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 factor1 (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 missin

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 manne

Dumpy-30 family members as determinants of male fertility and interaction partners of metal-responsive transcription factor 1 (MTF-1) in Drosophila

BACKGROUND: Metal-responsive transcription factor 1 (MTF-1), which binds to metal response elements (MREs), plays a central role in transition metal detoxification and homeostasis. A Drosophila interactome analysis revealed two candidate dMTF-1 interactors, both of which are related to the small regulatory protein Dumpy-30 (Dpy-30) of the worm C. elegans. Dpy-30 is the founding member of a protein family involved in chromatin modifications, notably histone methylation. Mutants affect mating type in yeast and male mating in C. elegans. RESULTS: Constitutive expression of the stronger interactor, Dpy-30L1 (CG6444), in transgenic flies inhibits MTF-1 activity and results in elevated sensitivity to Cd(II) and Zn(II), an effect that could be rescued by co-overexpression of dMTF-1. Electrophoretic mobility shift assays (EMSA) suggest that Dpy-30L1 interferes with the binding of MTF-1 to its cognate MRE binding site. Dpy-30L1 is expressed in the larval brain, gonads, imaginal discs, salivary glands and in the brain, testes, ovaries and salivary glands of adult flies. Expression of the second interactor, Dpy-30L2 (CG11591), is restricted to larval male gonads, and to the testes of adult males. Consistent with these findings, dpy-30-like transcripts are also prominently expressed in mouse testes. Targeted gene disruption by homologous recombination revealed that dpy-30L1 knockout flies are viable and show no overt disruption of metal homeostasis. In contrast, the knockout of the male-specific dpy-30L2 gene results in male sterility, as does the double knockout of dpy-30L1 and dpy-30L2. A closer inspection showed that Dpy-30L2 is expressed in elongated spermatids but not in early or mature sperm. Mutant sperm had impaired motility and failed to accumulate in sperm storage organs of females. CONCLUSION: Our studies help to elucidate the physiological roles of the Dumpy-30 proteins, which are conserved from yeast to humans and typically act in concert with other nuclear proteins to modify chromatin structure and gene expression. The results from these studies reveal an inhibitory effect of Dpy-30L1 on MTF-1 and an essential role for Dpy-30L2 in male fertility

Validation of 34betaE12 immunoexpression in clear cell papillary renal cell carcinoma as a sensitive biomarker

Clear cell papillary renal cell carcinoma (CCPRCC) is a recently recognised neoplasm with a broad spectrum of morphological characteristics, thus representing a challenging differential diagnosis, especially with the low malignant potential multicystic renal cell neoplasms and clear cell renal cell carcinoma. We selected 14 cases of CCPRCC with a wide spectrum of morphological features diagnosed on morphology and CK7 immunoreactivity and analysed them using a panel of immunohistochemical markers, focusing on 34 beta E12 and related CKs 1,5,10 and 14 and several molecular analyses such as fluorescence in situ hybridisation (FISH), array comparative genomic hybridisation (aCGH), VHL methylation, VHL and TCEB1 sequencing and multiplex ligation-dependent probe amplification (MLPA). Twelve of 13 (92%) CCPRCC tumours were positive for 34 beta E12. One tumour without 3p alteration by FISH revealed VHL mutation and 3p deletion at aCGH; thus, it was re-classified as clear cell RCC. We concluded that: (1) immunohistochemical expression of CK7 is necessary for diagnostic purposes, but may not be sufficient to identify CCPRCC, while 34 beta E12, in part due to the presence of CK14 antigen expression, can be extremely useful for the recognition of this tumour; and (2) further molecular analysis of chromosome 3p should be considered to support of CCPRCC diagnosis, when FISH analysis does not evidence the common loss of chromosome 3p.Peer reviewe

A clinical case of neonatal diabetes caused by INS gene mutation

Neonatal diabetes mellitus (NDM) is a severe endocrine pathology diagnosed in children during the first months of life. It comprises rare (1:300 000–1:400 000 newborns) metabolic disorders with postnatal pancreatic β-cell dysfunction, manifested by hyperglycaemia and hypoinsulinaemia. It is currently established that molecular genetic diagnosis of neonatal diabetes forms can influence treatment and prognosis. Interestingly, most identified mutations in the insulin gene are not inherited, but are sporadic. There is evidence that, in addition to heterozygous INS mutations, NDM can be caused by homozygous or compound-heterozygous mutations. The present article presents the clinical case of a girl with NDM associated with an INS gene mutation. INS gene mutations cause permanent diabetes and require children to undergo genetic examination, especially patients with type 1 diabetes in the absence of antibodies. Currently, there are no data that allow to determine a phenotypic and genotypic ‘portrait’ of NDM forms or to explain the factors determining their occurrence. Further studies of clinical cases of neonatal diabetes are therefore required to determine the characteristics of NDM subtypes with subsequent disease prognosis

Polyglutamine-mediated and heavy metal-induced transcription from yeast to humans

During my PhD thesis I was following two major projects, namely, the effect of polyglutamine tracts on the activity of a specific transcription factor, and on regulation of heavy metal homeostasis and detoxification. Project I: Many proteins, especially regulatory proteins of gene expression, contain homopolymeric repeats of single amino acids, such as glutamine, asparagine, serine, glycine, proline and alanine. Especially polyamino acid tracts that are encoded by repeats of a same codon are genetically unstable, due to polymerase slippage and out- of-register recombination, and thus subject to expansion and shrinkage. No less than 18 diseases are known to date to be caused by polyglutamine or polyalanine expansions. Since polyamino acid tracts are found from yeast to humans and even in bacteria the question arose whether they might also exert some positive effects, i.e., whether they might confer some kind of selective advantage. In our lab it had been shown before that a polyglutamine tract in a synthetic transcription factor can contribute to transcriptional activation, and it was postulated that expansion and shrinkage are useful to reversibly alter the activity of transcription factors in short- term evolution [Gerber HP et al, Science 1994, 263:808-11]. I have confirmed and extended these results by showing a clear, positive correlation between the length of a polyglutamine stretch and the transcriptional activation by the factor Gal4DBD- polyQ-VP16AD (Gal4DBD = DNA binding domain of the yeast Gal4 transcription factor, polyQ = polyglutamine stretch, VP16AD = activation domain of the viral protein VP16) in widely divergent species, namely, human cells, transgenic flies, and baker’s yeast. I also wished to test the hypothesis that homopolymeric codons are genetically less stable than a mixture of codons for a homopolymeric amino acid tract, but the number of generations was probably too small to observe differences. Project II. It is now widely recognized that a major challenge for any cell is to keep the right balance of essential trace metals and at the same time minimize the effect of non-essential ones such as cadmium, mercury, lead and silver. This is achieved by a variety of mechanisms, notably metal-specific import or export, binding to specific chaperones, storage, and detoxification by scavenging and export. Even essential metals such as copper and iron can have adverse effects if in excess, by interfering with metabolic functions via misincorporation into proteins, or by generation of reactive oxygen species due to redox cycling. Metallothioneins are small, cysteine-rich proteins which avidly bind a number of essential and non-essential heavy metals. Here I characterize metallothionein E (MtnE), the fifth and apparently ultimate member of the Drosophila metallothionein family. It is strongly expressed in the intestinal tract, notably in the so-called copper cells and in the iron cells of the midgut. I was also involved in the characterization of two related, small Drosophila proteins named Dumpy-30L1 and Dumpy-30L2. Dumpy-30L1 binds to, and thereby downregulates, the activity of MTF-1 (metal-responsive transcription factor-1). Accordingly, overexpression of Dumpy-30L1 rendered flies more sensitive to an excess of dietary copper or zinc. Targeted disruption of the gene for Dumpy-30L2 revealed a different phenotype in that male fertility was compromised. Furthermore, I was involved in a study led by D. Steiger on the characterization of Ctr1C, a copper importer which is strongly expressed in male gonads and contributes, together with the importer Ctr1B, to male fertility [Steiger et al, JBC 2010, 285(22):17089-97]. All these studies have led to a deeper understanding of various aspects of cellular metal homeostasis. Die vorliegende Arbeit beschäftigt sich mit zwei Themengebieten: Im ersten Teil wird die Auswirkung von Polyglutamin-Einheiten auf die Aktivität eines Transkriptions-faktors untersucht. Während im zweiten Teil verschiedene Aspekte der Regulation von Schwermetallhomöostase und -entgiftung behandelt werden. Projekt I: Viele Proteine, vor allem Regulatorproteine der Genexpression, enthalten Bereiche von Polyaminosäuren zum Beispiel von Glutamin, Asparagin, Serin, Glycin, Prolin und Alanin. Polyaminosäuren, welche durch Wiederholungen des selben Codons kodiert werden, sind genetisch instabil. Sich wiederholende Codons können ein Verrutschen der DNA-Polymerase oder eine fehlerhafte genetische Rekombination verursachen und führen somit zu einer Verlängerung oder einer Verkürzung der DNA-Sequenz und folglich auch der Polyaminosäure-Einheiten. Bislang sind 18 Krankheiten bekannt, welche durch eine ausufernde Verlängerung eines Polyglutamin- oder Polyalanin-Abschnitts verursacht werden. Da Polyaminosäuren von der Hefe bis zum Menschen vorkommen, stellte sich die Frage, ob Transkriptionsfaktoren, welche einen solchen Polyaminosäuren-Abschnitt enthalten, einen selektiven Vorteil haben. In unserem Labor wurde gezeigt, das ein Polyglutamin-Abschnitt in einem synthetischen Transkriptionsfaktor zur transkriptionellen Aktivität beiträgt. Es wurde vorgeschlagen, dass eine im Prinzip umkehrbare Veränderung der Polyglutaminlänge es möglich macht, die Aktivität eines Transkriptionsfaktors im Sinne einer Kurzzeit-Evolution zu modulieren [Gerber HP et al., Science 1994, 263:808-11]. In der vorliegenden Arbeit konnte ich diese Ergebnisse bestätigen und durch weitere Experimente vertiefen. In den evolutionär weit auseinander stehenden Arten, Mensch (Zellkultur), Fliege und Bäckerhefe, wird eine eindeutige positive Korrelation zwischen der Länge einer Polyglutamin-Einheit und der Aktivität des synthetischen Transkriptionsfaktors Gal4DBD-polyQ-VP16AD gezeigt (Gal4DBD: DNA-Bindedomaine des Transkriptionsfaktors Gal4 der Bäckerhefe, polyQ: Polyglutamin-Abschnitt, VP16AD: Aktivierungsdomäne des viralen Aktivatorproteins VP16). Unter anderem wollte ich auch die Hypothese testen, dass sich selbst wiederholende Codons im Vergleich zu einer Mischung von Codons, welche einen Polyaminosäure-Abschnitt kodieren, genetisch weniger stabil sind. Die Anzahl der Generationen reichte jedoch vermutlich nicht aus, um ein aussagekräftiges Ergebnis zu erhalten. Projekt II: Jede Zelle steht vor der Herausforderung die Konzentration an essentiellen Spurenelementen auf einem bestimmten Niveau zu halten und gleichzeitig eine Schädigung durch nicht-essentielle, giftige Metalle, wie Cadmium, Quecksilber, Blei und Silber, zu vermeiden. Eine Vielzahl von zellulären Mechanismen spielen hierbei eine Rolle: metall-spezifischer zellulärer Import, Bindung an spezifische intrazelluläre Bindeproteine („Chaperone“), Speicherung und Entgiftung durch Bindung und Export. In zu hohen Konzentrationen können sogar essentielle Metalle wie Kupfer und Eisen die Zelle schädigen, indem sie unspezifisch an Proteine binden und deren Funktion beeinträchtigen und/oder Sauerstoffradikale erzeugen. Metallothioneine sind kleine, cysteinreiche Proteine, welche eine Vielzahl von Metallen mit hoher Affinität binden. In dieser Arbeit charakterisiere ich MtnE, das letzte von fünf Mitgliedern der Metallothionein-Familie in Drosophila. Dieses Protein wird im Magen/Darm-Trakt stark exprimiert, und ist vor allem in den sogenannten Kupfer- und Eisenzellen des Mitteldarmes angereichert. Zudem war ich bei einer Studie beteiligt, in der zwei nah verwandte, kleine Proteine, Dumpy-30L1 und Dumpy-30L2, in Drosophila untersucht wurden. Es wurde gezeigt, dass Dumpy-30L1 an den Transkriptionsfaktor MTF-1 („metal-responsive transcription factor-1“) bindet und dadurch dessen Aktivität hemmt. Dementsprechend erhöht die Überexpression von Dumpy-30L1 in Drosophila die Sensitivität der Fliegen gegenüber Kupfer und Zink. Im Gegensatz dazu führte die Entfernung des Gens für Dumpy30L2, zu einer verminderten Fertilität männlicher Fliegen. In einer anderen Studie habe ich in Zusammenarbeit mit Dr. Dominik Steiger das Kupferimportprotein Ctr1C untersucht. Dieses Protein wird vor allem in den Keimdrüsen männlicher Fliegen exprimiert und ist neben Ctr1B, einem homologen Kupferimportprotein, für die Fertilität der Männchen notwendig [Steiger et al., JBC 2010, 285(22):17089-97]. Die Studien zu diesem Projekt haben zu einem besseren Verständnis der zellulären Metallhomöostase beigetragen

Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA)

This chapter describes a method for the rapid assessment of promoter hypermethylation levels or methylation of imprinted regions in human genomic DNA extracted from various sources using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Multiplex ligation-dependent probe amplification (MLPA) is a powerful and easy-to-perform PCR-based technique that can identify gains, amplifications, losses, deletions, methylation and mutations of up to 55 targets in a single reaction, while requiring only minute quantities of DNA (about 50 ng) extracted from blood, fresh frozen or formalin-fixed paraffin-embedded materials. Methylation-specific MLPA (MS-MLPA) is a variant of MLPA, which does not require sodium bisulfite conversion of unmethylated cytosine residues, but instead makes use of the methylation-sensitive endonuclease HhaI. MS-MLPA probes are designed to contain a HhaI recognition site (GCGC) and thus target one CpG dinucleotide within a CpG island. If the HhaI recognition site is not methylated, HhaI will cut the probe–sample DNA hybrid and no PCR product will be formed. If the target DNA is methylated, HhaI is not able to cut, and the fragment will be amplified during subsequent PCR. For data analysis, MS-MLPA peak patterns of the HhaI-treated and -untreated reactions are compared, leading to calculation of a methylation percentage. The methylation profile of a test sample is assessed by comparing the probe methylation percentages obtained on the test sample to the percentages of the reference samples. MS-MLPA can be combined with copy number and point mutation detection in the same reaction

Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA

OBJECTIVES: Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods. METHODS: Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared. RESULTS: Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance. CONCLUSIONS: FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen