title: Bloodstream form-specific up-regulation of silent vsg expression sites and procyclin in Trypanosoma brucei after inhibition of DNA synthesis or DNA damage.
abstract: The African trypanosome Trypanosoma brucei transcribes the active variant surface glycoprotein (VSG) gene from one of about 20 VSG expression sites (ESs). In order to study ES control, we made reporter lines with a green fluorescent protein gene inserted behind the promoter of different ESs. We attempted to disrupt the silencing machinery, and we used fluorescence-activated cell sorter analysis for the rapid and sensitive detection of ES up-regulation. We find that a range of treatments that either block nuclear DNA synthesis, like aphidicolin, or modify DNA-like cisplatin and 1-methyl-3-nitro-1-nitrosoguanidine results in up-regulation of silent ESs. Aphidicolin treatment was the most effective, with almost 80% of the cells expressing green fluorescent protein from a silent ES. All of these treatments blocked the cells in S phase. In contrast, a range of toxic chemicals had little or no effect on expression. These included berenil and pentamidine, which selectively cleave the mitochondrial kinetoplast DNA, the metabolic inhibitors suramin and difluoromethylornithine, and the mitotic inhibitor rhizoxin. Up-regulation also affected other RNA polymerase I (pol I) transcription units, as procyclin genes were also up-regulated after cells were treated with either aphidicolin or DNA-modifying agents. Strikingly, this up-regulation of silent pol I transcription units was bloodstream form-specific and was not observed in insect form T. brucei. We postulate that the redistribution of a limiting bloodstream form-specific factor involved in both silencing and DNA repair results in the derepression of normally silenced pol I transcription units after DNA damage.
authors.affiliation: The Peter Medawar Building for Pathogen Research, University of Oxford, South Parks Road, Oxford OX1 3SY, United Kingdom.
authors.forename: Karen
authors.initials: K
authors.lastname: Sheader
authors.forename: Daniëlle
authors.initials: D
authors.lastname: te Vruchte
authors.forename: Gloria
authors.initials: G
authors.lastname: Rudenko
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D016590:Aphidicolin / Q000494:pharmacology; D001769:Blood; D004249:DNA Damage; D004133:Diminazene / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020868:Gene Silencing; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics*; D010419:Pentamidine / Q000494:pharmacology; D015800:Protozoan Proteins; D012318:RNA Polymerase I / Q000378:metabolism; D016196:S Phase / Q000187:drug effects; D013498:Suramin / Q000494:pharmacology; D014158:Transcription, Genetic; D014344:Trypanocidal Agents / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D015854:Up-Regulation; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics*
chemical_list: D000981:Antiprotozoal Agents; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D014344:Trypanocidal Agents; D014643:Variant Surface Glycoproteins, Trypanosoma; C053277:procyclic acidic repetitive protein, Trypanosoma; D049452:Green Fluorescent Proteins; D016590:Aphidicolin; D013498:Suramin; D010419:Pentamidine; D012318:RNA Polymerase I; C003915:diminazene aceturate; D004133:Diminazene