Abstract
Monoallelic exclusion ensures that the African trypanosome Trypanosoma brucei exclusively expresses only 1 of thousands of different variant surface glycoprotein (VSG) coat genes. The active VSG is transcribed from 1 of 15 polycistronic bloodstream-form VSG expression sites (ESs), which are controlled in a mutually exclusive fashion. Unusually, T. brucei uses RNA polymerase I (Pol I) to transcribe the active ES, which is unprecedented among eukaryotes. This active ES is located within a unique extranucleolar Pol I body called the expression-site body (ESB). A stringent restriction mechanism prevents T. brucei from expressing multiple ESs at the same time, although how this is mediated is unclear. By using drug-selection pressure, we generated VSG double-expresser T. brucei lines, which have disrupted monoallelic exclusion, and simultaneously express 2 ESs in a dynamic fashion. The 2 unstably active ESs appear epigenetically similar to fully active ESs as determined by using chromatin immunoprecipitation for multiple epigenetic marks (histones H3 and H1, TDP1, and DNA base J). We find that the double-expresser cells, similar to wild-type single-expresser cells, predominantly contain 1 subnuclear ESB, as determined using Pol I or the ESB marker VEX1. Strikingly, simultaneous transcription of the 2 dynamically transcribed ESs is normally observed only when the 2 ESs are both located within this single ESB. This colocalization is reversible in the absence of drug selection. This discovery that simultaneously active ESs dynamically share a single ESB demonstrates the importance of this unique subnuclear body in restricting the monoallelic expression of VSG.
Highlights
The monoallelic expression of 1 gene out of a large assortment of highly similar variants is a poorly understood phenomenon
Selection for reactivation of BES1 resulted in generation of the VSG221-expressing (221+)PG-VHC line
The growth rate of the DE KW01 line was equivalent to the parental line, whereas the DE BH03 line appeared to show a minor reduction in growth rate (SI Appendix, Fig. S1C)
Summary
The monoallelic expression of 1 gene out of a large assortment of highly similar variants is a poorly understood phenomenon. The African trypanosome Trypanosoma brucei expresses a single variant surface glycoprotein (VSG) gene from one of multiple VSG expression sites (ESs) in a stringent monoallelic fashion. Bloodstream-form T. brucei expresses only a single ES at a time, perturbation of ES monoallelic exclusion can be achieved by using drug-selection pressure [26, 27] We used this approach to generate independently derived T. brucei double-expresser strains that unstably and transiently express 2 ESs at the same time. We see simultaneous transcription of 2 ESs only when these are colocalized within a single ESB These results showing that the 2 dynamically transcribed ESs appear to continuously transition between a single shared ESB demonstrate the importance of this unique subnuclear body in restricting the monoallelic expression of VSG
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