underlying heavy metal responsiveness and transcriptional activation mediated by mammalian or Drosophila MTF-1. This article is part of a Special Issue entitled: Cell Biology of Metals.

A conserved cysteine cluster, essential for transcriptional activity, mediates homodimerization of human metal-responsive transcription factor-1 (MTF-1).

Metal-responsive transcription factor-1 (MTF-1) is a zinc finger protein that activates transcription in response to heavy metals such as Zn(II), Cd(II) and Cu(I) and is also involved in the response to hypoxia and oxidative stress. MTF-1 recognizes a specific DNA sequence motif termed the metal response element (MRE), located in the promoter/enhancer region of its target genes. The functional domains of MTF-1 include, besides the DNA-binding and activation domains and signals for subcellular localization (NLS and NES), a cysteine cluster 632CQCQCAC638 located near the C-terminus. Here we show that this cysteine cluster mediates homodimerization of human MTF-1, and that dimer formation in vivo is important for basal and especially metal-induced transcriptional activity. Neither nuclear translocation nor DNA binding is impaired in a mutant protein in which these cysteines are replaced by alanines. Although zinc supplementation induces MTF-1 dependent transcription it does not per se enhance dimerization, implying that actual zinc sensing is mediated by another domain. By contrast copper, which on its own activates MTF-1 only weakly in the cell lines tested, stabilizes the dimer by inducing intermolecular disulfide bond formation and synergizes with zinc to boost MTF-1 dependent transcription.

Simian virus 40 strains with novel properties generated by replacing the viral enhancer with synthetic oligonucleotides.

Typical enhancers of viral or cellular genes are approximately 100 to 400 bp long and contain several transcription factor binding sites. Previously, we have shown that simian virus 40 (SV40) genomic DNA that lacks its own enhancer can be used as an "enhancer trap" since it reacquires infectivity upon incorporation of heterologous enhancers. Here, we show that SV40 infectivity can be restored with synthetic enhancers that are assembled by the host cell. We found that several oligonucleotides, cotransfected with enhancerless SV40 DNA into host cells, were incorporated into the viral genome via cellular DNA end joining. The oligonucleotides tested included metal response elements (MREs), the binding sites for the transcription factor MTF-1, which induces gene activity in response to heavy metals. These recombinant SV40 strains showed preferential growth on cells overloaded with zinc or cadmium. We also cotransfected enhancerless SV40 DNA with oligonucleotides corresponding to enhancer motifs of human and mouse cytomegalovirus (HCMV and MCMV, respectively). In contrast to SV40 wild type, the viruses with cytomegalovirus-derived patchwork enhancers strongly expressed T-antigen in human HEK293 cells, accompanied by viral DNA replication. Occasionally, we also observed the assembly of functional viral genomes by incorporation of fragments of bovine DNA, an ingredient of the fetal calf serum in the medium. These fragments contained, among other sites, binding sites for AP-1 and CREB transcription factors. Taken together, our studies show that viruses with novel properties can be generated by intracellular incorporation of synthetic enhancer DNA motifs.

Polyglutamine tracts as modulators of transcriptional activation from yeast to mammals.

Microsatellite repeats are genetically unstable and subject to expansion and shrinkage. A subset of them, triplet repeats, can occur within the coding region and specify homomeric tracts of amino acids. Polyglutamine (polyQ) tracts are enriched in eukaryotic regulatory proteins, notably transcription factors, and we had shown before that they can contribute to transcriptional activation in mammalian cells. Here we generalize this finding by also including evolutionarily divergent organisms, namely, Drosophila and baker's yeast. In all three systems, Gal4-based model transcription factors were more active if they harbored a polyQ tract, and the activity depended on the length of the tract. By contrast, a polyserine tract was inactive. PolyQs acted from either an internal or a C-terminal position, thus ruling out a merely structural 'linker' effect. Finally, a two-hybrid assay in mammalian cells showed that polyQ tracts can interact with each other, supporting the concept that a polyQ-containing transcription factor can recruit other factors with polyQ tracts or glutamine-rich activation domains. The widespread occurrence of polyQ repeats in regulatory proteins suggests a beneficial role; in addition to the contribution to transcriptional activity, their genetic instability might help a species to adapt to changing environmental conditions in a potentially reversible manner.

Characterization of MtnE, the fifth metallothionein member in Drosophila.

Metallothioneins (MTs) constitute a family of cysteine-rich, low molecular weight metal-binding proteins which occur in almost all forms of life. They bind physiological metals, such as zinc and copper, as well as nonessential, toxic heavy metals, such as cadmium, mercury, and silver. MT expression is regulated at the transcriptional level by metal-regulatory transcription factor 1 (MTF-1), which binds to the metal-response elements (MREs) in the enhancer/promoter regions of MT genes. Drosophila was thought to have four MT genes, namely, MtnA, MtnB, MtnC, and MtnD. Here we characterize a new fifth member of Drosophila MT gene family, coding for metallothionein E (MtnE). The MtnE transcription unit is located head-to-head with the one of MtnD. The intervening sequence contains four MREs which bind, with different affinities, to MTF-1. Both of the divergently transcribed MT genes are completely dependent on MTF-1, whereby MtnE is consistently more strongly transcribed. MtnE expression is induced in response to heavy metals, notably copper, mercury, and silver, and is upregulated in a genetic background where the other four MTs are missing.

Distorted copper homeostasis with decreased sensitivity to cisplatin upon chaperone Atox1 deletion in Drosophila.

Copper is an integral part of a number of proteins and thus an essential trace metal. However, free copper ions can be highly toxic and every organism has to carefully control its bioavailability. Eukaryotes contain three copper chaperones; Atx1p/Atox1 which delivers copper to ATP7 transporters located in the trans-Golgi network, Cox17 which provides copper to the mitochondrial cytochrome c oxidase, and CCS which is a copper chaperone for superoxide dismutase 1. Here we describe the knockout phenotype of the Drosophila homolog of mammalian Atox1 (ATX1 in yeast). Atox1-/- flies develop normally, though at reduced numbers, and the eclosing flies are fertile. However, the mutants are unable to develop on low-copper food. Furthermore, the intestinal copper importer Ctr1B, which is regulated by copper demand, fails to be induced upon copper starvation in Atox1-/- larvae. At the same time, intestinal metallothionein is upregulated. This phenotype, which resembles the one of the ATP7 mutant, is best explained by intestinal copper accumulation, combined with insufficient delivery to the rest of the body. In addition, compared to controls, Drosophila Atox1 mutants are relatively insensitive to the anticancer drug cisplatin, a compound which is also imported via Ctr1 copper transporters and was recently found to bind mammalian Atox1.

Using Business Intelligence Tools to Support Medical Validation of Laboratory Tests.

Modern clinical laboratories have to confirm that the procedures used for specific tests are reliable and valid. There are several sources of errors and interferences that can invalidate the results. Medical validation refers to the plausibility check of the test results. Implausible results indicate that something might went wrong with a sample retrieved from the patient, e.g., the blood sample got contaminated with another fluid, which requires re-examination. Here, we describe how an integrated R-based business intelligence (BI) tool can be developed that increase the efficiency of the medical validation at the Institute of Clinical Chemistry (ICC) of the University Hospital Zurich. A BI software environment allowed us to digitalize steps in the validation process that were manually done in Excel worksheets, e.g., importing the data, calculating percentiles, and producing graphical outputs.

A splice-supporting intronic mutation in the last bp position of a cryptic exon within intron 6 of the CYBB gene induces its incorporation into the mRNA causing chronic granulomatous disease (CGD).

Chronic granulomatous disease (CGD) is caused by a defect in both the host's defenses and its regulation of inflammation normally provided by phagocytes and other leukocytes. As in the case described here, it is not uncommon that CGD patients are diagnosed late, only after organ-damaging manifestations have already progressed. In this patient, we found that CGD arose due to a splice-supporting mutation in the last position of a cryptic exon towards the middle of intron 6 of the CYBB (gp91-phox) gene. The mutation led to the insertion of 56 bp into most of the CYBB mRNA of leukocytes causing a frame shift and a premature stop codon. The normal cryptic exon was also found to be mildly active in some tissues other than leukocytes in healthy donors, to be conserved in many primates, and to a lesser degree in other mammals. Some sequence similarity suggests that the cryptic exon may have originated from a mammalian interspersed repetitive (MIR) element. Taken together, we clarify an unusual disease-causing mutation, indicate its evolutionary background and emphasize the importance of a timely diagnosis of CGD.

Dissection of Drosophila MTF-1 reveals a domain for differential target gene activation upon copper overload vs. copper starvation.

Metal-responsive transcription factor-1 (MTF-1) is a zinc finger protein conserved from mammals to insects. It mediates protection against heavy metal load by activating the expression of metallothionein and other genes. In Drosophila, MTF-1 serves a dual function in that it not only helps to protect against heavy metal load but also induces the expression of Ctr1B, the gene for an intestinal copper importer, upon copper starvation. By dissecting Drosophila MTF-1 function, we have identified determinants for nuclear import and export, and characterized a phosphorylation site mutant (T127A) that differentially affects MTF-1 target genes. Further, by generating a series of fusion proteins with the heterologous DNA binding domain of Gal4 we identified a strong, constitutive activation domain in the central region of MTF-1 (aa 352-540). By contrast, an extended fusion protein that includes MTF-1's C-terminus (aa 352-791) is not active in standard conditions but induced by copper load. The paramount regulatory importance of the C-terminal part, that harbors a cysteine-rich "metallothionein-like" domain, was corroborated by different experiments. Transgenic flies expressing C-terminally truncated MTF-1 variants displayed high constitutive transcription of both, the genes for metallothioneins and the copper importer Ctr1B. The indiscriminate activation of these genes that are normally induced under opposite conditions of copper load and copper starvation manifested itself in a shortened lifespan, crippled wings, and female sterility.