• t(8;9)(p22;p24)/PCM1-JAK2 Activates SOCS2 and SOCS3 via STAT5.

      Ehrentraut, Stefan; Nagel, Stefan; Scherr, Michaela E; Schneider, Björn; Quentmeier, Hilmar; Geffers, Robert; Kaufmann, Maren; Meyer, Corinna; Prochorec-Sobieszek, Monika; Ketterling, Rhett P; et al. (2013)
      Fusions of the tyrosine kinase domain of JAK2 with multiple partners occur in leukemia/lymphoma where they reportedly promote JAK2-oligomerization and autonomous signalling, Affected entities are promising candidates for therapy with JAK2 signalling inhibitors. While JAK2-translocations occur in myeloid, B-cell and T-cell lymphoid neoplasms, our findings suggest their incidence among the last group is low. Here we describe the genomic, transcriptional and signalling characteristics of PCM1-JAK2 formed by t(8;9)(p22;p24) in a trio of cell lines established at indolent (MAC-1) and aggressive (MAC-2A/2B) phases of a cutaneous T-cell lymphoma (CTCL). To investigate signalling, PCM1-JAK2 was subjected to lentiviral knockdown which inhibited 7 top upregulated genes in t(8;9) cells, notably SOCS2/3. SOCS3, but not SOCS2, was also upregulated in a chronic eosinophilic leukemia bearing PCM1-JAK2, highlighting its role as a central signalling target of JAK2 translocation neoplasia. Conversely, expression of GATA3, a key T-cell developmental gene silenced in aggressive lymphoma cells, was partially restored by PCM1-JAK2 knockdown. Treatment with a selective JAK2 inhibitor (TG101348) to which MAC-1/2A/2B cells were conspicuously sensitive confirmed knockdown results and highlighted JAK2 as the active moiety. PCM1-JAK2 signalling required pSTAT5, supporting a general paradigm of STAT5 activation by JAK2 alterations in lymphoid malignancies. MAC-1/2A/2B - the first JAK2-translocation leukemia/lymphoma cell lines described - display conspicuous JAK/STAT signalling accompanied by T-cell developmental and autoimmune disease gene expression signatures, confirming their fitness as CTCL disease models. Our data support further investigation of SOCS2/3 as signalling effectors, prognostic indicators and potential therapeutic targets in cancers with JAK2 rearrangements.
    • Th17 cytokine differentiation and loss of plasticity after SOCS1 inactivation in a cutaneous T-cell lymphoma.

      Ehrentraut, Stefan; Schneider, Björn; Nagel, Stefan; Pommerenke, Claudia; Quentmeier, Hilmar; Geffers, Robert; Feist, Maren; Kaufmann, Maren; Meyer, Corinna; Kadin, Marshall E; et al. (2016-04-28)
      We propose that deregulated T-helper-cell (Th) signaling underlies evolving Th17 cytokine expression seen during progression of cutaneous T-cell lymphoma (CTCL). Accordingly, we developed a lymphoma progression model comprising cell lines established at indolent (MAC-1) and aggressive (MAC-2A) CTCL stages. We discovered activating JAK3 (V722I) mutations present at indolent disease, reinforced in aggressive disease by novel compound heterozygous SOCS1 (G78R/D105N) JAK-binding domain inactivating mutations. Though isogenic, indolent and aggressive-stage cell lines had diverged phenotypically, the latter expressing multiple Th17 related cytokines, the former a narrower profile. Importantly, indolent stage cells remained poised for Th17 cytokine expression, readily inducible by treatment with IL-2 - a cytokine which mitigates Th17 differentiation in mice. In indolent stage cells JAK3 expression was boosted by IL-2 treatment. Th17 conversion of MAC-1 cells by IL-2 was blocked by pharmacological inhibition of JAK3 or STAT5, implicating IL2RG - JAK3 - STAT5 signaling in plasticity responses. Like IL-2 treatment, SOCS1 knockdown drove indolent stage cells to mimic key aggressive stage properties, notably IL17F upregulation. Co-immunoprecipitation experiments showed that SOCS1 mutations abolished JAK3 binding, revealing a key role for SOCS1 in regulating JAK3/STAT5 signaling. Collectively, our results show how JAK/STAT pathway mutations contribute to disease progression in CTCL cells by potentiating inflammatory cytokine signaling, widening the potential therapeutic target range for this intractable entity. MAC-1/2A cells also provide a candidate human Th17 laboratory model for identifying potentally actionable CTCL markers or targets and testing their druggability in vitro.
    • Therapeutic modulation of RNA-binding protein Rbm38 facilitates re-endothelialization after arterial injury.

      Sonnenschein, Kristina; Fiedler, Jan; Pfanne, Angelika; Just, Annette; Mitzka, Saskia; Geffers, Robert Robert; Pich, Andreas; Bauersachs, Johann; Thum, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Oxford Academic, 2019-03-07)
      Aims Delayed re-endothelialization after balloon angioplasty in patients with coronary or peripheral artery disease impairs vascular healing and leads to neointimal proliferation. In the present study, we examined the effect of RNA-binding motif protein 38 (Rbm38) during re-endothelialization in a murine model of experimental vascular injury. Methods and results Left common carotid arteries of C57BL/6 mice were electrically denudated and endothelial regeneration was evaluated. Profiling of RNA-binding proteins revealed dysregulated expression of Rbm38 in the denudated and regenerated areas. We next tested the importance of Rbm38 in human umbilical vein endothelial cells (HUVECS) and analysed its effects on cellular proliferation, migration and apoptosis. Rbm38 silencing in vitro demonstrated important beneficial functional effects on migratory capacity and proliferation of endothelial cells. In vivo, local silencing of Rbm38 also improved re-endothelialization of denuded carotid arteries. Luciferase reporter assay identified miR-98 and let-7f to regulate Rbm38 and the positive proliferative properties of Rbm38 silencing in vitro and in vivo were mimicked by therapeutic overexpression of these miRNAs. Conclusion The present data identified Rbm38 as an important factor of the regulation of various endothelial cell functions. Local inhibition of Rbm38 as well as overexpression of the upstream regulators miR-98 and let-7f improved endothelial regeneration in vivo and thus may be a novel therapeutic entry point to avoid endothelial damage after balloon angioplasty.
    • Three-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding.

      Agren, Daniel; Stehr, Matthias; Berthold, Catrine L; Kapoor, Shobhna; Oehlmann, Wulf; Singh, Mahavir; Schneider, Gunter; Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden. (2008-04-04)
      L-alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to L-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 A resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16 degrees toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism.
    • Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs

      Wiemann, Stefan; Weil, Bernd; Wellenreuther, Ruth; Gassenhuber, Johannes; Glassl, Sabine; Ansorge, Wilhelm; Böcher, Michael; Blöcker, Helmut; Bauersachs, Stefan; Blum, Helmut; et al. (Cold Spring Harbor Laboratory Press, 2001-03)
    • Tracking HCV protease population diversity during transmission and susceptibility of founder populations to antiviral therapy.

      Khera, Tanvi; Todt, Daniel; Vercauteren, Koen; McClure, C Patrick; Verhoye, Lieven; Farhoudi, Ali; Bhuju, Sabin; Geffers, Robert; Baumert, Thomas F; Steinmann, Eike; et al. (2017-03)
      Due to the highly restricted species-tropism of Hepatitis C virus (HCV) a limited number of animal models exist for pre-clinical evaluation of vaccines and antiviral compounds. The human-liver chimeric mouse model allows heterologous challenge with clinically relevant strains derived from patients. However, to date, the transmission and longitudinal evolution of founder viral populations in this model have not been characterized in-depth using state-of-the-art sequencing technologies. Focusing on NS3 protease encoding region of the viral genome, mutant spectra in a donor inoculum and individual recipient mice were determined via Illumina sequencing and compared, to determine the effects of transmission on founder viral population complexity. In all transmissions, a genetic bottleneck was observed, although diverse viral populations were transmitted in each case. A low frequency cloud of mutations (<1%) was detectable in the donor inoculum and recipient mice, with single nucleotide variants (SNVs) > 1% restricted to a subset of nucleotides. The population of SNVs >1% was reduced upon transmission while the low frequency SNV cloud remained stable. Fixation of multiple identical synonymous substitutions was apparent in independent transmissions, and no evidence for reversion of T-cell epitopes was observed. In addition, susceptibility of founder populations to antiviral therapy was assessed. Animals were treated with protease inhibitor (PI) monotherapy to track resistance associated substitution (RAS) emergence. Longitudinal analyses revealed a decline in population diversity under therapy, with no detectable RAS >1% prior to therapy commencement. Despite inoculation from a common source and identical therapeutic regimens, unique RAS emergence profiles were identified in different hosts prior to and during therapeutic failure, with complex mutational signatures at protease residues 155, 156 and 168 detected. Together these analyses track viral population complexity at high-resolution in the human-liver chimeric mouse model post-transmission and under therapeutic intervention, revealing novel insights into the evolutionary processes which shape viral protease population composition at various critical stages of the viral life-cycle.
    • TRANSCompel®: a database on composite regulatory elements in eukaryotic genes

      Kel-Margoulis, Olga V.; Kel, Alexander E.; Reuter, Ingmar; Deineko, Igor V.; Wingender, Edgar (Oxford University Press, 2002-01-01)
    • Transcriptional activation of prostate specific homeobox gene NKX3-1 in subsets of T-cell lymphoblastic leukemia (T-ALL).

      Nagel, Stefan; Ehrentraut, Stefan; Tomasch, Jürgen; Lienenklaus, Stefan; Schneider, Björn; Geffers, Robert; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F; et al. (2012)
      Homeobox genes encode transcription factors impacting key developmental processes including embryogenesis, organogenesis, and cell differentiation. Reflecting their tight transcriptional control, homeobox genes are often embedded in large non-coding, cis-regulatory regions, containing tissue specific elements. In T-cell acute lymphoblastic leukemia (T-ALL) homeobox genes are frequently deregulated by chromosomal aberrations, notably translocations adding T-cell specific activatory elements. NKX3-1 is a prostate specific homeobox gene activated in T-ALL patients expressing oncogenic TAL1 or displaying immature T-cell characteristics. After investigating regulation of NKX3-1 in primary cells and cell lines, we report its ectopic expression in T-ALL cells independent of chromosomal rearrangements. Using siRNAs and expression profiling, we exploited NKX3-1 positive T-ALL cell lines as tools to investigate aberrant activatory mechanisms. Our data confirmed NKX3-1 activation by TAL1/GATA3/LMO and identified LYL1 as an alternative activator in immature T-ALL cells devoid of GATA3. Moreover, we showed that NKX3-1 is directly activated by early T-cell homeodomain factor MSX2. These activators were regulated by MLL and/or by IL7-, BMP4- and IGF2-signalling. Finally, we demonstrated homeobox gene SIX6 as a direct leukemic target of NKX3-1 in T-ALL. In conclusion, we identified three major mechanisms of NKX3-1 regulation in T-ALL cell lines which are represented by activators TAL1, LYL1 and MSX2, corresponding to particular T-ALL subtypes described in patients. These results may contribute to the understanding of leukemic transcriptional networks underlying disturbed T-cell differentiation in T-ALL.
    • The transcriptional regulator LysG (Rv1985c) of Mycobacterium tuberculosis activates lysE (Rv1986) in a lysine-dependent manner.

      Schneefeld, Marie; Busche, Tobias; Geffers, Robert; Kalinowski, Jörn; Bange, Franz-Christoph; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      The Mycobacterium tuberculosis protein encoded by the Rv1986 gene is a target for memory T cells in patients with tuberculosis, and shows strong similarities to a lysine exporter LysE of Corynebacterium glutamicum. During infection, the pathogen Mycobacterium tuberculosis adapts its metabolism to environmental changes. In this study, we found that the expression of Rv1986 is controlled by Rv1985c. Rv1985c is located directly upstream of Rv1986 with an overlapping promoter region between both genes. Semiquantitative reverse transcription PCR using an isogenic mutant of Mycobacterium tuberculosis lacking Rv1985c showed that in the presence of lysine, Rv1985c protein positively upregulated the expression of Rv1986. RNA sequencing revealed the transcription start points for both transcripts and overlapping promoters. An inverted repeat in the center of the intergenic region was identified, and binding of Rv1985c protein to the intergenic region was confirmed by electrophoretic mobility shift assays. Whole transcriptome expression analysis and RNAsequencing showed downregulated transcription of ppsBCD in the Rv1985c-mutant compared to the wild type strain. Taken together, our findings characterize the regulatory network of Rv1985c in Mycobacterium tuberculosis. Due to their similarity of an orthologous gene pair in Corynebacterium glutamicum, we suggest to rename Rv1985c to lysG(Mt), and Rv1986 to lysE(Mt).
    • Transcriptomic Analysis Reveals Selective Metabolic Adaptation of Streptococcus suis to Porcine Blood and Cerebrospinal Fluid.

      Koczula, Anna; Jarek, Michael; Visscher, Christian; Valentin-Weigand, Peter; Goethe, Ralph; Willenborg, Jörg; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02-15)
      Streptococcus suis is a zoonotic pathogen that can cause severe pathologies such as septicemia and meningitis in its natural porcine host as well as in humans. Establishment of disease requires not only virulence of the infecting strain but also an appropriate metabolic activity of the pathogen in its host environment. However, it is yet largely unknown how the streptococcal metabolism adapts to the different host niches encountered during infection. Our previous isotopologue profiling studies on S. suis grown in porcine blood and cerebrospinal fluid (CSF) revealed conserved activities of central carbon metabolism in both body fluids. On the other hand, they suggested differences in the de novo amino acid biosynthesis. This prompted us to further dissect S. suis adaptation to porcine blood and CSF by RNA deep sequencing (RNA-seq). In blood, the majority of differentially expressed genes were associated with transport of alternative carbohydrate sources and the carbohydrate metabolism (pentose phosphate pathway, glycogen metabolism). In CSF, predominantly genes involved in the biosynthesis of branched-chain and aromatic amino acids were differentially expressed. Especially, isoleucine biosynthesis seems to be of major importance for S. suis in CSF because several related biosynthetic genes were more highly expressed. In conclusion, our data revealed niche-specific metabolic gene activity which emphasizes a selective adaptation of S. suis to host environments.
    • TRANSFAC, TRRD and COMPEL: towards a federated database system on transcriptional regulation.

      Wingender, E; Kel, A E; Kel, O V; Karas, H; Heinemeyer, T; Dietze, P; Knüppel, R; Romaschenko, A G; Kolchanov, N A (1997-01-01)
    • TRANSFAC: an integrated system for gene expression regulation

      Wingender, E.; Chen, X.; Hehl, R.; Karas, H.; Liebich, I.; Matys, V.; Meinhardt, T.; Prüß, M.; Reuter, I.; Schacherer, F. (Oxford University Press, 2000-01-01)
    • TRANSFAC®: transcriptional regulation, from patterns to profiles

      Matys, V.; Fricke, E.; Geffers, Robert; Gößling, E.; Haubrock, M.; Hehl, R.; Hornischer, K.; Karas, D.; Kel, A. E.; Kel-Margoulis, O. V.; et al. (Oxford University Press, 2003-01-01)
    • TRANSPATH®: an integrated database on signal transduction and a tool for array analysis

      Krull, Mathias; Voss, Nico; Choi, Claudia; Pistor, Susanne; Potapov, Anatolij; Wingender, Edgar (Oxford University Press, 2003-01-01)
    • Worlds Apart - Transcriptome Profiles of Key Oral Microbes in the Periodontal Pocket Compared to Single Laboratory Culture Reflect Synergistic Interactions.

      Deng, Zhi-Luo; Sztajer, Helena; Jarek, Michael; Bhuju, Sabin; Wagner-Döbler, Irene; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2018-02-06)
      Periodontitis is a worldwide prevalent oral disease which results from dysbiosis of the periodontal microbiome. Some of the most active microbial players, e.g., Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum, have extensively been studied in the laboratory, but it is unclear to which extend these findings can be transferred to in vivo conditions. Here we show that the transcriptional profiles of P. gingivalis, T. denticola, and F. nucleatum in the periodontal niche are distinct from those in single laboratory culture and exhibit functional similarities. GO (gene ontology) term enrichment analysis showed up-regulation of transporters, pathogenicity related traits and hemin/heme uptake mechanisms for all three species in vivo. Differential gene expression analysis revealed that cysteine proteases, transporters and hemin/heme-binding proteins were highly up-regulated in the periodontal niche, while genes involved in DNA modification were down-regulated. The data suggest strong interactions between those three species regarding protein degradation, iron up-take, and mobility in vivo, explaining their enhanced synergistic pathogenicity. We discovered a strikingly high frequency of Single Nucleotide Polymorphisms (SNPs) in vivo. For F. nucleatum we discovered a total of 127,729 SNPs in periodontal niche transcripts, which were found in similar frequency in health and disease and covered the entire genome, suggesting continuous evolution in the host. We conclude that metabolic interactions shape gene expression in vivo. Great caution is required when inferring pathogenicity of microbes from laboratory data, and microdiversity is an important adaptive trait of natural communities.