• The Activation of IL-1-Induced Enhancers Depends on TAK1 Kinase Activity and NF-κB p65.

      Jurida, Liane; Soelch, Johanna; Bartkuhn, Marek; Handschick, Katja; Müller, Helmut; Newel, Doris; Weber, Axel; Dittrich-Breiholz, Oliver; Schneider, Heike; Bhuju, Sabin; Saul, Vera V; Schmitz, M Lienhard; Kracht, Michael; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015-02-04)
      The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-κB subunit p65 in relation to active enhancers and promoters of transcribed genes by chromatin immunoprecipitation sequencing (ChIP-seq) experiments. Out of 35,000 active enhancer regions, 410 H3K4me1-positive enhancers show interleukin 1 (IL-1)-induced H3K27ac and p65 binding. Inhibition of TAK1 or IKK2 or depletion of p65 blocked inducible enhancer activation and gene expression. As exemplified by the CXC chemokine cluster located on chromosome 4, the TAK1-p65 pathway also regulates the recruitment kinetics of the histone acetyltransferase CBP, of NF-κB p50, and of AP-1 transcription factors to both promoters and enhancers. This study provides a high-resolution view of epigenetic changes occurring during the IL-1 response and allows the genome-wide identification of a distinct class of inducible p65 NF-κB-dependent enhancers in epithelial cells.
    • AgNPs Change Microbial Community Structures of Wastewater.

      Guo, Yuting; Cichocki, Nicolas; Schattenberg, Florian; Geffers, Robert; Harms, Hauke; Müller, Susann; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2018-01-01)
      Due to their strong antimicrobial activity, silver nanoparticles (AgNPs) are massively produced, applied, consumed and, as a negative consequence, released into wastewater treatment plants. Most AgNPs are assumed to be bound by sludge, and thus bear potential risk for microbial performance and stability. In this lab-scale study, flow cytometry as a high-throughput method and 16S rRNA gene amplicon Illumina MiSeq sequencing were used to track microbial community structure changes when being exposed to AgNPs. Both methods allowed deeper investigation of the toxic impact of chemicals on microbial communities than classical EC50 determination. In addition, ecological metrics were used to quantify microbial community variations depending on AgNP types (10 and 30 nm) and concentrations. Only low changes in α- and intra-community β-diversity values were found both in successive negative and positive control batches and batches that were run with AgNPs below the EC50 value. Instead, AgNPs at EC50 concentrations caused upcoming of certain and disappearance of formerly dominant subcommunities. Flavobacteriia were among those that almost disappeared, while phylotypes affiliated with Gammaproteobacteria (3.6-fold) and Bacilli (8.4-fold) increased in cell abundance in comparison to the negative control. Thus, silver amounts at the EC50 value affected community structure suggesting a potential negative impact on functions in wastewater treatment systems.
    • Alloantigen-Induced Regulatory T Cells Generated in Presence of Vitamin C Display Enhanced Stability of Foxp3 Expression and Promote Skin Allograft Acceptance.

      Nikolouli, Eirini; Hardtke-Wolenski, Matthias; Hapke, Martin; Beckstette, Michael; Geffers, Robert; Floess, Stefan; Jaeckel, Elmar; Huehn, Jochen; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      Regulatory T cells (Tregs) are critical for the maintenance of immune homeostasis and self-tolerance and can be therapeutically used for prevention of unwanted immune responses such as allotransplant rejection. Tregs are characterized by expression of the transcription factor Foxp3, and recent work suggests that epigenetic imprinting of Foxp3 and other Treg-specific epigenetic signatures genes is crucial for the stabilization of both Foxp3 expression and immunosuppressive properties within Tregs. Lately, vitamin C was reported to enhance the activity of enzymes of the ten-eleven translocation family, thereby fostering the demethylation of Foxp3 and other Treg-specific epigenetic signatures genes in developing Tregs. Here, we in vitro generated alloantigen-induced Foxp3(+) Tregs (allo-iTregs) in presence of vitamin C. Although vitamin C hardly influenced the transcriptome of allo-iTregs as revealed by RNA-seq, those vitamin C-treated allo-iTregs showed a more pronounced demethylation of Foxp3 and other Treg-specific epigenetic signatures genes accompanied with an enhanced stability of Foxp3 expression. Accordingly, when being tested in vivo in an allogeneic skin transplantation model, vitamin C-treated allo-iTregs showed a superior suppressive capacity. Together, our results pave the way for the establishment of novel protocols for the in vitro generation of alloantigen-induced Foxp3(+) Tregs for therapeutic use in transplantation medicine.
    • Amphibian gut microbiota shifts differentially in community structure but converges on habitat-specific predicted functions.

      Bletz, Molly C; Goedbloed, Daniel J; Sanchez, Eugenia; Reinhardt, Timm; Tebbe, Christoph C; Bhuju, Sabin; Geffers, Robert; Jarek, Michael; Vences, Miguel; Steinfartz, Sebastian; Helmholtz Centre for infection research. Inhoffenstr. 7. 38124 Braunschweig, Germany. (2016-12-15)
      Complex microbial communities inhabit vertebrate digestive systems but thorough understanding of the ecological dynamics and functions of host-associated microbiota within natural habitats is limited. We investigate the role of environmental conditions in shaping gut and skin microbiota under natural conditions by performing a field survey and reciprocal transfer experiments with salamander larvae inhabiting two distinct habitats (ponds and streams). We show that gut and skin microbiota are habitat-specific, demonstrating environmental factors mediate community structure. Reciprocal transfer reveals that gut microbiota, but not skin microbiota, responds differentially to environmental change. Stream-to-pond larvae shift their gut microbiota to that of pond-to-pond larvae, whereas pond-to-stream larvae change to a community structure distinct from both habitat controls. Predicted functions, however, match that of larvae from the destination habitats in both cases. Thus, microbial function can be matched without taxonomic coherence and gut microbiota appears to exhibit metagenomic plasticity.
    • Analysis and Design of Stimulus Response Curves of E. coli.

      Kremling, Andreas; Goehler, Anna; Jahreis, Knut; Nees, Markus; Auerbach, Benedikt; Schmidt-Heck, Wolfgang; Kökpinar, Oznur; Geffers, Robert; Rinas, Ursula; Bettenbrock, Katja; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2012-11-12)
      Metabolism and signalling are tightly coupled in bacteria. Combining several theoretical approaches, a core model is presented that describes transcriptional and allosteric control of glycolysis in Escherichia coli. Experimental data based on microarrays, signalling components and extracellular metabolites are used to estimate kinetic parameters. A newly designed strain was used that adjusts the incoming glucose flux into the system and allows a kinetic analysis. Based on the results, prediction for intracelluar metabolite concentrations over a broad range of the growth rate could be performed and compared with data from literature.
    • Antibodies against C-reactive protein cross-react with 60-kilodalton heat shock proteins.

      Udvarnoki, Katalin; Cervenak, László; Uray, Katalin; Hudecz, Ferenc; Kacskovics, Imre; Spallek, Ralf; Singh, Mahavir; Füst, George; Prohászka, Zoltán; Third Department of Medicine, Semmelweis University, H-1125 Budapest, Kútvölgyi st. 4, Hungary. (2007-04)
      C-reactive protein (CRP) is an acute-phase reactant frequently used in histochemistry as a marker of ongoing inflammation. Furthermore, CRP is a powerful biomarker for the prediction of coronary artery disease risk. Heat-shock protein 60 (Hsp60) and CRP are complement-activating molecules, and the effect of their interactions on the regulation of complement activation was studied. However, during the first experiments, we learned that polyclonal anti-CRP antibodies cross-react with Hsp60. Therefore, the aim of our present study was to analyze the cross-reactivity of anti-CRP antibodies (Ab) with Hsp60 in solid-phase enzyme immune assays, in epitope studies using a series of overlapping synthetic peptides, and in Ouchterlony analyses. We found that three different commercial rabbit polyclonal antibodies and two monoclonal (9C9 and CRP-8) anti-CRP antibodies specifically recognize recombinant human Hsp60 and recombinant Mycobacterium tuberculosis Hsp65, respectively. Hsp60 was found to inhibit the binding of anti-CRP polyclonal Ab to Hsp60. Six epitope regions of Hsp60 were recognized by the anti-CRP antibodies, and one region (amino acids [AA] 218 to 232) was recognized by monoclonal antibodies CRP-8 and 9C9. This epitope region of Hsp60 displays 26.6% amino acid identity to CRP AA region 77 to 90. These data suggest that the B-cell epitopes shared between CRP and Hsp60 give rise to a true mimicry-based cross-reaction and the induction of cross-reactive antibodies. Our study underlines the importance of thorough study design and careful interpretation of results while using polyclonal anti-CRP antibodies for histochemistry, especially at low dilutions. Furthermore, analytical interference with Hsp60 in CRP assays should also be tested.
    • Archival bone marrow trephines are suitable for high-throughput mutation analysis using next generation sequencing technology.

      Hasemeier, Britta; Geffers, Robert; Bartels, Stephan; Schlegelberger, Brigitte; Kreipe, Hans; Lehmann, Ulrich; Institute of Pathology, Medizinische Hochschule Hannover, Hannover, Germany (Ferrata Storti Foundation, 2013-09)
    • Assessment of an APOBEC3B truncating mutation, c.783delG, in patients with breast cancer.

      Radmanesh, Hoda; Spethmann, Tessa; Enßen, Julia; Schürmann, Peter; Bhuju, Sabin; Geffers, Robert; Antonenkova, Natalia; Khusnutdinova, Elza; Sadr-Nabavi, Ariane; Shandiz, Fatemeh Homaei; Park-Simon, Tjoung-Won; Hillemanns, Peter; Christiansen, Hans; Bogdanova, Natalia; Dörk, Thilo; Helmholtz Centre for infection research, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-02)
      APOBEC3B belongs to the family of DNA-editing enzymes. A copy number variant targeting the genomic APOBEC3A-APOBEC3B locus has a significant impact on breast cancer risk, but the relative contribution of APOBEC3B is uncertain. In this study, we investigate a loss-of-function mutation that selectively targets APOBEC3B, for its association with breast cancer risk.
    • ATP inhibits the generation and function of regulatory T cells through the activation of purinergic P2X receptors.

      Schenk, Ursula; Frascoli, Michela; Proietti, Michele; Geffers, Robert; Traggiai, Elisabetta; Buer, Jan; Ricordi, Camillo; Westendorf, Astrid M; Grassi, Fabio; Institute for Research in Biomedicine, Bellinzona, Switzerland. (2011)
      Extracellular nucleotides are pleiotropic regulators of mammalian cell function. Adenosine triphosphate (ATP) released from CD4(+) helper T cells upon stimulation of the T cell receptor (TCR) contributes in an autocrine manner to the activation of mitogen-activated protein kinase (MAPK) signaling through purinergic P2X receptors. Increased expression of p2rx7, which encodes the purinergic receptor P2X7, is part of the transcriptional signature of immunosuppressive CD4(+)CD25(+) regulatory T cells (T(regs)). Here, we show that the activation of P2X7 by ATP inhibits the suppressive potential and stability of T(regs). The inflammatory cytokine interleukin-6 (IL-6) increased ATP synthesis and P2X7-mediated signaling in T(regs), which induced their conversion to IL-17-secreting T helper 17 (T(H)17) effector cells in vivo. Moreover, pharmacological antagonism of P2X receptors promoted the cell-autonomous conversion of naïve CD4(+) T cells into T(regs) after TCR stimulation. Thus, ATP acts as an autocrine factor that integrates stimuli from the microenvironment and cellular energetics to tune the developmental and immunosuppressive program of the T cell in adaptive immune responses.
    • BCL6--regulated by AhR/ARNT and wild-type MEF2B--drives expression of germinal center markers MYBL1 and LMO2.

      Ding, Jie; Dirks, Wilhelm G; Ehrentraut, Stefan; Geffers, Robert; MacLeod, Roderick A F; Nagel, Stefan; Pommerenke, Claudia; Romani, Julia; Scherr, Michaela; Vaas, Lea A I; Zaborski, Margarete; Drexler, Hans G; Quentmeier, Hilmar; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2015-06)
      Genetic heterogeneity is widespread in tumors, but poorly documented in cell lines. According to immunoglobulin hypermutation analysis, the diffuse large B-cell lymphoma cell line U-2932 comprises two subpopulations faithfully representing original tumor subclones. We set out to identify molecular causes underlying subclone-specific expression affecting 221 genes including surface markers and the germinal center oncogenes BCL6 and MYC. Genomic copy number variations explained 58/221 genes differentially expressed in the two U-2932 clones. Subclone-specific expression of the aryl-hydrocarbon receptor (AhR) and the resulting activity of the AhR/ARNT complex underlaid differential regulation of 11 genes including MEF2B. Knock-down and inhibitor experiments confirmed that AhR/ARNT regulates MEF2B, a key transcription factor for BCL6. AhR, MEF2B and BCL6 levels correlated not only in the U-2932 subclones but in the majority of 23 cell lines tested, indicting overexpression of AhR as a novel mechanism behind BCL6 diffuse large B-cell lymphoma. Enforced modulation of BCL6 affected 48/221 signature genes. Although BCL6 is known as a transcriptional repressor, 28 genes were up-regulated, including LMO2 and MYBL1 which, like BCL6, signify germinal center diffuse large B-cell lymphoma. Supporting the notion that BCL6 can induce gene expression, BCL6 and the majority of potential targets were co-regulated in a series of B-cell lines. In conclusion, genomic copy number aberrations, activation of AhR/ARNT, and overexpression of BCL6 are collectively responsible for differential expression of more than 100 genes in subclones of the U-2932 cell line. It is particularly interesting that BCL6 - regulated by AhR/ARNT and wild-type MEF2B - may drive expression of germinal center markers in diffuse large B-cell lymphoma.
    • The BH3-only protein BID impairs the p38-mediated stress response and promotes hepatocarcinogenesis during chronic liver injury in mice.

      Orlik, Johanna; Schüngel, Sven; Buitrago-Molina, Laura Elisa; Marhenke, Silke; Geffers, Robert; Endig, Jessica; Lobschat, Katharina; Rössler, Stephanie; Goeppert, Benjamin; Manns, Michael P; Gross, Atan; Vogel, Arndt; Hannover Medical School, Hannover, Germany. (2015-09)
      Apoptosis is critical for maintaining tissue homeostasis, and apoptosis evasion is considered as a hallmark of cancer. However, increasing evidence also suggests that proapoptotic molecules can contribute to the development of cancer, including liver cancer. The aim of this study was to further clarify the role of the proapoptotic B-cell lymphoma 2 homology domain 3 (BH3)-only protein BH3 interacting-domain death agonist (BID) for chronic liver injury (CLI) and hepatocarcinogenesis (HCG). Loss of BID significantly delayed tumor development in two mouse models of Fah-mediated and HBsTg-driven HCG, suggesting a tumor-promoting effect of BID. Liver injury as well as basal and mitogen-stimulated hepatocyte proliferation were not modulated by BID. Moreover, there was no in vivo or in vitro evidence that BID was involved in DNA damage response in hepatocytes and hepatoma cells. Our data revealed that CLI was associated with strong activation of oxidative stress (OS) response and that BID impaired full activation of p38 after OS.
    • The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of Escherichia coli.

      Donner, Jannik; Reck, Michael; Bunk, Boyke; Jarek, Michael; App, Constantin Benjamin; Meier-Kolthoff, Jan P; Overmann, Jörg; Müller, Rolf; Kirschning, Andreas; Wagner-Döbler, Irene; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr, 7,38124 Braunschweig, Germany. (2017-11-01)
      The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditis-associated pathogen Streptococcus mutans at nanomolar concentrations. Here, we studied the response to carolacton of an Escherichia coli strain that lacked the outer membrane protein TolC. Whole-genome sequencing of the laboratory E. coli strain TolC revealed the integration of an insertion element, IS5, at the tolC locus and a close phylogenetic relationship to the ancient E. coli K-12. We demonstrated via transcriptome sequencing (RNA-seq) and determination of MIC values that carolacton penetrates the phospholipid bilayer of the Gram-negative cell envelope and inhibits growth of E. coli TolC at similar concentrations as for streptococci. This inhibition is completely lost for a C-9 (R) epimer of carolacton, a derivative with an inverted stereocenter at carbon atom 9 [(S) → (R)] as the sole difference from the native molecule, which is also inactive in S. pneumoniae and S. mutans, suggesting a specific interaction of native carolacton with a conserved cellular target present in bacterial phyla as distantly related as Firmicutes and Proteobacteria. The efflux pump inhibitor (EPI) phenylalanine arginine β-naphthylamide (PAβN), which specifically inhibits AcrAB-TolC, renders E. coli susceptible to carolacton. Our data indicate that carolacton has potential for use in antimicrobial chemotherapy against Gram-negative bacteria, as a single drug or in combination with EPIs. Strain E. coli TolC has been deposited at the DSMZ; together with the associated RNA-seq data and MIC values, it can be used as a reference during future screenings for novel bioactive compounds. IMPORTANCE The emergence of pathogens resistant against most or all of the antibiotics currently used in human therapy is a global threat, and therefore the search for antimicrobials with novel targets and modes of action is of utmost importance. The myxobacterial secondary metabolite carolacton had previously been shown to inhibit biofilm formation and growth of streptococci. Here, we investigated if carolacton could act against Gram-negative bacteria, which are difficult targets because of their double-layered cytoplasmic envelope. We found that the model organism Escherichia coli is susceptible to carolacton, similar to the Gram-positive Streptococcus pneumoniae, if its multidrug efflux system AcrAB-TolC is either inactivated genetically, by disruption of the tolC gene, or physiologically by coadministering an efflux pump inhibitor. A carolacton epimer that has a different steric configuration at carbon atom 9 is completely inactive, suggesting that carolacton may interact with the same molecular target in both Gram-positive and Gram-negative bacteria.
    • Birth, evolution, and transmission of satellite-free mammalian centromeric domains.

      Nergadze, Solomon G; Piras, Francesca M; Gamba, Riccardo; Corbo, Marco; Cerutti, Federico; McCarter, Joseph G W; Cappelletti, Eleonora; Gozzo, Francesco; Harman, Rebecca M; Antczak, Douglas F; Miller, Donald; Scharfe, Maren; Pavesi, Giulio; Raimondi, Elena; Sullivan, Kevin F; Giulotto, Elena; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-01-01)
      Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on
    • CD25+ FoxP3+ Memory CD4 T Cells Are Frequent Targets of HIV Infection In Vivo.

      Chachage, Mkunde; Pollakis, Georgios; Kuffour, Edmund Osei; Haase, Kerstin; Bauer, Asli; Nadai, Yuka; Podola, Lilli; Clowes, Petra; Schiemann, Matthias; Henkel, Lynette; Hoffmann, Dieter; Joseph, Sarah; Bhuju, Sabin; Maboko, Leonard; Sarfo, Fred Stephen; Eberhardt, Kirsten; Hoelscher, Michael; Feldt, Torsten; Saathoff, Elmar; Geldmacher, Christof; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-10-15)
      Interleukin 2 (IL-2) signaling through the IL-2 receptor alpha chain (CD25) facilitates HIV replication in vitro and facilitates homeostatic proliferation of CD25(+) FoxP3(+) CD4(+) T cells. CD25(+) FoxP3(+) CD4(+) T cells may therefore constitute a suitable subset for HIV infection and plasma virion production. CD25(+) FoxP3(+) CD4(+) T cell frequencies, absolute numbers, and the expression of CCR5 and cell cycle marker Ki67 were studied in peripheral blood from HIV(+) and HIV(-) study volunteers. Different memory CD4(+) T cell subsets were then sorted for quantification of cell-associated HIV DNA and phylogenetic analyses of the highly variable EnvV1V3 region in comparison to plasma-derived virus sequences. In HIV(+) subjects, 51% (median) of CD25(+) FoxP3(+) CD4(+) T cells expressed the HIV coreceptor CCR5. Very high frequencies of Ki67(+) cells were detected in CD25(+) FoxP3(+) memory CD4(+) T cells (median, 27.6%) in comparison to CD25(-) FoxP3(-) memory CD4(+) T cells (median, 4.1%; P < 0.0001). HIV DNA content was 15-fold higher in CD25(+) FoxP3(+) memory CD4(+) T cells than in CD25(-) FoxP3(-) T cells (P = 0.003). EnvV1V3 sequences derived from CD25(+) FoxP3(+) memory CD4(+) T cells did not preferentially cluster with plasma-derived sequences. Quasi-identical cell-plasma sequence pairs were rare, and their proportion decreased with the estimated HIV infection duration. These data suggest that specific cellular characteristics of CD25(+) FoxP3(+) memory CD4(+) T cells might facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. The contribution of this cell population to plasma virion production remains unclear.
    • Cellular retinaldehyde-binding protein (CRALBP) is a direct downstream target of transcription factor Pax6.

      Boppana, Sridhar; Scheglov, Alexander; Geffers, Robert; Tarabykin, Victor; Max-Planck-Institute for Experimental Medicine, Hermann-Rein Strasse 3, 37075 Göttingen, Germany. boppansr@umdnj.edu (2012-02)
      Transcription factor Pax6 plays an essential role in the expression of other transcription factors, cell adhesion molecules and is crucial for neurogenesis in the developing forebrain. Analysis of gene expression profiles through microarray experiments in Pax6 mutants allowed us to focus on CRALBP, one of the many genes that were downregulated.
    • Characterization of the p53 cistrome--DNA binding cooperativity dissects p53's tumor suppressor functions.

      Schlereth, Katharina; Heyl, Charlotte; Krampitz, Anna-Maria; Mernberger, Marco; Finkernagel, Florian; Scharfe, Maren; Jarek, Michael; Leich, Ellen; Rosenwald, Andreas; Stiewe, Thorsten; Molecular Oncology, Philipps-University, Marburg, Germany. (2013-08)
      p53 protects us from cancer by transcriptionally regulating tumor suppressive programs designed to either prevent the development or clonal expansion of malignant cells. How p53 selects target genes in the genome in a context- and tissue-specific manner remains largely obscure. There is growing evidence that the ability of p53 to bind DNA in a cooperative manner prominently influences target gene selection with activation of the apoptosis program being completely dependent on DNA binding cooperativity. Here, we used ChIP-seq to comprehensively profile the cistrome of p53 mutants with reduced or increased cooperativity. The analysis highlighted a particular relevance of cooperativity for extending the p53 cistrome to non-canonical binding sequences characterized by deletions, spacer insertions and base mismatches. Furthermore, it revealed a striking functional separation of the cistrome on the basis of cooperativity; with low cooperativity genes being significantly enriched for cell cycle and high cooperativity genes for apoptotic functions. Importantly, expression of high but not low cooperativity genes was correlated with superior survival in breast cancer patients. Interestingly, in contrast to most p53-activated genes, p53-repressed genes did not commonly contain p53 binding elements. Nevertheless, both the degree of gene activation and repression were cooperativity-dependent, suggesting that p53-mediated gene repression is largely indirect and mediated by cooperativity-dependently transactivated gene products such as CDKN1A, E2F7 and non-coding RNAs. Since both activation of apoptosis genes with non-canonical response elements and repression of pro-survival genes are crucial for p53's apoptotic activity, the cistrome analysis comprehensively explains why p53-induced apoptosis, but not cell cycle arrest, strongly depends on the intermolecular cooperation of p53 molecules as a possible safeguard mechanism protecting from accidental cell killing.
    • ChIP-on-chip analysis identifies IL-22 as direct target gene of ectopically expressed FOXP3 transcription factor in human T cells

      Jeron, Andreas; Hansen, Wiebke; Ewert, Franziska; Buer, Jan; Geffers, Robert; Bruder, Dunja (2012-12-17)
      Abstract Background The transcription factor (TF) forkhead box P3 (FOXP3) is constitutively expressed at high levels in naturally occurring CD4+CD25+ regulatory T cells (nTregs). It is not only the most accepted marker for that cell population but is also considered lineage determinative. Chromatin immunoprecipitation (ChIP) of TFs in combination with genomic tiling microarray analysis (ChIP-on-chip) has been shown to be an appropriate tool for identifying FOXP3 transcription factor binding sites (TFBSs) on a genome-wide scale. In combination with microarray expression analysis, the ChIP-on-chip technique allows identification of direct FOXP3 target genes. Results ChIP-on-chip analysis of the human FOXP3 expressed in resting and PMA/ionomycin–stimulated Jurkat T cells revealed several thousand putative FOXP3 binding sites and demonstrated the importance of intronic regions for FOXP3 binding. The analysis of expression data showed that the stimulation-dependent down-regulation of IL-22 was correlated with direct FOXP3 binding in the IL-22 promoter region. This association was confirmed by real-time PCR analysis of ChIP-DNA. The corresponding ChIP-region also contained a matching FOXP3 consensus sequence. Conclusions Knowledge of the general distribution patterns of FOXP3 TFBSs in the human genome under resting and activated conditions will contribute to a better understanding of this TF and its influence on direct target genes, as well as its importance for the phenotype and function of Tregs. Moreover, FOXP3-dependent repression of Th17-related IL-22 may be relevant to an understanding of the phenomenon of Treg/Th17 cell plasticity.
    • Chromatin binding of Gcn5 in Drosophila is largely mediated by CP190.

      Ali, Tamer; Krüger, Marcus; Bhuju, Sabin; Jarek, Michael; Bartkuhn, Marek; Renkawitz, Rainer; Hel,holtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-11-29)
      Centrosomal 190 kDa protein (CP190) is a promoter binding factor, mediates long-range interactions in the context of enhancer-promoter contacts and in chromosomal domain formation. All Drosophila insulator proteins bind CP190 suggesting a crucial role in insulator function. CP190 has major effects on chromatin, such as depletion of nucleosomes, high nucleosomal turnover and prevention of heterochromatin expansion. Here, we searched for enzymes, which might be involved in CP190 mediated chromatin changes. Eighty percent of the genomic binding sites of the histone acetyltransferase Gcn5 are colocalizing with CP190 binding. Depletion of CP190 reduces Gcn5 binding to chromatin. Binding dependency was further supported by Gcn5 mediated co-precipitation of CP190. Gcn5 is known to activate transcription by histone acetylation. We used the dCas9 system to target CP190 or Gcn5 to a Polycomb repressed and H3K27me3 marked gene locus. Both, CP190 as well as Gcn5, activate this locus, thus supporting the model that CP190 recruits Gcn5 and thereby activates chromatin.
    • Chromera velia, endosymbioses and the rhodoplex hypothesis--plastid evolution in cryptophytes, alveolates, stramenopiles, and haptophytes (CASH lineages).

      Petersen, Jörn; Ludewig, Ann-Kathrin; Michael, Victoria; Bunk, Boyke; Jarek, Michael; Baurain, Denis; Brinkmann, Henner (2014-03)
      The discovery of Chromera velia, a free-living photosynthetic relative of apicomplexan pathogens, has provided an unexpected opportunity to study the algal ancestry of malaria parasites. In this work, we compared the molecular footprints of a eukaryote-to-eukaryote endosymbiosis in C. velia to their equivalents in peridinin-containing dinoflagellates (PCD) to reevaluate recent claims in favor of a common ancestry of their plastids. To this end, we established the draft genome and a set of full-length cDNA sequences from C. velia via next-generation sequencing. We documented the presence of a single coxI gene in the mitochondrial genome, which thus represents the genetically most reduced aerobic organelle identified so far, but focused our analyses on five "lucky genes" of the Calvin cycle. These were selected because of their known support for a common origin of complex plastids from cryptophytes, alveolates (represented by PCDs), stramenopiles, and haptophytes (CASH) via a single secondary endosymbiosis with a red alga. As expected, our broadly sampled phylogenies of the nuclear-encoded Calvin cycle markers support a rhodophycean origin for the complex plastid of Chromera. However, they also suggest an independent origin of apicomplexan and dinophycean (PCD) plastids via two eukaryote-to-eukaryote endosymbioses. Although at odds with the current view of a common photosynthetic ancestry for alveolates, this conclusion is nonetheless in line with the deviant plastome architecture in dinoflagellates and the morphological paradox of four versus three plastid membranes in the respective lineages. Further support for independent endosymbioses is provided by analysis of five additional markers, four of them involved in the plastid protein import machinery. Finally, we introduce the "rhodoplex hypothesis" as a convenient way to designate evolutionary scenarios where CASH plastids are ultimately the product of a single secondary endosymbiosis with a red alga but were subsequently horizontally spread via higher-order eukaryote-to-eukaryote endosymbioses.
    • Clinical and Biological Manifestation of RNF168 Deficiency in Two Polish Siblings.

      Pietrucha, Barbara; Heropolitańska-Pliszka, Edyta; Geffers, Robert; Enßen, Julia; Wieland, Britta; Bogdanova, Natalia Valerijevna; Dörk, Thilo; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      Germline mutations in the RING finger protein gene RNF168 have been identified in a combined immunodeficiency disorder called RIDDLE syndrome. Since only two patients have been described with somewhat different phenotypes, there is need to identify further patients. Here, we report on two Polish siblings with RNF168 deficiency due to homozygosity for a novel frameshift mutation, c.295delG, that was identified through exome sequencing. Both patients presented with immunoglobulin deficiency, telangiectasia, cellular radiosensitivity, and increased alpha-fetoprotein (AFP) levels. The younger sibling had a more pronounced neurological and morphological phenotype, and she also carried an ATM gene mutation in the heterozygous state. Immunoblot analyses showed absence of RNF168 protein, whereas ATM levels and function were proficient in lymphoblastoid cells from both patients. Consistent with the absence of RNF168 protein, 53BP1 recruitment to DNA double-strand breaks (DSBs) after irradiation was undetectable in lymphoblasts or primary fibroblasts from either of the two patients. γH2AX foci accumulated normally but they disappeared with significant delay, indicating a severe defect in DSB repair. A comparison with the two previously identified patients indicates immunoglobulin deficiency, cellular radiosensitivity, and increased AFP levels as hallmarks of RNF168 deficiency. The variability in its clinical expression despite similar cellular phenotypes suggests that some manifestations of RNF168 deficiency may be modified by additional genetic or epidemiological factors.