Dokument-ID Dokumenttyp Verfasser/Autoren Herausgeber Haupttitel Abstract Auflage Verlagsort Verlag Erscheinungsjahr Seitenzahl Schriftenreihe Titel Schriftenreihe Bandzahl ISBN Quelle der Hochschulschrift Konferenzname Quelle:Titel Quelle:Jahrgang Quelle:Heftnummer Quelle:Erste Seite Quelle:Letzte Seite URN DOI Abteilungen OPUS4-13144 Wissenschaftlicher Artikel Gassen, Alwine; Brechtefeld, Doris; Schandry, Niklas; Arteaga-Salas, J. Manuel; Israel, Lars; Imhof, Axel; Janzen, Christian J. DOT1A-dependent H3K76 methylation is required for replication regulation in Trypanosoma brucei Cell-cycle progression requires careful regulation to ensure accurate propagation of genetic material to the daughter cells. Although many cell-cycle regulators are evolutionarily conserved in the protozoan parasite Trypanosoma brucei, novel regulatory mechanisms seem to have evolved. Here, we analyse the function of the histone methyltransferase DOT1A during cell-cycle progression. Over-expression of DOT1A generates a population of cells with aneuploid nuclei as well as enucleated cells. Detailed analysis shows that DOT1A over-expression causes continuous replication of the nuclear DNA. In contrast, depletion of DOT1A by RNAi abolishes replication but does not prevent karyokinesis. As histone H3K76 methylation has never been associated with replication control in eukaryotes before, we have discovered a novel function of DOT1 enzymes, which might not be unique to trypanosomes. 2012 10302 - 10311 Nucleic Acids Research 40 20 urn:nbn:de:bvb:20-opus-131449 10.1093/nar/gks801 Theodor-Boveri-Institut für Biowissenschaften OPUS4-11972 Wissenschaftlicher Artikel Batram, Christopher; Jones, Nivola G.; Janzen, Christian J.; Markert, Sebastian M.; Engstler, Markus Expression site attenuation mechanistically links antigenic variation and development in Trypanosoma brucei We have discovered a new mechanism of monoallelic gene expression that links antigenic variation, cell cycle, and development in the model parasite Trypanosoma brucei. African trypanosomes possess hundreds of variant surface glycoprotein (VSG) genes, but only one is expressed from a telomeric expression site (ES) at any given time. We found that the expression of a second VSG alone is sufficient to silence the active VSG gene and directionally attenuate the ES by disruptor of telomeric silencing-1B (DOT1B)-mediated histone methylation. Three conserved expression-site-associated genes (ESAGs) appear to serve as signal for ES attenuation. Their depletion causes G1-phase dormancy and reversible initiation of the slender-to-stumpy differentiation pathway. ES-attenuated slender bloodstream trypanosomes gain full developmental competence for transformation to the tsetse fly stage. This surprising connection between antigenic variation and developmental progression provides an unexpected point of attack against the deadly sleeping sickness. 2014 eLife 3 e02324 urn:nbn:de:bvb:20-opus-119727 10.7554/eLife.02324 Theodor-Boveri-Institut für Biowissenschaften OPUS4-11722 Wissenschaftlicher Artikel Pascoalino, Bruno; Dindar, Gülcin; Vieira-da-Rocha, João P.; Machado, Carlos Renato; Janzen, Christian J.; Schenkman, Sergio Characterization of two different Asf1 histone chaperones with distinct cellular localizations and functions in Trypanosoma brucei The anti-silencing function protein 1 (Asf1) is a chaperone that forms a complex with histones H3 and H4 facilitating dimer deposition and removal from chromatin. Most eukaryotes possess two different Asf1 chaperones but their specific functions are still unknown. Trypanosomes, a group of early-diverged eukaryotes, also have two, but more divergent Asf1 paralogs than Asf1 of higher eukaryotes. To unravel possible different functions, we characterized the two Asf1 proteins in Trypanosoma brucei. Asf1A is mainly localized in the cytosol but translocates to the nucleus in S phase. In contrast, Asf1B is predominantly localized in the nucleus, as described for other organisms. Cytosolic Asf1 knockdown results in accumulation of cells in early S phase of the cell cycle, whereas nuclear Asf1 knockdown arrests cells in S/G2 phase. Overexpression of cytosolic Asf1 increases the levels of histone H3 and H4 acetylation. In contrast to cytosolic Asf1, overexpression of nuclear Asf1 causes less pronounced growth defects in parasites exposed to genotoxic agents, prompting a function in chromatin remodeling in response to DNA damage. Only the cytosolic Asf1 interacts with recombinant H3/H4 dimers in vitro. These findings denote the early appearance in evolution of distinguishable functions for the two Asf1 chaperons in trypanosomes. 2014 2906-2918 Nucleic Acids Research 42 5 urn:nbn:de:bvb:20-opus-117220 10.1093/nar/gkt1267 Theodor-Boveri-Institut für Biowissenschaften OPUS4-22518 Wissenschaftlicher Artikel Reis, Helena; Schwebs, Marie; Dietz, Sabrina; Janzen, Christian J.; Butter, Falk TelAP1 links telomere complexes with developmental expression site silencing in African trypanosomes During its life cycle, Trypanosoma brucei shuttles between a mammalian host and the tsetse fly vector. In the mammalian host, immune evasion of T. brucei bloodstream form (BSF) cells relies on antigenic variation, which includes monoallelic expression and periodic switching of variant surface glycoprotein (VSG) genes. The active VSG is transcribed from only 1 of the 15 subtelomeric expression sites (ESs). During differentiation from BSF to the insect-resident procyclic form (PCF), the active ES is transcriptionally silenced. We used mass spectrometry-based interactomics to determine the composition of telomere protein complexes in T. brucei BSF and PCF stages to learn more about the structure and functions of telomeres in trypanosomes. Our data suggest a different telomere complex composition in the two forms of the parasite. One of the novel telomere-associated proteins, TelAP1, forms a complex with telomeric proteins TbTRF, TbRAP1 and TbTIF2 and influences ES silencing kinetics during developmental differentiation. 2018 2820-2833 Nucleic Acids Research 46 6 urn:nbn:de:bvb:20-opus-225180 10.1093/nar/gky028 Theodor-Boveri-Institut für Biowissenschaften OPUS4-22628 Wissenschaftlicher Artikel Rinaldetti, Sébastien; Pfirrmann, Markus; Manz, Kirsi; Guilhot, Joelle; Dietz, Christian; Panagiotidis, Panayiotidis; Spiess, Birgit; Seifarth, Wolfgang; Fabarius, Alice; Müller, Martin; Pagoni, Maria; Dimou, Maria; Dengler, Jolanta; Waller, Cornelius F.; Brümmendorf, Tim H.; Herbst, Regina; Burchert, Andreas; Janßen, Carsten; Goebeler, Maria Elisabeth; Jost, Philipp J.; Hanzel, Stefan; Schafhausen, Philippe; Prange-Krex, Gabriele; Illmer, Thomas; Janzen, Viktor; Klausmann, Martine; Eckert, Robert; Büschel, Gerd; Kiani, Alexander; Hofmann, Wolf-Karsten; Mahon, François-Xavier; Saussele, Susanne Effect of ABCG2, OCT1, and ABCB1 (MDR1) Gene Expression on Treatment-Free Remission in a EURO-SKI Subtrial Within the EURO-SKI trial, 132 chronic phase CML patients discontinued imatinib treatment. RNA was isolated from peripheral blood in order to analyze the expression of MDR1, ABCG2 and OCT1. ABCG2 was predictive for treatment-free remission in Cox regression analysis. High transcript levels of the ABCG2 efflux transporter (>4.5 parts per thousand) were associated with a twofold higher risk of relapse. Introduction: Tyrosine kinase inhibitors (TKIs) can safely be discontinued in chronic myeloid leukemia (CML) patients with sustained deep molecular response. ABCG2 (breast cancer resistance protein), OCT1 (organic cation transporter 1), and ABCB1 (multidrug resistance protein 1) gene products are known to play a crucial role in acquired pharmacogenetic TKI resistance. Their influence on treatment-free remission (TFR) has not yet been investigated. Materials and Methods: RNA was isolated on the last day of TKI intake from peripheral blood leukocytes of 132 chronic phase CML patients who discontinued TKI treatment within the European Stop Tyrosine Kinase Inhibitor Study trial. Plasmid standards were designed including subgenic inserts of OCT1, ABCG2, and ABCB1 together with GUSB as reference gene. For expression analyses, quantitative real-time polymerase chain reaction was used. Multiple Cox regression analysis was performed. In addition, gene expression cutoffs for patient risk stratification were investigated. Results: The TFR rate of 132 patients, 12 months after TKI discontinuation, was 54% (95% confidence interval [CI], 46%-62%). ABCG2 expression (parts per thousand) was retained as the only significant variable (P=.02; hazard ratio, 1.04; 95% CI, 1.01-1.07) in multiple Cox regression analysis. Only for the ABCG2 efflux transporter, a significant cutoff was found (P=.04). Patients with an ABCG2/GUSB transcript level >4.5 parts per thousand (n=93) showed a 12-month TFR rate of 47% (95% CI, 37%-57%), whereas patients with low ABCG2 expression (<= 4.5 parts per thousand; n=39) had a 12-month TFR rate of 72% (95% CI, 55%-82%). Conclusion: In this study, we investigated the effect of pharmacogenetics in the context of a CML treatment discontinuation trial. The transcript levels of the efflux transporter ABCG2 predicted TFR after TKI discontinuation. (C) 2018 The Authors. Published by Elsevier Inc. 2018 266-271 Clinical Lymphoma, Myeloma & Leukemia 18 4 urn:nbn:de:bvb:20-opus-226281 10.1016/j.clml.2018.02.004 Medizinische Klinik und Poliklinik II OPUS4-18102 Wissenschaftlicher Artikel Cicova, Zdenka; Dejung, Mario; Skalicky, Tomas; Eisenhuth, Nicole; Hanselmann, Steffen; Morriswood, Brooke; Figueiredo, Luisa M.; Butter, Falk; Janzen, Christian J. Two flagellar BAR domain proteins in Trypanosoma brucei with stage-specific regulation Trypanosomes are masters of adaptation to different host environments during their complex life cycle. Large-scale proteomic approaches provide information on changes at the cellular level, and in a systematic way. However, detailed work on single components is necessary to understand the adaptation mechanisms on a molecular level. Here, we have performed a detailed characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation factor Tb927.11.2400, identified previously in a SILAC-based comparative proteome study. Tb927.11.2400 shares 38% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form (PCF) stage-specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin-like (TbFlabarinL), and demonstrate that it originates from a gene duplication event, which occurred in the African trypanosomes. TbFlabarinL is not essential for the growth of the parasites under cell culture conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We generated TbFlabarinL-specific antibodies, and showed that it localizes in the flagellum. Co-immunoprecipitation experiments together with a biochemical cell fractionation suggest a dual association of TbFlabarinL with the flagellar membrane and the components of the paraflagellar rod. 2016 Scientific Reports 6 urn:nbn:de:bvb:20-opus-181021 10.1038/srep35826 Julius-von-Sachs-Institut für Biowissenschaften OPUS4-30137 Wissenschaftlicher Artikel Vellmer, Tim; Hartleb, Laura; Fradera Sola, Albert; Kramer, Susanne; Meyer-Natus, Elisabeth; Butter, Falk; Janzen, Christian J. A novel SNF2 ATPase complex in Trypanosoma brucei with a role in H2A.Z-mediated chromatin remodelling A cascade of histone acetylation events with subsequent incorporation of a histone H2A variant plays an essential part in transcription regulation in various model organisms. A key player in this cascade is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant H2A.Z. Transcriptional regulation of polycistronic transcription units in the unicellular parasite Trypanosoma brucei has been shown to be highly dependent on acetylation of H2A.Z, which is mediated by the histone-acetyltransferase HAT2. The chromatin remodelling complex which mediates H2A.Z incorporation is not known and an SWR1 orthologue in trypanosomes has not yet been reported. In this study, we identified and characterised an SWR1-like remodeller complex in T. brucei that is responsible for Pol II-dependent transcriptional regulation. Bioinformatic analysis of potential SNF2 DEAD/Box helicases, the key component of SWR1 complexes, identified a 1211 amino acids-long protein that exhibits key structural characteristics of the SWR1 subfamily. Systematic protein-protein interaction analysis revealed the existence of a novel complex exhibiting key features of an SWR1-like chromatin remodeller. RNAi-mediated depletion of the ATPase subunit of this complex resulted in a significant reduction of H2A.Z incorporation at transcription start sites and a subsequent decrease of steady-state mRNA levels. Furthermore, depletion of SWR1 and RNA-polymerase II (Pol II) caused massive chromatin condensation. The potential function of several proteins associated with the SWR1-like complex and with HAT2, the key factor of H2A.Z incorporation, is discussed. 2022 PLoS Pathogens 18 6 urn:nbn:de:bvb:20-opus-301372 10.1371/journal.ppat.1010514 Theodor-Boveri-Institut für Biowissenschaften OPUS4-23057 Wissenschaftlicher Artikel Leal, Andrea Zurita; Schwebs, Marie; Briggs, Emma; Weisert, Nadine; Reis, Helena; Lemgruber, Leondro; Luko, Katarina; Wilkes, Jonathan; Butter, Falk; McCulloch, Richard; Janzen, Christian J. Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to off-spring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation. 2020 9660-9680 Nucleic Acids Research 48 17 urn:nbn:de:bvb:20-opus-230579 10.1093/nar/gkaa686 Theodor-Boveri-Institut für Biowissenschaften OPUS4-15857 Wissenschaftlicher Artikel Goos, Carina; Dejung, Mario; Janzen, Christian J.; Butter, Falk; Kramer, Susanne The nuclear proteome of Trypanosoma brucei Trypanosoma brucei is a protozoan flagellate that is transmitted by tsetse flies into the mammalian bloodstream. The parasite has a huge impact on human health both directly by causing African sleeping sickness and indirectly, by infecting domestic cattle. The biology of trypanosomes involves some highly unusual, nuclear-localised processes. These include polycistronic transcription without classical promoters initiated from regions defined by histone variants, trans-splicing of all transcripts to the exon of a spliced leader RNA, transcription of some very abundant proteins by RNA polymerase I and antigenic variation, a switch in expression of the cell surface protein variants that allows the parasite to resist the immune system of its mammalian host. Here, we provide the nuclear proteome of procyclic Trypanosoma brucei, the stage that resides within the tsetse fly midgut. We have performed quantitative label-free mass spectrometry to score 764 significantly nuclear enriched proteins in comparison to whole cell lysates. A comparison with proteomes of several experimentally characterised nuclear and non-nuclear structures and pathways confirmed the high quality of the dataset: the proteome contains about 80% of all nuclear proteins and less than 2% false positives. Using motif enrichment, we found the amino acid sequence KRxR present in a large number of nuclear proteins. KRxR is a sub-motif of a classical eukaryotic monopartite nuclear localisation signal and could be responsible for nuclear localization of proteins in Kinetoplastida species. As a proof of principle, we have confirmed the nuclear localisation of six proteins with previously unknown localisation by expressing eYFP fusion proteins. While proteome data of several T. brucei organelles have been published, our nuclear proteome closes an important gap in knowledge to study trypanosome biology, in particular nuclear-related processes. 2017 e0181884 PLoS ONE 12 7 urn:nbn:de:bvb:20-opus-158572 10.1371/journal.pone.0181884 Theodor-Boveri-Institut für Biowissenschaften OPUS4-15823 Wissenschaftlicher Artikel Zimmermann, Henriette; Subota, Ines; Batram, Christopher; Kramer, Susanne; Janzen, Christian J.; Jones, Nicola G.; Engstler, Markus A quorum sensing-independent path to stumpy development in Trypanosoma brucei For persistent infections of the mammalian host, African trypanosomes limit their population size by quorum sensing of the parasite-excreted stumpy induction factor (SIF), which induces development to the tsetse-infective stumpy stage. We found that besides this cell density-dependent mechanism, there exists a second path to the stumpy stage that is linked to antigenic variation, the main instrument of parasite virulence. The expression of a second variant surface glycoprotein (VSG) leads to transcriptional attenuation of the VSG expression site (ES) and immediate development to tsetse fly infective stumpy parasites. This path is independent of SIF and solely controlled by the transcriptional status of the ES. In pleomorphic trypanosomes varying degrees of ES-attenuation result in phenotypic plasticity. While full ES-attenuation causes irreversible stumpy development, milder attenuation may open a time window for rescuing an unsuccessful antigenic switch, a scenario that so far has not been considered as important for parasite survival. 2017 e1006324 PLoS Pathogens 13 4 urn:nbn:de:bvb:20-opus-158230 10.1371/journal.ppat.1006324 Theodor-Boveri-Institut für Biowissenschaften OPUS4-26069 Wissenschaftlicher Artikel Eisenhuth, Nicole; Vellmer, Tim; Rauh, Elisa T.; Butter, Falk; Janzen, Christian J. A DOT1B/Ribonuclease H2 Protein Complex Is Involved in R-Loop Processing, Genomic Integrity, and Antigenic Variation in Trypanosoma brucei The parasite Trypanosoma brucei periodically changes the expression of protective variant surface glycoproteins (VSGs) to evade its host's immune sys-tem in a process known as antigenic variation. One route to change VSG expres-sion is the transcriptional activation of a previously silent VSG expression site (ES), a subtelomeric region containing the VSG genes. Homologous recombination of a different VSG from a large reservoir into the active ES represents another route. The conserved histone methyltransferase DOT1B is involved in transcriptional silencing of inactive ES and influences ES switching kinetics. The molecular machin-ery that enables DOT1B to execute these regulatory functions remains elusive, however. To better understand DOT1B-mediated regulatory processes, we purified DOT1B-associated proteins using complementary biochemical approaches. We iden-tified several novel DOT1B interactors. One of these was the RNase H2 complex, previously shown to resolve RNA-DNA hybrids, maintain genome integrity, and play a role in antigenic variation. Our study revealed that DOT1B depletion results in an increase in RNA-DNA hybrids, accumulation of DNA damage, and ES switch-ing events. Surprisingly, a similar pattern of VSG deregulation was observed in RNase H2 mutants. We propose that both proteins act together in resolving R-loops to ensure genome integrity and contribute to the tightly regulated process of anti-genic variation. 2021 e01352-21 mbio 12 6 urn:nbn:de:bvb:20-opus-260698 10.1128/mBio.01352-21 Theodor-Boveri-Institut für Biowissenschaften