Publications by authors named "Falk Butter"

72 Publications

Identification of the transcription factor MAZ as a regulator of erythropoiesis.

Blood Adv 2021 08;5(15):3002-3015

The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.

Erythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors (TFs). Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a TF that binds to the promoter of the erythroid-specific human α-globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes. Consistent with an important role during erythropoiesis, knockdown of MAZ reduces α-globin expression in K562 cells and impairs differentiation in primary human erythroid cells. Genetic variants in the MAZ locus are associated with changes in clinically important human erythroid traits. Taken together, these findings reveal the zinc-finger TF MAZ to be a previously unrecognized regulator of the erythroid differentiation program.
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http://dx.doi.org/10.1182/bloodadvances.2021004609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361462PMC
August 2021

Oncogenic Kinase Cascades Induce Molecular Mechanisms That Protect Leukemic Cell Models from Lethal Effects of De Novo dNTP Synthesis Inhibition.

Cancers (Basel) 2021 Jul 10;13(14). Epub 2021 Jul 10.

Department of Toxicology, University Medical Center, 55131 Mainz, Germany.

The ribonucleotide reductase inhibitor hydroxyurea suppresses de novo dNTP synthesis and attenuates the hyperproliferation of leukemic blasts. Mechanisms that determine whether cells undergo apoptosis in response to hydroxyurea are ill-defined. We used unbiased proteomics to uncover which pathways control the transition of the hydroxyurea-induced replication stress into an apoptotic program in chronic and acute myeloid leukemia cells. We noted a decrease in the serine/threonine kinase RAF1/c-RAF in cells that undergo apoptosis in response to clinically relevant doses of hydroxyurea. Using the RAF inhibitor LY3009120, we show that RAF activity determines the sensitivity of leukemic cells toward hydroxyurea. We further disclose that pharmacological inhibition of the RAF downstream target BCL-XL with the drug navitoclax and RNAi combine favorably with hydroxyurea against leukemic cells. BCR-ABL1 and hyperactive FLT3 are tyrosine kinases that causally contribute to the development of leukemia and induce RAF1 and BCL-XL. Accordingly, the ABL inhibitor imatinib and the FLT3 inhibitor quizartinib sensitize leukemic cells to pro-apoptotic effects of hydroxyurea. Moreover, hydroxyurea and navitoclax kill leukemic cells with mutant FLT3 that are resistant to quizartinib. These data reveal cellular susceptibility factors toward hydroxyurea and how they can be exploited to eliminate difficult-to-treat leukemic cells with clinically relevant drug combinations.
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http://dx.doi.org/10.3390/cancers13143464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8304262PMC
July 2021

The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1.

Nat Commun 2021 05 11;12(1):2668. Epub 2021 May 11.

Institute of Molecular Biology (IMB), Mainz, Germany.

Telomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.
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http://dx.doi.org/10.1038/s41467-021-22861-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113555PMC
May 2021

The In Vitro Interaction of 12-Oxophytodienoic Acid and Related Conjugated Carbonyl Compounds with Thiol Antioxidants.

Biomolecules 2021 03 18;11(3). Epub 2021 Mar 18.

Department of Biochemistry and Physiology of Plants, Faculty of Biology, University of Bielefeld, 33615 Bielefeld, Germany.

α,β-unsaturated carbonyls interfere with numerous plant physiological processes. One mechanism of action is their reactivity toward thiols of metabolites like cysteine and glutathione (GSH). This work aimed at better understanding these interactions. Both 12-oxophytodienoic acid (12-OPDA) and abscisic acid (ABA) conjugated with cysteine. It was found that the reactivity of α,β-unsaturated carbonyls with GSH followed the sequence trans-2-hexenal < 12-OPDA ≈ 12-OPDA-ethylester < 2-cyclopentenone << methyl vinylketone (MVK). Interestingly, GSH, but not ascorbate (vitamin C), supplementation ameliorated the phytotoxic potential of MVK. In addition, 12-OPDA and 12-OPDA-related conjugated carbonyl compounds interacted with proteins, e.g., with members of the thioredoxin (TRX)-fold family. 12-OPDA modified two cysteinyl residues of chloroplast TRX-f1. The OPDAylated TRX-f1 lost its activity to activate the Calvin-Benson-cycle enzyme fructose-1,6-bisphosphatase (FBPase). Finally, we show that 12-OPDA interacts with cyclophilin 20-3 (Cyp20-3) non-covalently and affects its peptidyl-prolyl-cis/trans isomerase activity. The results demonstrate the high potential of 12-OPDA as a diverse interactor and cellular regulator and suggest that OPDAylation may occur in plant cells and should be investigated as novel regulatory mechanism.
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http://dx.doi.org/10.3390/biom11030457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003295PMC
March 2021

TbSAP is a novel chromatin protein repressing metacyclic variant surface glycoprotein expression sites in bloodstream form Trypanosoma brucei.

Nucleic Acids Res 2021 04;49(6):3242-3262

Sir Alexander Fleming Building, Department of Life Sciences, Imperial College London, South Kensington, London SW7 2AZ, UK.

The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective variant surface glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of ∼15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more 'metacyclic-like'. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei.
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http://dx.doi.org/10.1093/nar/gkab109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034637PMC
April 2021

Characterization of the novel mitochondrial genome segregation factor TAP110 in .

J Cell Sci 2021 03 8;134(5). Epub 2021 Mar 8.

Institute of Cell Biology, University of Bern, 3012 Bern, Switzerland

Proper mitochondrial genome inheritance is important for eukaryotic cell survival. , a protozoan parasite, contains a singular mitochondrial genome, the kinetoplast (k)DNA. The kDNA is anchored to the basal body via the tripartite attachment complex (TAC) to ensure proper segregation. Several components of the TAC have been described; however, the connection of the TAC to the kDNA remains elusive. Here, we characterize the TAC-associated protein TAP110. We find that both depletion and overexpression of TAP110 leads to a delay in the separation of the replicated kDNA networks. Proteome analysis after TAP110 overexpression identified several kDNA-associated proteins that changed in abundance, including a TEX-like protein that dually localizes to the nucleus and the kDNA, potentially linking replication and segregation in the two compartments. The assembly of TAP110 into the TAC region seems to require the TAC but not the kDNA itself; however, once TAP110 has been assembled, it also interacts with the kDNA. Finally, we use ultrastructure expansion microscopy in trypanosomes for the first time, and reveal the precise position of TAP110 between TAC102 and the kDNA, showcasing the potential of this approach.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.254300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7970207PMC
March 2021

The RNA polymerase II subunit RPB-9 recruits the integrator complex to terminate Caenorhabditis elegans piRNA transcription.

EMBO J 2021 Mar 3;40(5):e105565. Epub 2021 Feb 3.

Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK.

PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. We show that rpb-9 initiates heritable piRNA-mediated gene silencing at two DNA transposon families and at a subset of somatic genes in the germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis by recruiting the Integrator complex at piRNA genes, hence promoting transcriptional termination. We conclude that, as a part of its rapid evolution, the piRNA pathway has co-opted an ancient machinery for high-fidelity transcription.
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http://dx.doi.org/10.15252/embj.2020105565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917558PMC
March 2021

Parasite Presence Induces Gene Expression Changes in an Ant Host Related to Immunity and Longevity.

Genes (Basel) 2021 01 13;12(1). Epub 2021 Jan 13.

Institute of Organismic and Molecular Evolution, Johannes Gutenberg University Mainz, 55128 Mainz, Germany.

Most species are either parasites or exploited by parasites, making parasite-host interactions a driver of evolution. Parasites with complex life cycles often evolve strategies to facilitate transmission to the definitive host by manipulating their intermediate host. Such manipulations could explain phenotypic changes in the ant , the intermediate host of the cestode . In addition to behavioral and morphological alterations, infected workers exhibit prolonged lifespans, comparable to that of queens, which live up to two decades. We used transcriptomic data from cestodes and ants of different castes and infection status to investigate the molecular underpinnings of phenotypic alterations in infected workers and explored whether the extended lifespan of queens and infected workers has a common molecular basis. Infected workers and queens commonly upregulated only six genes, one of them with a known anti-aging function. Both groups overexpressed immune genes, although not the same ones. Our findings suggest that the lifespan extension of infected workers is not achieved via the expression of queen-specific genes. The analysis of the cestodes' transcriptome revealed dominant expression of genes of the mitochondrial respiratory transport chain, which indicates an active metabolism and shedding light on the physiology of the parasite in its cysticercoid stage.
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http://dx.doi.org/10.3390/genes12010095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828512PMC
January 2021

Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila.

EMBO J 2021 Feb 11;40(4):e104975. Epub 2021 Jan 11.

Center for Integrative Genomics, Génopode Building, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland.

N6-methyladenosine (m A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m A alters fly behavior, albeit the underlying molecular mechanism and the role of m A during nervous system development have remained elusive. Here we find that impairment of the m A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.
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http://dx.doi.org/10.15252/embj.2020104975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883056PMC
February 2021

Chromatin modifiers and recombination factors promote a telomere fold-back structure, that is lost during replicative senescence.

PLoS Genet 2020 12 28;16(12):e1008603. Epub 2020 Dec 28.

Institute of Developmental Biology and Neurobiology (IDN), Johannes Gutenberg-Universität, Mainz, Germany.

Telomeres have the ability to adopt a lariat conformation and hence, engage in long and short distance intra-chromosome interactions. Budding yeast telomeres were proposed to fold back into subtelomeric regions, but a robust assay to quantitatively characterize this structure has been lacking. Therefore, it is not well understood how the interactions between telomeres and non-telomeric regions are established and regulated. We employ a telomere chromosome conformation capture (Telo-3C) approach to directly analyze telomere folding and its maintenance in S. cerevisiae. We identify the histone modifiers Sir2, Sin3 and Set2 as critical regulators for telomere folding, which suggests that a distinct telomeric chromatin environment is a major requirement for the folding of yeast telomeres. We demonstrate that telomeres are not folded when cells enter replicative senescence, which occurs independently of short telomere length. Indeed, Sir2, Sin3 and Set2 protein levels are decreased during senescence and their absence may thereby prevent telomere folding. Additionally, we show that the homologous recombination machinery, including the Rad51 and Rad52 proteins, as well as the checkpoint component Rad53 are essential for establishing the telomere fold-back structure. This study outlines a method to interrogate telomere-subtelomere interactions at a single unmodified yeast telomere. Using this method, we provide insights into how the spatial arrangement of the chromosome end structure is established and demonstrate that telomere folding is compromised throughout replicative senescence.
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http://dx.doi.org/10.1371/journal.pgen.1008603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793543PMC
December 2020

Fertility Relevance Probability Analysis Shortlists Genetic Markers for Male Fertility Impairment.

Cytogenet Genome Res 2020 25;160(9):506-522. Epub 2020 Nov 25.

Anthropology, Institute of Organismic and Molecular Evolution (iomE), Johannes Gutenberg University Mainz, Mainz, Germany,

Impairment of male fertility is one of the major public health issues worldwide. Nevertheless, genetic causes of male sub- and infertility can often only be suspected due to the lack of reliable and easy-to-use routine tests. Yet, the development of a marker panel is complicated by the large quantity of potentially predictive markers. Actually, hundreds or even thousands of genes could have fertility relevance. Thus, a systematic method enabling a selection of the most predictive markers out of the many candidates is required. As a criterion for marker selection, we derived a gene-specific score, which we refer to as fertility relevance probability (FRP). For this purpose, we first categorized 2,753 testis-expressed genes as either candidate markers or non-candidates, according to phenotypes in male knockout mice. In a parallel approach, 2,502 genes were classified as candidate markers or non-candidates based on phenotypes in men. Subsequently, we conducted logistic regression analyses with evolutionary rates of genes (dN/dS), transcription levels in testis relative to other organs, and connectivity of the encoded proteins in a protein-protein interaction network as covariates. In confirmation of the procedure, FRP values showed the expected pattern, thus being overall higher in genes with known relevance for fertility than in their counterparts without corresponding evidence. In addition, higher FRP values corresponded with an increased dysregulation of protein abundance in spermatozoa of 37 men with normal and 38 men with impaired fertility. Present analyses resulted in a ranking of genes according to their probable predictive power as candidate markers for male fertility impairment. Thus, AKAP4, TNP1, DAZL, BRDT, DMRT1, SPO11, ZPBP, HORMAD1, and SMC1B are prime candidates toward a marker panel for male fertility impairment. Additional candidate markers are DDX4, SHCBP1L, CCDC155, ODF1, DMRTB1, ASZ1, BOLL, FKBP6, SLC25A31, PRSS21, and RNF17. FRP inference additionally provides clues for potential new markers, thereunder TEX37 and POU4F2. The results of our logistic regression analyses are freely available at the PreFer Genes website (https://prefer-genes.uni-mainz.de/).
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http://dx.doi.org/10.1159/000511117DOI Listing
December 2020

Intrinsically disordered protein PID-2 modulates Z granules and is required for heritable piRNA-induced silencing in the Caenorhabditis elegans embryo.

EMBO J 2021 Feb 24;40(3):e105280. Epub 2020 Nov 24.

Biology of Non-coding RNA Group, Institute of Molecular Biology (IMB), Mainz, Germany.

In Caenorhabditis elegans, the piRNA (21U RNA) pathway is required to establish proper gene regulation and an immortal germline. To achieve this, PRG-1-bound 21U RNAs trigger silencing mechanisms mediated by RNA-dependent RNA polymerase (RdRP)-synthetized 22G RNAs. This silencing can become PRG-1-independent and heritable over many generations, a state termed RNA-induced epigenetic gene silencing (RNAe). How and when RNAe is established, and how it is maintained, is not known. We show that maternally provided 21U RNAs can be sufficient for triggering RNAe in embryos. Additionally, we identify PID-2, a protein containing intrinsically disordered regions (IDRs), as a factor required for establishing and maintaining RNAe. PID-2 interacts with two newly identified and partially redundant eTudor domain-containing proteins, PID-4 and PID-5. PID-5 has an additional domain related to the X-prolyl aminopeptidase APP-1, and binds APP-1, implicating potential N-terminal proteolysis in RNAe. All three proteins are required for germline immortality, localize to perinuclear foci, affect size and appearance of RNA inheritance-linked Z granules, and are required for balancing of 22G RNA populations. Overall, our study identifies three new proteins with crucial functions in C. elegans small RNA silencing.
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http://dx.doi.org/10.15252/embj.2020105280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849312PMC
February 2021

Correction to: Proteotranscriptomics assisted gene annotation and spatial proteomics of Bombyx mori BmN4 cell line.

BMC Genomics 2020 Nov 12;21(1):790. Epub 2020 Nov 12.

Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany.

An amendment to this paper has been published and can be accessed via the original article.
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http://dx.doi.org/10.1186/s12864-020-07211-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659087PMC
November 2020

PAR-TERRA is the main contributor to telomeric repeat-containing RNA transcripts in normal and cancer mouse cells.

RNA 2021 01 30;27(1):106-121. Epub 2020 Oct 30.

Genetic and Epigenetic Alterations of Genomes, de Duve Institute, Université catholique de Louvain (UCLouvain), 1200 Brussels, Belgium.

Telomeric repeat-containing RNA (TERRA) molecules play important roles at telomeres, from heterochromatin regulation to telomerase activity control. In human cells, TERRA is transcribed from subtelomeric promoters located on most chromosome ends and associates with telomeres. The origin of mouse TERRA molecules is, however, unclear, as transcription from the pseudoautosomal PAR locus was recently suggested to account for the vast majority of TERRA in embryonic stem cells (ESC). Here, we confirm the production of TERRA from both the chromosome 18q telomere and the PAR locus in mouse embryonic fibroblasts, ESC, and various mouse cancer and immortalized cell lines, and we identify two novel sources of TERRA on mouse chromosome 2 and X. Using various approaches, we show that PAR-TERRA molecules account for the majority of TERRA transcripts, displaying an increase of two to four orders of magnitude compared to the telomeric 18q transcript. Finally, we present a SILAC-based pull-down screen revealing a large overlap between TERRA-interacting proteins in human and mouse cells, including PRC2 complex subunits, chromatin remodeling factors, DNA replication proteins, Aurora kinases, shelterin complex subunits, Bloom helicase, Coilin, and paraspeckle proteins. Hence, despite originating from distinct genomic regions, mouse and human TERRA are likely to play similar functions in cells.
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http://dx.doi.org/10.1261/rna.076281.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749631PMC
January 2021

Proteotranscriptomics assisted gene annotation and spatial proteomics of Bombyx mori BmN4 cell line.

BMC Genomics 2020 Oct 6;21(1):690. Epub 2020 Oct 6.

Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany.

Background: The process of identifying all coding regions in a genome is crucial for any study at the level of molecular biology, ranging from single-gene cloning to genome-wide measurements using RNA-seq or mass spectrometry. While satisfactory annotation has been made feasible for well-studied model organisms through great efforts of big consortia, for most systems this kind of data is either absent or not adequately precise.

Results: Combining in-depth transcriptome sequencing and high resolution mass spectrometry, we here use proteotranscriptomics to improve gene annotation of protein-coding genes in the Bombyx mori cell line BmN4 which is an increasingly used tool for the analysis of piRNA biogenesis and function. Using this approach we provide the exact coding sequence and evidence for more than 6200 genes on the protein level. Furthermore using spatial proteomics, we establish the subcellular localization of thousands of these proteins. We show that our approach outperforms current Bombyx mori annotation attempts in terms of accuracy and coverage.

Conclusions: We show that proteotranscriptomics is an efficient, cost-effective and accurate approach to improve previous annotations or generate new gene models. As this technique is based on de-novo transcriptome assembly, it provides the possibility to study any species also in the absence of genome sequence information for which proteogenomics would be impossible.
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http://dx.doi.org/10.1186/s12864-020-07088-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541253PMC
October 2020

Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation.

Nucleic Acids Res 2020 09;48(17):9660-9680

Department of Cell & Developmental Biology, Biocenter, University of Würzburg, Würzburg, Germany.

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring 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.
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http://dx.doi.org/10.1093/nar/gkaa686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515707PMC
September 2020

Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei.

PLoS Biol 2020 06 10;18(6):e3000741. Epub 2020 Jun 10.

Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Ceske Budejovice, Czech Republic.

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.
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http://dx.doi.org/10.1371/journal.pbio.3000741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307792PMC
June 2020

The RNA fold interactome of evolutionary conserved RNA structures in S. cerevisiae.

Nat Commun 2020 06 3;11(1):2789. Epub 2020 Jun 3.

Institute of Molecular Biology, Ackermannweg 4, 55128, Mainz, Germany.

RNA-binding proteins play key roles in regulation of gene expression via recognition of structural features in RNA molecules. Here we apply a quantitative RNA pull-down approach to 186 evolutionary conserved RNA structures and report 162 interacting proteins. Unlike global RNA interactome capture, we associate individual RNA structures within messenger RNA with their interacting proteins. Of our binders 69% are known RNA-binding proteins, whereas some are previously unrelated to RNA binding and do not harbor canonical RNA-binding domains. While current knowledge about RNA-binding proteins relates to their functions at 5' or 3'-UTRs, we report a significant number of them binding to RNA folds in the coding regions of mRNAs. Using an in vivo reporter screen and pulsed SILAC, we characterize a subset of mRNA-RBP pairs and thus connect structural RNA features to functionality. Ultimately, we here present a generic, scalable approach to interrogate the increasing number of RNA structural motifs.
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http://dx.doi.org/10.1038/s41467-020-16555-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270185PMC
June 2020

Misactivation of multiple starvation responses in yeast by loss of tRNA modifications.

Nucleic Acids Res 2020 07;48(13):7307-7320

Institut für Biologie, Fachgebiet Mikrobiologie, Universität Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany.

Previously, combined loss of different anticodon loop modifications was shown to impair the function of distinct tRNAs in Saccharomyces cerevisiae. Surprisingly, each scenario resulted in shared cellular phenotypes, the basis of which is unclear. Since loss of tRNA modification may evoke transcriptional responses, we characterized global transcription patterns of modification mutants with defects in either tRNAGlnUUG or tRNALysUUU function. We observe that the mutants share inappropriate induction of multiple starvation responses in exponential growth phase, including derepression of glucose and nitrogen catabolite-repressed genes. In addition, autophagy is prematurely and inadequately activated in the mutants. We further demonstrate that improper induction of individual starvation genes as well as the propensity of the tRNA modification mutants to form protein aggregates are diminished upon overexpression of tRNAGlnUUG or tRNALysUUU, the tRNA species that lack the modifications of interest. Hence, our data suggest that global alterations in mRNA translation and proteostasis account for the transcriptional stress signatures that are commonly triggered by loss of anticodon modifications in different tRNAs.
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http://dx.doi.org/10.1093/nar/gkaa455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367188PMC
July 2020

Autophagy interferes with human cytomegalovirus genome replication, morphogenesis, and progeny release.

Autophagy 2021 03 1;17(3):779-795. Epub 2020 Mar 1.

Institute for Virology , University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany.

Viral infections are often accompanied by the induction of autophagy as an intrinsic cellular defense mechanism. Herpesviruses have developed strategies to evade autophagic degradation and to manipulate autophagy of the host cells to their benefit. Here we addressed the role of macroautophagy/autophagy in human cytomegalovirus replication and for particle morphogenesis. We found that proteins of the autophagy machinery localize to cytoplasmic viral assembly compartments and enveloped virions in the cytoplasm. Surprisingly, the autophagy receptor SQSTM1/p62 was also found to colocalize with HCMV capsids in the nucleus of infected cells. This finding indicates that the autophagy machinery interacts with HCMV already at the early nuclear stages of particle morphogenesis. The membrane-bound form of LC3 and several autophagy receptors were packaged into extracellular HCMV virions. This suggested that autophagic membranes were included during secondary envelopment of HCMV virions. To further address the importance of autophagy in HCMV infection, we generated an HCMV mutant that expressed a dominant-negative version of the protease ATG4B (BAD-ATG4B). The proteolytic activity of ATG4B is required for LC3 cleavage, priming it for membrane conjugation. Surprisingly, both genome replication and virus release were enhanced in cells infected with BAD-ATG4B, compared to control strains. These results show that autophagy operates as an antiviral process during HCMV infection but is dispensable for secondary HCMV particle envelopment. ATG: autophagy-related; BAC: bacterial artificial chromosome; BECN1: beclin 1; CPE: cytopathic effect; cVACs: cytoplasmic viral assembly compartments; d.p.i.: days post-infection; DB: dense body; EBV: Epstein-Barr virus; galK: galactokinase; HCMV: human cytomegalovirus; HFF: human foreskin fibroblasts; IE: immediate-early; IRS: internal repeat short; LC3: MAP1LC3A/B; m.o.i.; multiplicity of infection; MCP: major capsid protein; Pp: phosphoprotein; sCP/UL48a: smallest capsid protein; TRS: terminal repeat short; UL: unique long; US: unique short.
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http://dx.doi.org/10.1080/15548627.2020.1732686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032242PMC
March 2021

Quantitative Proteomics to Identify Nuclear RNA-Binding Proteins of Malat1.

Int J Mol Sci 2020 Feb 10;21(3). Epub 2020 Feb 10.

Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany.

The long non-coding RNA Malat1 has been implicated in several human cancers, while the mechanism of action is not completely understood. As RNAs in cells function together with RNA-binding proteins (RBPs), the composition of their RBP complex can shed light on their functionality. We here performed quantitative interactomics of 14 non-overlapping fragments covering the full length of Malat1 to identify possible nuclear interacting proteins. Overall, we identified 35 candidates including 14 already known binders, which are able to interact with Malat1 in the nucleus. Furthermore, the use of fragments along the full-length RNA allowed us to reveal two hotspots for protein binding, one in the 5'-region and one in the 3'-region of Malat1. Our results provide confirmation on previous RNA-protein interaction studies and suggest new candidates for functional investigations.
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http://dx.doi.org/10.3390/ijms21031166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037011PMC
February 2020

Npl3 stabilizes R-loops at telomeres to prevent accelerated replicative senescence.

EMBO Rep 2020 03 6;21(3):e49087. Epub 2020 Feb 6.

Institute of Molecular Biology (IMB), Mainz, Germany.

Telomere shortening rates must be regulated to prevent premature replicative senescence. TERRA R-loops become stabilized at critically short telomeres to promote their elongation through homology-directed repair (HDR), thereby counteracting senescence onset. Using a non-bias proteomic approach to detect telomere binding factors, we identified Npl3, an RNA-binding protein previously implicated in multiple RNA biogenesis processes. Using chromatin immunoprecipitation and RNA immunoprecipitation, we demonstrate that Npl3 interacts with TERRA and telomeres. Furthermore, we show that Npl3 associates with telomeres in an R-loop-dependent manner, as changes in R-loop levels, for example, at short telomeres, modulate the recruitment of Npl3 to chromosome ends. Through a series of genetic and biochemical approaches, we reveal that Npl3 binds to TERRA and stabilizes R-loops at short telomeres, which in turn promotes HDR and prevents premature replicative senescence onset. This may have implications for diseases associated with excessive telomere shortening.
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http://dx.doi.org/10.15252/embr.201949087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054685PMC
March 2020

Histone deacetylase inhibitors dysregulate DNA repair proteins and antagonize metastasis-associated processes.

J Cancer Res Clin Oncol 2020 Feb 13;146(2):343-356. Epub 2020 Jan 13.

Department of Toxicology, University Medical Center of the Johannes Gutenberg University Mainz, Obere Zahlbacher Straße 67, 55131, Mainz, Germany.

Purpose: We set out to determine whether clinically tested epigenetic drugs against class I histone deacetylases (HDACs) affect hallmarks of the metastatic process.

Methods: We treated permanent and primary renal, lung, and breast cancer cells with the class I histone deacetylase inhibitors (HDACi) entinostat (MS-275) and valproic acid (VPA), the replicative stress inducer hydroxyurea (HU), the DNA-damaging agent cis-platinum (L-OHP), and the cytokine transforming growth factor-β (TGFβ). We used proteomics, quantitative PCR, immunoblot, single cell DNA damage assays, and flow cytometry to analyze cell fate after drug exposure.

Results: We show that HDACi interfere with DNA repair protein expression and trigger DNA damage and apoptosis alone and in combination with established chemotherapeutics. Furthermore, HDACi disrupt the balance of cell adhesion protein expression and abrogate TGFβ-induced cellular plasticity of transformed cells.

Conclusion: HDACi suppress the epithelial-mesenchymal transition (EMT) and compromise the DNA integrity of cancer cells. These data encourage further testing of HDACi against tumor cells.
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http://dx.doi.org/10.1007/s00432-019-03118-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985217PMC
February 2020

The PHIST protein GEXP02 targets the host cytoskeleton during sexual development of Plasmodium falciparum.

Cell Microbiol 2020 02 4;22(2):e13123. Epub 2019 Dec 4.

Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland.

A hallmark of the biology of Plasmodium falciparum blood stage parasites is their extensive host cell remodelling, facilitated by parasite proteins that are exported into the erythrocyte. Although this area has received extensive attention, only a few exported parasite proteins have been analysed in detail, and much of this remodelling process remains unknown, particularly for gametocyte development. Recent advances to induce high rates of sexual commitment enable the production of large numbers of gametocytes. We used this approach to study the Plasmodium helical interspersed subtelomeric (PHIST) protein GEXP02, which is expressed during sexual development. We show by immunofluorescence that GEXP02 is exported to the gametocyte-infected host cell periphery. Co-immunoprecipitation revealed potential interactions between GEXP02 and components of the erythrocyte cytoskeleton as well as other exported parasite proteins. This indicates that GEXP02 targets the erythrocyte cytoskeleton and is likely involved in its remodelling. GEXP02 knock-out parasites show no obvious phenotype during gametocyte maturation, transmission through mosquitoes, and hepatocyte infection, suggesting auxiliary or redundant functions for this protein. In summary, we performed a detailed cellular and biochemical analysis of a sexual stage-specific exported parasite protein using a novel experimental approach that is broadly applicable to study the biology of P. falciparum gametocytes.
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http://dx.doi.org/10.1111/cmi.13123DOI Listing
February 2020

PETISCO is a novel protein complex required for 21U RNA biogenesis and embryonic viability.

Genes Dev 2019 07 30;33(13-14):857-870. Epub 2019 May 30.

Biology of Non-coding RNA Group, Institute of Molecular Biology, 55128 Mainz, Germany.

Piwi proteins are important for germ cell development in most animals. These proteins are guided to specific targets by small guide RNAs, referred to as piRNAs or 21U RNAs in In this organism, even though genetic screens have uncovered 21U RNA biogenesis factors, little is known about how these factors interact or what they do. Based on the previously identified 21U biogenesis factor PID-1 (piRNA-induced silencing-defective 1), we here define a novel protein complex, PETISCO (PID-3, ERH-2, TOFU-6, and IFE-3 small RNA complex), that is required for 21U RNA biogenesis. PETISCO contains both potential 5' cap and 5' phosphate RNA-binding domains and interacts with capped 21U precursor RNA. We resolved the architecture of PETISCO and revealed a second function for PETISCO in embryonic development. This essential function of PETISCO is mediated not by PID-1 but by the novel protein TOST-1 (twenty-one U pathway antagonist). In contrast, TOST-1 is not essential for 21U RNA biogenesis. Both PID-1 and TOST-1 interact directly with ERH-2 using a conserved sequence motif. Finally, our data suggest a role for TOST-1:PETISCO in SL1 homeostasis in the early embryo. Our work describes a key complex for 21U RNA processing in and strengthens the view that 21U RNA biogenesis is built on an snRNA-related pathway.
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http://dx.doi.org/10.1101/gad.322446.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601512PMC
July 2019

PU.1 controls fibroblast polarization and tissue fibrosis.

Nature 2019 02 30;566(7744):344-349. Epub 2019 Jan 30.

Department of Chemistry, Georgia State University, Atlanta, GA, USA.

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs.
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http://dx.doi.org/10.1038/s41586-019-0896-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526281PMC
February 2019

ZBTB10 binds the telomeric variant repeat TTGGGG and interacts with TRF2.

Nucleic Acids Res 2019 02;47(4):1896-1907

Quantitative Proteomics, Institute of Molecular Biology, 55128 Mainz, Germany.

Telomeres are nucleoprotein structures at the ends of linear chromosomes and present an essential feature for genome integrity. Vertebrate telomeres usually consist of hexameric TTAGGG repeats, however, in cells that use the alternative lengthening of telomeres (ALT) mechanism, variant repeat sequences are interspersed throughout telomeres. Previously, it was shown that NR2C/F transcription factors bind to TCAGGG variant repeats and contribute to telomere maintenance in ALT cells. While specific binders to other variant repeat sequences have been lacking to date, we here identify ZBTB10 as the first TTGGGG-binding protein and demonstrate direct binding via the two zinc fingers with affinity in the nanomolar range. Concomitantly, ZBTB10 co-localizes with a subset of telomeres in ALT-positive U2OS cells and interacts with TRF2/RAP1 via the N-terminal region of TRF2. Our data establishes ZBTB10 as a novel variant repeat binding protein at ALT telomeres.
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http://dx.doi.org/10.1093/nar/gky1289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393293PMC
February 2019

Human platelet lysate as validated replacement for animal serum to assess chemosensitivity.

ALTEX 2019 14;36(2):277-288. Epub 2018 Dec 14.

Department of Toxicology, University Medical Center, Mainz, Germany.

Experiments with cultured mammalian cells represent an in vitro alternative to animal experiments. Fetal calf serum (FCS) is the most commonly used media supplement worldwide. FCS contains a mixture of largely undefined growth factors and cytokines, which support cell proliferation. This undefined nature of FCS is a source of experimental variation, undesired immune responses, possible contaminations, and because of its way of production an ethical concern. Thus, alternative, defined, valid, and reliable media supplements should be characterized in a large number of experiments. Human platelet lysate (hPL) is increasingly appreciated as an alternative to FCS. Since it is unclear whether cells respond differentially to clinically relevant chemotherapeutics inducing replicative stress and DNA damage (Hydroxyurea, Irinotecan), induction of reactive oxygen species (ROS), the tyrosine kinase inhibitor (TKi) Imatinib, and novel epigenetic modifiers belonging to the group of histone deacetylase inhibitors (HDACi), we investigated these issues. Here we show that cancer cells derived from leukemia and colon cancer grow very similarly in culture media with FCS or outdated hPL. Notably, cells have practically identical proteomes under both culture conditions. Moreover, cells grown with FCS or hPL respond equally to all types of drugs and stress conditions that we have tested. In addition, the transfection of blood cells by electroporation can be achieved under both conditions. Furthermore, we reveal that class I HDACs, but not HDAC6, are required for the expression of the pan-leukemic marker WT1 under various culture conditions. Hence, hPL is a moderately priced substitute for FCS in various experimental settings.
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http://dx.doi.org/10.14573/altex.1809211DOI Listing
August 2019

A DNA methylation reader complex that enhances gene transcription.

Science 2018 12;362(6419):1182-1186

Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA.

DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression. In either case, the downstream factors remain largely unknown. By using comparative interactomics, we isolated proteins in that associate with methylated DNA. Two SU(VAR)3-9 homologs, the transcriptional antisilencing factor SUVH1, and SUVH3, were among the methyl reader candidates. SUVH1 and SUVH3 bound methylated DNA in vitro, were associated with euchromatic methylation in vivo, and formed a complex with two DNAJ domain-containing homologs, DNAJ1 and DNAJ2. Ectopic recruitment of DNAJ1 enhanced gene transcription in plants, yeast, and mammals. Thus, the SUVH proteins bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes.
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http://dx.doi.org/10.1126/science.aar7854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353633PMC
December 2018

Quantifying post-transcriptional regulation in the development of Drosophila melanogaster.

Nat Commun 2018 11 26;9(1):4970. Epub 2018 Nov 26.

Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany.

Even though proteins are produced from mRNA, the correlation between mRNA levels and protein abundances is moderate in most studies, occasionally attributed to complex post-transcriptional regulation. To address this, we generate a paired transcriptome/proteome time course dataset with 14 time points during Drosophila embryogenesis. Despite a limited mRNA-protein correlation (ρ = 0.54), mathematical models describing protein translation and degradation explain 84% of protein time-courses based on the measured mRNA dynamics without assuming complex post transcriptional regulation, and allow for classification of most proteins into four distinct regulatory scenarios. By performing an in-depth characterization of the putatively post-transcriptionally regulated genes, we postulate that the RNA-binding protein Hrb98DE is involved in post-transcriptional control of sugar metabolism in early embryogenesis and partially validate this hypothesis using Hrb98DE knockdown. In summary, we present a systems biology framework for the identification of post-transcriptional gene regulation from large-scale, time-resolved transcriptome and proteome data.
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http://dx.doi.org/10.1038/s41467-018-07455-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6255845PMC
November 2018
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