• Rare heterozygous GDF6 variants in patients with renal anomalies.

      Martens, Helge; Hennies, Imke; Getwan, Maike; Christians, Anne; Weiss, Anna-Carina; Brand, Frank; Gjerstad, Ann Christin; Christians, Arne; Gucev, Zoran; Geffers, Robert; et al. (Springer Nature, 2020-07-31)
      Although over 50 genes are known to cause renal malformation if mutated, the underlying genetic basis, most easily identified in syndromic cases, remains unsolved in most patients. In search of novel causative genes, whole-exome sequencing in a patient with renal, i.e., crossed fused renal ectopia, and extrarenal, i.e., skeletal, eye, and ear, malformations yielded a rare heterozygous variant in the GDF6 gene encoding growth differentiation factor 6, a member of the BMP family of ligands. Previously, GDF6 variants were reported to cause pleiotropic defects including skeletal, e.g., vertebral, carpal, tarsal fusions, and ocular, e.g., microphthalmia and coloboma, phenotypes. To assess the role of GDF6 in the pathogenesis of renal malformation, we performed targeted sequencing in 193 further patients identifying rare GDF6 variants in two cases with kidney hypodysplasia and extrarenal manifestations. During development, gdf6 was expressed in the pronephric tubule of Xenopus laevis, and Gdf6 expression was observed in the ureteric tree of the murine kidney by RNA in situ hybridization. CRISPR/Cas9-derived knockout of Gdf6 attenuated migration of murine IMCD3 cells, an effect rescued by expression of wild-type but not mutant GDF6, indicating affected variant function regarding a fundamental developmental process. Knockdown of gdf6 in Xenopus laevis resulted in impaired pronephros development. Altogether, we identified rare heterozygous GDF6 variants in 1.6% of all renal anomaly patients and 5.4% of renal anomaly patients additionally manifesting skeletal, ocular, or auricular abnormalities, adding renal hypodysplasia and fusion to the phenotype spectrum of GDF6 variant carriers and suggesting an involvement of GDF6 in nephrogenesis.
    • Recruitment of the ATP-dependent chromatin remodeler dMi-2 to the transcribed region of active heat shock genes.

      Mathieu, Eve-Lyne; Finkernagel, Florian; Murawska, Magdalena; Scharfe, Maren; Jarek, Michael; Brehm, Alexander; Institute for Molecular Biology and Tumor Research, Philipps-University, Emil-Mannkopff-Strasse 2, 35037 Marburg, Germany. (2012-06)
      The ATP-dependent chromatin remodeler dMi-2 can play both positive and negative roles in gene transcription. Recently, we have shown that dMi-2 is recruited to the hsp70 gene in a heat shock-dependent manner, and is required to achieve high transcript levels. Here, we use chromatin immunoprecipitation sequencing (ChIP-Seq) to identify other chromatin regions displaying increased dMi-2 binding upon heat shock and to characterize the distribution of dMi-2 over heat shock genes. We show that dMi-2 is recruited to the body of at least seven heat shock genes. Interestingly, dMi-2 binding extends several hundred base pairs beyond the polyadenylation site into the region where transcriptional termination occurs. We find that dMi-2 does not associate with the entire nucleosome-depleted hsp70 locus 87A. Rather, dMi-2 binding is restricted to transcribed regions. Our results suggest that dMi-2 distribution over active heat shock genes are determined by transcriptional activity.
    • Recycling of Peptidyl-tRNAs by Peptidyl-tRNA Hydrolase Counteracts Azithromycin-Mediated Effects on Pseudomonas aeruginosa.

      Gödeke, Julia; Pustelny, Christian; Häussler, Susanne; Gödeke, Julia; Pustelny, Christian; Häussler, Susanne; Department of Molecular Bacteriology, Helmholtz Center for Infection Research, Braunschweig, Germany.; Department of Molecular Bacteriology, Helmholtz Center for Infection Research, Braunschweig, Germany. (2013-04)
      Acute and chronic infections caused by the opportunistic pathogen Pseudomonas aeruginosa pose a serious threat to human health worldwide, and its increasing resistance to antibiotics requires alternative treatments that are more effective than available strategies. Clinical studies have clearly demonstrated that cystic fibrosis (CF) patients with chronic P. aeruginosa infections benefit from long-term low-dose azithromycin (AZM) treatment. Immunomodulating activity, the impact of AZM on the expression of quorum-sensing-dependent virulence factors, type three secretion, and motility in P. aeruginosa seem to contribute to the therapeutic response. However, to date, the molecular mechanisms underlying these AZM effects have remained elusive. Our data indicate that the AZM-mediated phenotype is caused by a depletion of the intracellular pools of tRNAs available for protein synthesis. Overexpression of the P. aeruginosa peptidyl-tRNA hydrolase, which recycles the tRNA from peptidyl-tRNA drop-off during translation, counteracted the effects of AZM on stationary-phase cell killing, cytotoxicity, and the production of rhamnolipids and partially restored swarming motility. Intriguingly, the exchange of a rare for a frequent codon in rhlR also explicitly diminished the AZM-mediated decreased production of rhamnolipids. These results indicate that depletion of the tRNA pools by AZM seems to affect the translation of genes that use rare aminoacyl-tRNA isoacceptors to a great extent and might explain the selective activity of AZM on the P. aeruginosa proteome and possibly also on the protein expression profiles of other bacterial pathogens.
    • Regulation of Flagellum Biosynthesis in Response to Cell Envelope Stress in Serovar Typhimurium.

      Spöring, Imke; Felgner, Sebastian; Preuße, Matthias; Eckweiler, Denitsa; Rohde, M; Häussler, Susanne; Weiss, Siegfried; Erhardt, Marc (2018-05-01)
      Flagellum-driven motility of serovar Typhimurium facilitates host colonization. However, the large extracellular flagellum is also a prime target for the immune system. As consequence, expression of flagella is bistable within a population of , resulting in flagellated and nonflagellated subpopulations. This allows the bacteria to maximize fitness in hostile environments. The degenerate EAL domain protein RflP (formerly YdiV) is responsible for the bistable expression of flagella by directing the flagellar master regulatory complex FlhDC with respect to proteolytic degradation. Information concerning the environmental cues controlling expression of and thus about the bistable flagellar biosynthesis remains ambiguous. Here, we demonstrated that RflP responds to cell envelope stress and alterations of outer membrane integrity. Lipopolysaccharide (LPS) truncation mutants of Typhimurium exhibited increasing motility defects due to downregulation of flagellar gene expression. Transposon mutagenesis and genetic profiling revealed that σ (RpoE) and Rcs phosphorelay-dependent cell envelope stress response systems sense modifications of the lipopolysaccaride, low pH, and activity of the complement system. This subsequently results in activation of RflP expression and degradation of FlhDC via ClpXP. We speculate that the presence of diverse hostile environments inside the host might result in cell envelope damage and would thus trigger the repression of resource-costly and immunogenic flagellum biosynthesis via activation of the cell envelope stress response. Pathogenic bacteria such as Typhimurium sense and adapt to a multitude of changing and stressful environments during host infection. At the initial stage of gastrointestinal colonization, uses flagellum-mediated motility to reach preferred sites of infection. However, the flagellum also constitutes a prime target for the host's immune response. Accordingly, the pathogen needs to determine the spatiotemporal stage of infection and control flagellar biosynthesis in a robust manner. We found that uses signals from cell envelope stress-sensing systems to turn off production of flagella. We speculate that downregulation of flagellum synthesis after cell envelope damage in hostile environments aids survival of during late stages of infection and provides a means to escape recognition by the immune system.
    • A replication study for genome-wide gene expression levels in two layer lines elucidates differentially expressed genes of pathways involved in bone remodeling and immune responsiveness.

      Habig, Christin; Geffers, Robert; Distl, Ottmar (2014)
      The current replication study confirmed significant differences in gene expression profiles of the cerebrum among the two commercial layer lines Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB). Microarray analyses were performed for 30 LSL and another 30 LB laying hens kept in the small group housing system Eurovent German. A total of 14,103 microarray probe sets using customized Affymetrix ChiGene-1_0-st Arrays with 20,399 probe sets were differentially expressed among the two layer lines LSL and LB (FDR adjusted P-value <0.05). An at least 2-fold change in expression levels could be observed for 388 of these probe sets. In LSL, 214 of the 388 probe sets were down- and 174 were up-regulated and vice versa for the LB layer line. Among the 174 up-regulated probe sets in LSL, we identified 51 significantly enriched Gene ontology (GO) terms of the biological process category. A total of 63 enriched GO-terms could be identified for the 214 down-regulated probe sets of the layer line LSL. We identified nine genes significantly differentially expressed between the two layer lines in both microarray experiments. These genes play a crucial role in protection of neuronal cells from oxidative stress, bone mineral density and immune response among the two layer lines LSL and LB. Thus, the different regulation of these genes may significantly contribute to phenotypic trait differences among these layer lines. In conclusion, these novel findings provide a basis for further research to improve animal welfare in laying hens and these layer lines may be of general interest as an animal model.
    • Reprogramming of Small Noncoding RNA Populations in Peripheral Blood Reveals Host Biomarkers for Latent and Active Mycobacterium tuberculosis Infection.

      de Araujo, Leonardo Silva; Ribeiro-Alves, Marcelo; Leal-Calvo, Thyago; Leung, Janaína; Durán, Verónica; Samir, Mohamed; Talbot, Steven; Tallam, Aravind; Mello, Fernanda Carvalho de Queiroz; Geffers, Robert; et al. (America Society of Microbiology (ASM), 2019-12-03)
      In tuberculosis (TB), as in other infectious diseases, studies of small noncoding RNAs (sncRNA) in peripheral blood have focused on microRNAs (miRNAs) but have neglected the other major sncRNA classes in spite of their potential functions in host gene regulation. Using RNA sequencing of whole blood, we have therefore determined expression of miRNA, PIWI-interacting RNA (piRNA), small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA) in patients with TB (n = 8), latent TB infection (LTBI; n = 21), and treated LTBI (LTBItt; n = 6) and in uninfected exposed controls (ExC; n = 14). As expected, sncRNA reprogramming was greater in TB than in LTBI, with the greatest changes seen in miRNA populations. However, substantial dynamics were also evident in piRNA and snoRNA populations. One miRNA and 2 piRNAs were identified as moderately accurate (area under the curve [AUC] = 0.70 to 0.74) biomarkers for LTBI, as were 1 miRNA, 1 piRNA, and 2 snoRNAs (AUC = 0.79 to 0.91) for accomplished LTBI treatment. Logistic regression identified the combination of 4 sncRNA (let-7a-5p, miR-589-5p, miR-196b-5p, and SNORD104) as a highly sensitive (100%) classifier to discriminate TB from all non-TB groups. Notably, it reclassified 8 presumed LTBI cases as TB cases, 5 of which turned out to have features of Mycobacterium tuberculosis infection on chest radiographs. SNORD104 expression decreased during M. tuberculosis infection of primary human peripheral blood mononuclear cells (PBMC) and M2-like (P = 0.03) but not M1-like (P = 0.31) macrophages, suggesting that its downregulation in peripheral blood in TB is biologically relevant. Taken together, the results demonstrate that snoRNA and piRNA should be considered in addition to miRNA as biomarkers and pathogenesis factors in the various stages of TB.IMPORTANCE Tuberculosis is the infectious disease with the worldwide largest disease burden and there remains a great need for better diagnostic biomarkers to detect latent and active M. tuberculosis infection. RNA molecules hold great promise in this regard, as their levels of expression may differ considerably between infected and uninfected subjects. We have measured expression changes in the four major classes of small noncoding RNAs in blood samples from patients with different stages of TB infection. We found that, in addition to miRNAs (which are known to be highly regulated in blood cells from TB patients), expression of piRNA and snoRNA is greatly altered in both latent and active TB, yielding promising biomarkers. Even though the functions of many sncRNA other than miRNA are still poorly understood, our results strongly suggest that at least piRNA and snoRNA populations may represent hitherto underappreciated players in the different stages of TB infection.
    • RNASeq Based Transcriptional Profiling of Pseudomonas aeruginosa PA14 after Short- and Long-Term Anoxic Cultivation in Synthetic Cystic Fibrosis Sputum Medium.

      Tata, Muralidhar; Wolfinger, Michael T; Amman, Fabian; Roschanski, Nicole; Dötsch, Andreas; Sonnleitner, Elisabeth; Häussler, Susanne; Bläsi, Udo; Helmholtz Centre for infection research (HZI), Inhoffenstraße 7, 38124 Braunschweig, Germany. (2016)
      The opportunistic human pathogen Pseudomonas aeruginosa can thrive under microaerophilic to anaerobic conditions in the lungs of cystic fibrosis patients. RNASeq based comparative RNA profiling of the clinical isolate PA14 cultured in synthetic cystic fibrosis medium was performed after planktonic growth (OD600 = 2.0; P), 30 min after shift to anaerobiosis (A-30) and after anaerobic biofilm growth for 96h (B-96) with the aim to reveal differentially regulated functions impacting on sustained anoxic biofilm formation as well as on tolerance towards different antibiotics. Most notably, functions involved in sulfur metabolism were found to be up-regulated in B-96 cells when compared to A-30 cells. Based on the transcriptome studies a set of transposon mutants were screened, which revealed novel functions involved in anoxic biofilm growth.In addition, these studies revealed a decreased and an increased abundance of the oprD and the mexCD-oprJ operon transcripts, respectively, in B-96 cells, which may explain their increased tolerance towards meropenem and to antibiotics that are expelled by the MexCD-OprD efflux pump. The OprI protein has been implicated as a target for cationic antimicrobial peptides, such as SMAP-29. The transcriptome and subsequent Northern-blot analyses showed that the abundance of the oprI transcript encoding the OprI protein is strongly decreased in B-96 cells. However, follow up studies revealed that the susceptibility of a constructed PA14ΔoprI mutant towards SMAP-29 was indistinguishable from the parental wild-type strain, which questions OprI as a target for this antimicrobial peptide in strain PA14.
    • S/MARt DB: a database on scaffold/matrix attached regions

      Liebich, Ines; Bode, Jürgen; Frisch, Matthias; Wingender, Edgar (Oxford University Press, 2002-01-01)
    • Sepsis induces specific changes in histone modification patterns in human monocytes.

      Weiterer, Sebastian; Uhle, Florian; Lichtenstern, Christoph; Siegler, Benedikt H; Bhuju, Sabin; Jarek, Michael; Bartkuhn, Marek; Weigand, Markus A (2015)
      Sepsis is a global burden and the primary cause of death in intensive care units worldwide. The pathophysiological changes induced by the host's systemic inflammatory response to infection are not yet fully understood. During sepsis, the immune system is confronted with a variety of factors, which are integrated within the individual cells and result in changes of their basal state of responsiveness. Epigenetic mechanisms like histone modifications are known to participate in the control of immune reactions, but so far the situation during sepsis is unknown.
    • Serum Response Factor (SRF) Drives the Transcriptional Upregulation of the MDM4 Oncogene in HCC.

      Pellegrino, Rossella; Thavamani, Abhishek; Calvisi, Diego F; Budczies, Jan; Neumann, Ariane; Geffers, Robert; Kroemer, Jasmin; Greule, Damaris; Schirmacher, Peter; Nordheim, Alfred; et al. (MDPI, 2021-01-08)
      Different molecular mechanisms support the overexpression of the mouse double minute homolog 4 (MDM4), a functional p53 inhibitor, in human hepatocellular carcinoma (HCC). However, the transcription factors (TFs) leading to its transcriptional upregulation remain unknown. Following promoter and gene expression analyses, putative TFs were investigated using gene-specific siRNAs, cDNAs, luciferase reporter assays, chromatin immunoprecipitation, and XI-011 drug treatment in vitro. Additionally, MDM4 expression was investigated in SRF-VP16iHep transgenic mice. We observed a copy-number-independent upregulation of MDM4 in human HCCs. Serum response factor (SRF), ELK1 and ELK4 were identified as TFs activating MDM4 transcription. While SRF was constitutively detected in TF complexes at the MDM4 promoter, presence of ELK1 and ELK4 was cell-type dependent. Furthermore, MDM4 was upregulated in SRF-VP16-driven murine liver tumors. The pharmacological inhibitor XI-011 exhibited anti-MDM4 activity by downregulating the TFs driving MDM4 transcription, which decreased HCC cell viability and increased apoptosis. In conclusion, SRF drives transcriptional MDM4 upregulation in HCC, acting in concert with either ELK1 or ELK4. The transcriptional regulation of MDM4 may be a promising target for precision oncology of human HCC, as XI-011 treatment exerts anti-MDM4 activity independent from the MDM4 copy number and the p53 status.
    • Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

      Schulte-Schrepping, Jonas; Reusch, Nico; Paclik, Daniela; Baßler, Kevin; Schlickeiser, Stephan; Zhang, Bowen; Krämer, Benjamin; Krammer, Tobias; Brumhard, Sophia; Bonaguro, Lorenzo; et al. (Elsevier /Cell Press), 2020-08-05)
      Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
    • Simultaneous Presence of Bacteriochlorophyll and Xanthorhodopsin Genes in a Freshwater Bacterium.

      Kopejtka, Karel; Tomasch, Jürgen; Zeng, Yonghui; Selyanin, Vadim; Dachev, Marko; Piwosz, Kasia; Tichý, Martin; Bína, David; Gardian, Zdenko; Bunk, Boyke; et al. (ASM, 2020-12-22)
      Photoheterotrophic bacteria represent an important part of aquatic microbial communities. There exist two fundamentally different light-harvesting systems: bacteriochlorophyll-containing reaction centers or rhodopsins. Here, we report a photoheterotrophic Sphingomonas strain isolated from an oligotrophic lake, which contains complete sets of genes for both rhodopsin-based and bacteriochlorophyll-based phototrophy. Interestingly, the identified genes were not expressed when cultured in liquid organic media. Using reverse transcription quantitative PCR (RT-qPCR), RNA sequencing, and bacteriochlorophyll a quantification, we document that bacteriochlorophyll synthesis was repressed by high concentrations of glucose or galactose in the medium. Coactivation of photosynthesis genes together with genes for TonB-dependent transporters suggests the utilization of light energy for nutrient import. The photosynthetic units were formed by ring-shaped light-harvesting complex 1 and reaction centers with bacteriochlorophyll a and spirilloxanthin as the main light-harvesting pigments. The identified rhodopsin gene belonged to the xanthorhodopsin family, but it lacks salinixanthin antenna. In contrast to bacteriochlorophyll, the expression of xanthorhodopsin remained minimal under all experimental conditions tested. Since the gene was found in the same operon as a histidine kinase, we propose that it might serve as a light sensor. Our results document that photoheterotrophic Sphingomonas bacteria use the energy of light under carbon-limited conditions, while under carbon-replete conditions, they cover all their metabolic needs through oxidative phosphorylation.IMPORTANCE Phototrophic organisms are key components of many natural environments. There exist two main phototrophic groups: species that collect light energy using various kinds of (bacterio)chlorophylls and species that utilize rhodopsins. Here, we present a freshwater bacterium Sphingomonas sp. strain AAP5 which contains genes for both light-harvesting systems. We show that bacteriochlorophyll-based reaction centers are repressed by light and/or glucose. On the other hand, the rhodopsin gene was not expressed significantly under any of the experimental conditions. This may indicate that rhodopsin in Sphingomonas may have other functions not linked to bioenergetics.
    • Single-nucleotide polymorphism-based genetic diversity analysis of clinical Pseudomonas aeruginosa isolates.

      Muthukumarasamy, Uthayakumar; Preusse, Matthias; Kordes, Adrian; Koska, Michal; Schniederjans, Monika; Khaledi, Ariane; Häussler, Susanne; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Oxford Academic, 2020-03-20)
      Extensive use of next-generation sequencing has the potential to transform our knowledge on how genomic variation within bacterial species impacts phenotypic versatility. Since different environments have unique selection pressures, they drive divergent evolution. However, there is also parallel or convergent evolution of traits in independent bacterial isolates inhabiting similar environments. The application of tools to describe population-wide genomic diversity provides an opportunity to measure the predictability of genetic changes underlying adaptation. Here we describe patterns of sequence variations in the core genome among 99 individual Pseudomonas aeruginosa clinical isolates and identified single nucleotide polymorphisms (SNPs) that are the basis for branching of the phylogenetic tree. We also identified SNPs that were acquired independently, in separate lineages, and not through inheritance from a common ancestor. While our results demonstrate that the P. aeruginosa core genome is highly conserved and in general, not subject to adaptive evolution, instances of parallel evolution will provide an opportunity to uncover genetic changes that underlie phenotypic diversity.
    • Spatiotemporal control of FlgZ activity impacts Pseudomonas aeruginosa flagellar motility.

      Bense, Sarina; Bruchmann, Sebastian; Steffen, Anika; Stradal, Theresia E B; Häussler, Susanne; Düvel, Juliane; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (Wiley-Blackwell, 2019-03-12)
      The c-di-GMP-binding effector protein FlgZ has been demonstrated to control motility in the opportunistic pathogen Pseudomonas aeruginosa and it was suggested that c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator. To further understand how motility is downregulated in P. aeruginosa and to elucidate the general control mechanisms operating during bacterial growth, we examined the spatiotemporal activity of FlgZ. We re-annotated the P. aeruginosaflgZ open reading frame and demonstrated that FlgZ-mediated downregulation of motility is fine-tuned via three independent mechanisms. First, we found that flgZ gene is transcribed independently from flgMN in stationary growth phase to increase FlgZ protein levels in the cell. Second, FlgZ localizes to the cell pole upon c-di-GMP binding and third, we describe that FimV, a cell pole anchor protein, is involved in increasing the polar localized c-di-GMP bound FlgZ to inhibit both, swimming and swarming motility. Our results shed light on the complex dynamics and spatiotemporal control of c-di-GMP-dependent bacterial motility phenotypes and on how the polar anchor protein FimV, the motor brake FlgZ and the stator proteins function to repress flagella-driven swimming and swarming motility.
    • Strong interferon-inducing capacity of a highly virulent variant of influenza A virus strain PR8 with deletions in the NS1 gene.

      Kochs, Georg; Martínez-Sobrido, Luis; Lienenklaus, Stefan; Weiss, Siegfried; García-Sastre, Adolfo; Staeheli, Peter; Department of Virology, University of Freiburg, D-79008 Freiburg, Germany. georg.kochs@uniklinik-freiburg.de (2009-12)
      Influenza viruses lacking the interferon (IFN)-antagonistic non-structural NS1 protein are strongly attenuated. Here, we show that mutants of a highly virulent variant of A/PR/8/34 (H1N1) carrying either a complete deletion or C-terminal truncations of NS1 were far more potent inducers of IFN in infected mice than NS1 mutants derived from standard A/PR/8/34. Efficient induction of IFN correlated with successful initial virus replication in mouse lungs, indicating that the IFN response is boosted by enhanced viral activity. As the new NS1 mutants can be handled in standard biosafety laboratories, they represent convenient novel tools for studying virus-induced IFN expression in vivo.
    • Structural insights into catalysis and inhibition of O-acetylserine sulfhydrylase from Mycobacterium tuberculosis. Crystal structures of the enzyme alpha-aminoacrylate intermediate and an enzyme-inhibitor complex.

      Schnell, Robert; Oehlmann, Wulf; Singh, Mahavir; Schneider, Gunter; Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden. (2007-08-10)
      Cysteine biosynthetic genes are up-regulated in the persistent phase of Mycobacterium tuberculosis, and the corresponding enzymes are therefore of interest as potential targets for novel antibacterial agents. cysK1 is one of these genes and has been annotated as coding for an O-acetylserine sulfhydrylase. Recombinant CysK1 is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine to cysteine. The crystal structure of the enzyme was determined to 1.8A resolution. CysK1 belongs to the family of fold type II PLP enzymes and is similar in structure to other O-acetylserine sulfhydrylases. We were able to trap the alpha-aminoacrylate reaction intermediate and determine its structure by cryocrystallography. Formation of the aminoacrylate complex is accompanied by a domain rotation resulting in active site closure. The aminoacrylate moiety is bound in the active site via the covalent linkage to the PLP cofactor and by hydrogen bonds of its carboxyl group to several enzyme residues. The catalytic lysine residue is positioned such that it can protonate the Calpha-carbon atom of the aminoacrylate only from the si-face, resulting in the formation of L-cysteine. CysK1 is competitively inhibited by a four-residue peptide derived from the C-terminal of serine acetyl transferase. The crystallographic analysis reveals that the peptide binds to the enzyme active site, suggesting that CysK1 forms an bi-enzyme complex with serine acetyl transferase, in a similar manner to other bacterial and plant O-acetylserine sulfhydrylases. The structure of the enzyme-peptide complex provides a framework for the design of strong binding inhibitors.
    • Subclones in B-lymphoma cell lines: isogenic models for the study of gene regulation.

      Quentmeier, Hilmar; Pommerenke, Claudia; Ammerpohl, Ole; Geffers, Robert; Hauer, Vivien; MacLeod, Roderick Af; Nagel, Stefan; Romani, Julia; Rosati, Emanuela; Rosén, Anders; et al. (2016-08-23)
      Genetic heterogeneity though common in tumors has been rarely documented in cell lines. To examine how often B-lymphoma cell lines are comprised of subclones, we performed immunoglobulin (IG) heavy chain hypermutation analysis. Revealing that subclones are not rare in B-cell lymphoma cell lines, 6/49 IG hypermutated cell lines (12%) consisted of subclones with individual IG mutations. Subclones were also identified in 2/284 leukemia/lymphoma cell lines exhibiting bimodal CD marker expression. We successfully isolated 10 subclones from four cell lines (HG3, SU-DHL-5, TMD-8, U-2932). Whole exome sequencing was performed to molecularly characterize these subclones. We describe in detail the clonal structure of cell line HG3, derived from chronic lymphocytic leukemia. HG3 consists of three subclones each bearing clone-specific aberrations, gene expression and DNA methylation patterns. While donor patient leukemic cells were CD5+, two of three HG3 subclones had independently lost this marker. CD5 on HG3 cells was regulated by epigenetic/transcriptional mechanisms rather than by alternative splicing as reported hitherto. In conclusion, we show that the presence of subclones in cell lines carrying individual mutations and characterized by sets of differentially expressed genes is not uncommon. We show also that these subclones can be useful isogenic models for regulatory and functional studies.
    • SUMOylation of the polycomb group protein L3MBTL2 facilitates repression of its target genes.

      Stielow, Christina; Stielow, Bastian; Finkernagel, Florian; Scharfe, Maren; Jarek, Michael; Suske, Guntram (2013-12-24)
      Lethal(3) malignant brain tumour like 2 (L3MBTL2) is an integral component of the polycomb repressive complex 1.6 (PRC1.6) and has been implicated in transcriptional repression and chromatin compaction. Here, we show that L3MBTL2 is modified by SUMO2/3 at lysine residues 675 and 700 close to the C-terminus. SUMOylation of L3MBTL2 neither affected its repressive activity in reporter gene assays nor it's binding to histone tails in vitro. In order to analyse whether SUMOylation affects binding of L3MBTL2 to chromatin, we performed ChIP-Seq analysis with chromatin of wild-type HEK293 cells and with chromatin of HEK293 cells stably expressing either FLAG-tagged SUMOylation-competent or SUMOylation-defective L3MBTL2. Wild-type FLAG-L3MBTL2 and the SUMOylation-defective FLAG-L3MBTL2 K675/700R mutant essentially occupied the same sites as endogenous L3MBTL2 suggesting that SUMOylation of L3MBTL2 does not affect chromatin binding. However, a subset of L3MBTL2-target genes, particularly those with low L3MBTL2 occupancy including pro-inflammatory genes, was de-repressed in cells expressing the FLAG-L3MBTL2 K675/700R mutant. Finally, we provide evidence that SUMOylation of L3MBTL2 facilitates repression of these PRC1.6-target genes by balancing the local H2Aub1 levels established by the ubiquitinating enzyme RING2 and the de-ubiquitinating PR-DUB complex.
    • Susceptibility to experimental biliary atresia linked to different hepatic gene expression profiles in two mouse strains.

      Leonhardt, Johannes; Kuebler, Joachim F; Turowski, Carmen; Tschernig, Thomas; Geffers, Robert; Petersen, Claus; Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany. (2010-02)
      Aim: To compare hepatic gene expression during the development of experimental biliary atresia (BA) in two different mouse strains. Methods: Balb/c mice and C57Black/6 (Black/6) mice were infected with rhesus rotavirus (RRV) postpartum, clinical signs of BA and survival were noted. Liver sections were assessed for cluster of differentiation antigen (CD) 3, CD4 and CD8 expression, and the hepatic virus load was determined. Second, mice of both strains were sacrificed three days after infection. Isolated hepatic RNA was subjected to gene expression analysis using Affymetrix Gene Chip MOE 430 2.0. Results: The incidence of BA was significantly lower in Black/6 mice compared to Balb/c mice (13.5% vs. 67%, P < 0.05). The mean virus titers were higher in mice with BA compared to mice without BA. Different gene profiles three days after virus infection were noted, with differential expression of 201 genes, including those regulating apoptosis, nucleic acid binding, transport function and particularly the immune response (chemokine C-C motif ligand 2, toll-like receptor 3, CD antigen 14, chemokine (C-X-C motif) ligands 10 and 11). This correlated with a significant increase of CD4 positive cells only in Balb/c mice with BA compared to healthy mice (13.5 vs. 5.0; P < 0.05). Black/6 mice did not exhibit any significant increase of CD3 or CD4 leukocytes despite cholestasis. Conclusion: The different susceptibility to experimental BA was associated with an increase of CD4 T-cells in the liver of Balb/c mice, which is linked to different gene profiles at the onset of bile duct obstruction.