scholarly journals Dynamic colocalization of 2 simultaneously active VSG expression sites within a single expression-site body in Trypanosoma brucei

2019 ◽  
Vol 116 (33) ◽  
pp. 16561-16570 ◽  
Author(s):  
James Budzak ◽  
Louise E. Kerry ◽  
Aris Aristodemou ◽  
Belinda S. Hall ◽  
Kathrin Witmer ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Chromatin Immunoprecipitation ◽  
Surface Glycoprotein ◽  
Monoallelic Expression ◽  
Drug Selection ◽  
Wild Type ◽  
Pol I ◽  
Dna Base ◽  
Expression Site ◽  
Polymerase I

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.

scholarly journals Quantification of RNA Polymerase I transcriptional attenuation at the active VSG expression site in Trypanosoma brucei

2021 ◽  
Author(s):  
Nadine Weisert ◽  
Klara Thein ◽  
Helena Reis ◽  
Christian J Janzen
Keyword(s):  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Transcriptional Elongation ◽  
Pol I ◽  
Expression Site ◽  
Polymerase I ◽  
Large Repertoire

The cell surface of the extracellular pathogen Trypanosoma brucei consists of a dense coat of variant surface glycoprotein (VSG), which enables the parasite to evade the immune system of the vertebrate host. Only one VSG gene from a large repertoire is expressed from a so-called bloodstream form expression site (BES) at a given timepoint. There are several BES in every parasite but only one is transcriptionally active. Other BES are silenced by transcriptional attenuation. Periodic activation of a previously-silenced BES results in differential VSG transcription and escape from the immune response. A process called antigenic variation. In contrast to gene transcription in other eukaryotes, the BES is transcribed by RNA polymerase I (Pol I). It was proposed that this highly-processive polymerase is needed to provide a sufficiently high transcription rate at the VSG gene. Surprisingly, we discovered a position-dependent Pol I activity and attenuation of transcriptional elongation also at the active BES. Transcription rates at the VSG gene appear to be comparable to Pol II-mediated transcription of house-keeping genes. Although these findings are in contradiction to the long-standing concept of continuously high transcription rates at the active BES in Trypanosoma brucei, they are complementary to recent groundbreaking findings about transcriptional regulation of VSG genes.

scholarly journals An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
James Budzak ◽  
Robert Jones ◽  
Christian Tschudi ◽  
Nikolay G. Kolev ◽  
Gloria Rudenko
Keyword(s):  
Rna Polymerase I ◽  
Nuclear Bodies ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Cajal Bodies ◽  
African Trypanosomes ◽  
Spliced Leader ◽  
Pol I ◽  
Expression Site ◽  
Polymerase I

AbstractA Variant Surface Glycoprotein (VSG) coat protects bloodstream form Trypanosoma brucei. Prodigious amounts of VSG mRNA (~7-10% total) are generated from a single RNA polymerase I (Pol I) transcribed VSG expression site (ES), necessitating extremely high levels of localised splicing. We show that splicing is required for processive ES transcription, and describe novel ES-associated T. brucei nuclear bodies. In bloodstream form trypanosomes, the expression site body (ESB), spliced leader array body (SLAB), NUFIP body and Cajal bodies all frequently associate with the active ES. This assembly of nuclear bodies appears to facilitate the extraordinarily high levels of transcription and splicing at the active ES. In procyclic form trypanosomes, the NUFIP body and SLAB do not appear to interact with the Pol I transcribed procyclin locus. The congregation of a restricted number of nuclear bodies at a single active ES, provides an attractive mechanism for how monoallelic ES transcription is mediated.

scholarly journals Promoter occupancy of the basal class I transcription factor A differs strongly between active and silent VSG expression sites in Trypanosoma brucei

2013 ◽  
Vol 42 (5) ◽  
pp. 3164-3176 ◽  
Author(s):  
Tu N. Nguyen ◽  
Laura S. M. Müller ◽  
Sung Hee Park ◽  
T. Nicolai Siegel ◽  
Arthur Günzl
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Transcription Initiation ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Class I ◽  
Monoallelic Expression ◽  
Promoter Occupancy ◽  
Polymerase I

Abstract Monoallelic expression within a gene family is found in pathogens exhibiting antigenic variation and in mammalian olfactory neurons. Trypanosoma brucei, a lethal parasite living in the human bloodstream, expresses variant surface glycoprotein (VSG) from 1 of 15 bloodstream expression sites (BESs) by virtue of a multifunctional RNA polymerase I. The active BES is transcribed in an extranucleolar compartment termed the expression site body (ESB), whereas silent BESs, located elsewhere within the nucleus, are repressed epigenetically. The regulatory mechanisms, however, are poorly understood. Here we show that two essential subunits of the basal class I transcription factor A (CITFA) predominantly occupied the promoter of the active BES relative to that of a silent BES, a phenotype that was maintained after switching BESs in situ. In these experiments, high promoter occupancy of CITFA was coupled to high levels of both promoter-proximal RNA abundance and RNA polymerase I occupancy. Accordingly, fluorescently tagged CITFA-7 was concentrated in the nucleolus and the ESB. Because a ChIP-seq analysis found that along the entire BES, CITFA-7 is specifically enriched only at the promoter, our data strongly indicate that monoallelic BES transcription is activated by a mechanism that functions at the level of transcription initiation.

scholarly journals Monoallelic antigen expression in trypanosomes requires a stage-specific transcription activator

2021 ◽  
Author(s):  
Lara Lopez Escobar ◽  
Benjamin Hanisch ◽  
Clare Halliday ◽  
Samuel Dean ◽  
Jack Sunter ◽  
...  
Keyword(s):  
Antigenic Variation ◽  
Single Gene ◽  
Antigen Expression ◽  
Specific Protein ◽  
Surface Glycoprotein ◽  
Monoallelic Expression ◽  
Transcription Activator ◽  
Transcript Processing ◽  
Pol I ◽  
Expression Site

Monoallelic expression of a single gene family member underpins a molecular “arms race” between many pathogens and their host, through host monoallelic immunoglobulin and pathogen monoallelic antigen expression. In Trypanosoma brucei, a single, abundant, variant surface glycoprotein (VSG) covers the entire surface of the bloodstream parasite and monoallelic VSG transcription underpins their archetypal example of antigenic variation. It is vital for pathogenicity, only occurring in mammalian infectious forms. Transcription of one VSG gene is achieved by RNA polymerase I (Pol I) in a singular nuclear structure: the expression site body (ESB). How monoallelic expression of the single VSG is achieved is incompletely understood and no specific ESB components are known. Here, using a protein localisation screen in bloodstream parasites, we discovered the first ESB-specific protein: ESB1. It is specific to VSG-expressing life cycle stages where it is necessary for VSG expression, and its overexpression activates inactive VSG promoters. This showed monoallelic VSG transcription requires a stage-specific activator. Furthermore, ESB1 is necessary for Pol I recruitment to the ESB, however transcript processing and inactive VSG gene exclusion ESB sub-domains do not require ESB1. This shows that the cellular solution for monoallelic transcription is a complex factory of functionally distinct and separably assembled sub-domains.

scholarly journals Nuclear repositioning of the VSG promoter during developmental silencing in Trypanosoma brucei

2007 ◽  
Vol 176 (2) ◽  
pp. 133-139 ◽  
Author(s):  
David Landeira ◽  
Miguel Navarro
Keyword(s):  
Trypanosoma Brucei ◽  
Transcriptional Activation ◽  
Promoter Region ◽  
Chromatin Condensation ◽  
Surface Protein ◽  
Model System ◽  
Surface Glycoprotein ◽  
Pol I ◽  
Expression Site ◽  
Developmental Differentiation

Interphase nuclear repositioning of chromosomes has been implicated in the epigenetic regulation of RNA polymerase (pol) II transcription. However, little is known about the nuclear position–dependent regulation of RNA pol I–transcribed loci. Trypanosoma brucei is an excellent model system to address this question because its two main surface protein genes, procyclin and variant surface glycoprotein (VSG), are transcribed by pol I and undergo distinct transcriptional activation or downregulation events during developmental differentiation. Although the monoallelically expressed VSG locus is exclusively localized to an extranucleolar body in the bloodstream form, in this study, we report that nonmutually exclusive procyclin genes are located at the nucleolar periphery. Interestingly, ribosomal DNA loci and pol I transcription activity are restricted to similar perinucleolar positions. Upon developmental transcriptional downregulation, however, the active VSG promoter selectively undergoes a rapid and dramatic repositioning to the nuclear envelope. Subsequently, the VSG promoter region was subjected to chromatin condensation. We propose a model whereby the VSG expression site pol I promoter is selectively targeted by temporal nuclear repositioning during developmental silencing.

Trypanosoma brucei: posttranscriptional control of the variable surface glycoprotein gene expression site

1989 ◽  
Vol 9 (9) ◽  
pp. 4018-4021
Author(s):  
E Pays ◽  
H Coquelet ◽  
A Pays ◽  
P Tebabi ◽  
M Steinert
Keyword(s):  
Gene Expression ◽  
Trypanosoma Brucei ◽  
Transcription Initiation ◽  
Tsetse Fly ◽  
Surface Glycoprotein ◽  
Insect Vector ◽  
Variable Surface Glycoprotein ◽  
A Genome ◽  
Variable Surface ◽  
Expression Site

The arrest of variable surface glycoprotein (VSG) synthesis is one of the first events accompanying the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms, which are characteristic of the insect vector. This is because of a very fast inhibition of VSG gene transcription which occurs as soon as the temperature is lowered. We report that this effect is probably not controlled at the level of transcription initiation, since the beginning of the VSG gene expression site, about 45 kilobases upstream from the antigen gene, remains transcribed in procyclic forms. The permanent activity of the promoter readily accounts for the systematic reappearance, upon return to the bloodstream form after cyclical transmission, of the antigen type present before passage to the tsetse fly. The abortive transcription of the VSG gene expression site appears linked to RNA processing abnormalities. Such posttranscriptional controls may allow the modulation of gene expression in a genome organized in large multigenic transcription units.

scholarly journals In Vitro Generation of Human High-Density-Lipoprotein-Resistant Trypanosoma brucei brucei

2006 ◽  
Vol 5 (8) ◽  
pp. 1276-1286 ◽  
Author(s):  
Sara D. Faulkner ◽  
Monika W. Oli ◽  
Rudo Kieft ◽  
Laura Cotlin ◽  
Justin Widener ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Trypanosoma Brucei ◽  
Density Lipoprotein ◽  
High Density ◽  
Surface Glycoprotein ◽  
Trypanosoma Brucei Brucei ◽  
African Trypanosomes ◽  
Expression Site ◽  
Human High Density Lipoprotein

ABSTRACT The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei.

Frequent independent duplicative transpositions activate a single VSG gene

1987 ◽  
Vol 7 (1) ◽  
pp. 357-364
Author(s):  
M G Lee ◽  
L H Van der Ploeg
Keyword(s):  
Gene Conversion ◽  
Trypanosoma Brucei ◽  
Surface Antigen ◽  
Strong Evidence ◽  
Base Pair ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Gene Variants ◽  
Repeat Array ◽  
Expression Site

The expression of several surface antigen genes in Trypanosoma brucei is mediated by the duplicative transposition of a basic-copy variant surface glycoprotein (VSG) gene into an expression site. We determined that the appearance of variant 118, in a parasitemia, resulted from at least four independent duplicative transpositions of the same VSG 118 gene. Variants 117 and 118 both appeared at specific periods but resulted from multiple independent activations. Antigenic variants thus occur in an ordered manner. We show that in the duplicative transpositions of VSG genes, the ends of the transposed segments were homologous between the basic copy and the expression site. Sequences other than the previously reported 70-base-pair (bp) repeats could be involved. In one variant, 118 clone 1, the homology was between a sequence previously transposed into the expression site and a sequence located 6 kilobases upstream of the VSG 118 gene. In variant 118b the homology was presumably in 70-bp repeat arrays, while in a third 118 variant yet another sequence was involved. The possibility that the 70-bp repeats are important in the initial steps of the recombinational events was illustrated by a rearrangement involving a 70-bp repeat array. The data provide strong evidence for the notion that gene conversion mediates the duplicative transposition of VSG genes. We discuss a model that explains how the process of duplicative transposition can occur at random and still produce an ordered appearance of variants.

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