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  • 1
    Language: English
    In: Nucleic acids research, 12 October 2018, Vol.46(18), pp.e106
    Description: Despite their importance for most DNA-templated processes, the function of individual histone modifications has remained largely unknown because in vivo mutational analyses are lacking. The reason for this is that histone genes are encoded by multigene families and that tools to simultaneously edit multiple genomic loci with high efficiency are only now becoming available. To overcome these challenges, we have taken advantage of the power of CRISPR-Cas9 for precise genome editing and of the fact that most DNA repair in the protozoan parasite Trypanosoma brucei occurs via homologous recombination. By establishing an episome-based CRISPR-Cas9 system for T. brucei, we have edited wild type cells without inserting selectable markers, inserted a GFP tag between an ORF and its 3'UTR, deleted both alleles of a gene in a single transfection, and performed precise editing of genes that exist in multicopy arrays, replacing histone H4K4 with H4R4 in the absence of detectable off-target effects. The newly established genome editing toolbox allows for the generation of precise mutants without needing to change other regions of the genome, opening up opportunities to study the role of individual histone modifications, catalytic sites of enzymes or the regulatory potential of UTRs in their endogenous environments.
    Keywords: Crispr-Cas Systems ; Gene Editing -- Methods ; Histone Code -- Genetics ; Histones -- Metabolism
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 2
    Language: English
    In: Analytical Chemistry, Oct 6, 2015, Vol.87(19), p.9939(7)
    Description: The article introduces fragment ion patchwork quantification as a new mass spectrometry-based approach for the highly accurate quantification of site-specific acetylation degrees. This method merges 13C1-acetyl derivatization on the protein level, proteolysis by low-specificity proteases and quantification on the fragment ion level. Moreover, it is shown that this method enables determination of site-specific acetylation degrees of all lysine residues for all core histones of Trypanosoma brucei.
    Keywords: Acetylation – Analysis ; Mass Spectrometry – Usage ; Patchwork – Research
    ISSN: 0003-2700
    Source: Cengage Learning, Inc.
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  • 3
    In: EMBO Journal, 01 September 2017, Vol.36(17), pp.2581-2594
    Description: Genome‐wide transcription studies are revealing an increasing number of “dispersed promoters” that, unlike “focused promoters”, lack well‐conserved sequence motifs and tight regulation. Dispersed promoters are nevertheless marked by well‐defined chromatin structures, suggesting that specific sequence elements must exist in these unregulated promoters. Here, we have analyzed regions of transcription initiation in the eukaryotic parasite in which polymerase transcription initiation occurs over broad regions without distinct promoter motifs and lacks regulation. Using a combination of site‐specific and genome‐wide assays, we identified ‐rich promoters that can drive transcription and promote the targeted deposition of the histone variant H2A.Z in a genomic context‐dependent manner. In addition, upon mapping nucleosome occupancy at high resolution, we find nucleosome positioning to correlate with pol enrichment and gene expression, pointing to a role in maturation. Nucleosome positioning may thus represent a previously unrecognized layer of gene regulation in trypanosomes. Our findings show that even highly dispersed, unregulated promoters contain specific elements that are able to induce transcription and changes in chromatin structure. The lack of recognisable promoters in trypanosomes has limited our understanding of transcriptional control in this important pathogen. Via genome‐wide assays this study shows that transcription initiates broadly on ‐rich elements that control incorporation of activating histone variants. RNA pol II transcription initiates across a 2 kb‐wide region upstream of polycistronic transcription units. GT‐rich sequence elements enriched at transcription start sites can induce transcription and recruit the histone variant H2A.Z. Sites enriched in H2A.Z show increased sensitivity to MNase. Nucleosome occupancy upstream of genes correlates with RNA pol II enrichment and transcript levels. Composition of polyY tract affects nucleosome positioning and transcript levels. While trypanosomes lack classical promoter elements, they initiate transcription broadly over ‐rich sequences that control incorporation of activating histone variants.
    Keywords: Core Promoter ; Histone Variant ; Nucleosome Occupancy ; Trypanosoma Brucei
    ISSN: 0261-4189
    E-ISSN: 1460-2075
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  • 4
    Language: English
    In: Analytical chemistry, 06 October 2015, Vol.87(19), pp.9939-45
    Description: We introduce fragment ion patchwork quantification as a new mass spectrometry-based approach for the highly accurate quantification of site-specific acetylation degrees. This method combines (13)C1-acetyl derivatization on the protein level, proteolysis by low-specificity proteases and quantification on the fragment ion level. Acetylation degrees are determined from the isotope patterns of acetylated b and y ions. We show that this approach allows to determine site-specific acetylation degrees of all lysine residues for all core histones of Trypanosoma brucei. In addition, we demonstrate how this approach can be used to identify substrate sites of histone acetyltransferases.
    Keywords: Histones -- Chemistry ; Lysine -- Analysis ; Trypanosoma Brucei Brucei -- Chemistry
    ISSN: 00032700
    E-ISSN: 1520-6882
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  • 5
    Language: English
    In: Nature, 18 September 2014, Vol.513(7518), pp.431-5
    Description: Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria. The mechanism determining upsA activation remains unknown. Here we show that an entirely new type of gene silencing mechanism involving an exonuclease-mediated degradation of nascent RNA controls the silencing of genes linked to severe malaria. We identify a novel chromatin-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsA var genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsA var transcripts and intron-derived antisense long non-coding RNA. The presence of stable upsA var transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of PfRNase II and upsA-type var genes in parasites from severe malaria patients, implying a crucial role of PfRNase II in severe malaria. Our results uncover a previously unknown type of post-transcriptional gene silencing mechanism in malaria parasites with repercussions for other organisms. Additionally, the identification of RNase II as a parasite protein controlling the expression of virulence genes involved in pathogenesis in patients with severe malaria may provide new strategies for reducing malaria mortality.
    Keywords: Gene Silencing ; Exoribonucleases -- Metabolism ; Genes, Protozoan -- Genetics ; Malaria, Cerebral -- Parasitology ; Plasmodium Falciparum -- Enzymology ; RNA, Protozoan -- Metabolism
    ISSN: 00280836
    E-ISSN: 1476-4687
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  • 6
    Language: English
    In: Nature, November 2018, Vol.563(7729), pp.121-125
    Description: Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses-Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing-that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.
    Keywords: Antigenic Variation -- Genetics ; Chromatin -- Genetics ; DNA, Protozoan -- Metabolism ; Genome -- Genetics ; Trypanosoma Brucei Brucei -- Genetics
    ISSN: 00280836
    E-ISSN: 1476-4687
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  • 7
    Language: English
    In: Nucleic acids research, September 2014, Vol.42(15), pp.9717-29
    Description: Base J, β-d-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA polymerase (RNAP) II initiation and termination. Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in Leishmania major and Trypanosoma brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination at the end of polycistronic gene clusters and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription termination and increased expression of downstream genes. Thus, J regulation of RNAP II transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates termination and gene expression at specific sites within polycistronic gene clusters.
    Keywords: Gene Expression Regulation ; Transcription Termination, Genetic ; Leishmania Major -- Genetics ; Trypanosoma Brucei Brucei -- Genetics ; Uracil -- Analogs & Derivatives
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 8
    Language: English
    In: Current Opinion in Microbiology, August, 2014, Vol.20, p.153(9)
    Description: To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.mib.2014.06.013 Byline: Shruthi S Vembar, Artur Scherf, T Nicolai Siegel Abstract: acents Small and long noncoding RNAs are abundant in Plasmodium falciparum blood stages. acents Subtelomeric and intronic ncRNAs may determine singular var gene choice. acents Natural antisense transcripts may regulate gene expression in an epigenetic manner. acents Adaptation of new technologies to Plasmodium is vital to studying ncRNA function. Author Affiliation: (1) Biology of Host-Parasite Interactions Unit, Institut Pasteur, Paris, France (2) CNRS URA2581, Paris, France (3) Research Center for Infectious Diseases, University of Wuerzburg, Josef-Schneider-Str. 2/Bau D15, 97080 Wuerzburg, Germany
    Keywords: Malaria -- Genetic Aspects ; Plasmodium Falciparum ; Epigenetic Inheritance ; Genetic Research ; Genes ; Gene Expression
    ISSN: 1369-5274
    Source: Cengage Learning, Inc.
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  • 9
    Language: English
    In: Nucleic acids research, 18 September 2015, Vol.43(16), pp.8013-32
    Description: RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function.
    Keywords: Cytoplasmic Granules -- Chemistry ; Protozoan Proteins -- Analysis ; Ribonucleoproteins -- Analysis
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 10
    Language: English
    In: Trends in Parasitology, 2011, Vol.27(10), pp.434-441
    Description: undergoes major biochemical and morphological changes during its development from the bloodstream form in the mammalian host to the procyclic form in the midgut of its insect host. The underlying regulation of gene expression, however, is poorly understood. More than 60% of the predicted genes remain annotated as hypothetical, and the 5′ and 3′ untranslated regions important for regulation of gene expression are unknown for 〉90% of the genes. In this review, we compare the data from four recently published high-throughput RNA sequencing studies in light of the different experimental setups and discuss how these data can enhance genome annotation and give insights into the regulation of gene expression in .
    Keywords: Biology ; Veterinary Medicine
    ISSN: 1471-4922
    E-ISSN: 1471-5007
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