Bloodstream Form-specific Up-regulation of Silent VSG Expression Sites and Procyclin in Trypanosoma brucei after Inhibition of DNA Synthesis or DNA Damage

Sheader, Karen; Vruchte, Daniëlle te; Rudenko, Gloria

doi:10.1074/jbc.m312307200

Cited by 49 publications

(54 citation statements)

References 81 publications

(86 reference statements)

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“…Epitope tagging was performed in bloodstreamform T. brucei 221GPI(VO2ϩ) cells (49) and in wild-type procyclic T. brucei. In order to ensure functionality of the tagged TbISWI, cell lines were also made where the second allele of TbISWI was knocked out using the pSpot5KOPhleo vector.…”

Section: Methodsmentioning

confidence: 99%

“…Bloodstream-and procyclic-form T. brucei 427 cell lines were cultured as described previously (49), except that the bloodstream form was grown in the presence of 20% fetal calf serum without the addition of SerumPlus. The bloodstream-form T. brucei 121-SA1 cell has an active VSG121 ES, eGFP in the silent VSG221 ES, and a TbISWI RNA interference (RNAi) construct (described in reference 21).…”

Section: Methodsmentioning

confidence: 99%

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TbISWI Regulates Multiple Polymerase I (Pol I)-Transcribed Loci and Is Present at Pol II Transcription Boundaries in Trypanosoma brucei

et al. 2011

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The unicellular eukaryote Trypanosoma brucei is unusual in having very little transcriptional control. The bulk of the T. brucei genome is constitutively transcribed by RNA polymerase II (Pol II) as extensive polycistronic transcription units. Exceptions to this rule include several RNA Pol I transcription units such as the VSG expression sites (ESs), which are mono-allelically expressed. TbISWI, a member of the SWI2/SNF2 related chromatin remodeling ATPases, plays a role in repression of Pol I-transcribed ESs in both bloodstream-and procyclic-form T. brucei. We show that TbISWI binds both active and silent ESs but is depleted from the ES promoters themselves. TbISWI knockdown results in an increase in VSG transcripts from the silent VSG ESs. In addition to its role in the repression of the silent ESs, TbISWI also contributes to the downregulation of the Pol I-transcribed procyclin loci, as well as nontranscribed VSG basic copy arrays and minichromosomes. We also show that TbISWI is enriched at a number of strand switch regions which form the boundaries between Pol II transcription units. These strand switch regions are the presumed sites of Pol II transcription initiation and termination and are enriched in modified histones and histone variants. Our results indicate that TbISWI is a versatile chromatin remodeler that regulates transcription at multiple Pol I loci and is particularly abundant at many Pol II transcription boundaries in T. brucei.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

TbISWI Regulates Multiple Polymerase I (Pol I)-Transcribed Loci and Is Present at Pol II Transcription Boundaries in Trypanosoma brucei

et al. 2011

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“…Flow cytometry was performed according to ref. 8. Cell volume measurements were carried out by using the Coulter size exclusion method on a CASY cell counter model TT (Schärfe, Reutlingen, Germany) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…To ensure that the transformants did not switch away from the VSG221 expression site (ES), the 221GP1 construct containing GFP and puromycin (8) of FITC-tomato lectin. Endocytosis ceases at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Variant surface glycoprotein RNA interference triggers a precytokinesis cell cycle arrest in African trypanosomes

Sheader

Vaughan

Minchin

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness. T. brucei multiplies extracellularly in the bloodstream, relying on antigenic variation of a dense variant surface glycoprotein (VSG) coat to escape antibody-mediated lysis. We investigated the role of VSG in proliferation and pathogenicity by using inducible RNA interference to ablate VSG transcript down to 1-2% normal levels. Inhibiting VSG synthesis in vitro triggers a rapid and specific cell cycle checkpoint blocking cell division. Parasites arrest at a discrete precytokinesis stage with two fulllength flagella and opposing flagellar pockets, without undergoing additional rounds of S phase and mitosis. A subset (<10%) of the stalled cells have internal flagella, indicating that the progenitors of these cells were already committed to cytokinesis when VSG restriction was sensed. Although there was no obvious VSG depletion in vitro after 24-h induction of VSG RNA interference, there was rapid clearance of these cells in vivo. We propose that a stringent block in VSG synthesis produces stalled trypanosomes with a minimally compromised VSG coat, which can be targeted by the immune system. Our data indicate that VSG protein or transcript is monitored during cell cycle progression in bloodstreamform T. brucei and describes precise precytokinesis cell cycle arrest. This checkpoint before cell division provides a link between the protective VSG coat and cell cycle progression and could function as a novel parasite safety mechanism, preventing extensive dilution of the protective VSG coat in the absence of VSG synthesis. antigenic variation ͉ Trypanosoma bruceiA frican trypanosomes, including Trypanosoma brucei, are unicellular parasites transmitted by tsetse flies in subSaharan Africa, which are responsible for debilitating disease in humans and livestock. African sleeping sickness is resurgent, with an estimated 300,000-500,000 cases a year, and it is invariably fatal if untreated (1). T. brucei multiplies extracellularly in the bloodstream, exposing it to immune attack. Each bloodstream-form cell is coated with Ϸ10 7 variant surface glycoprotein (VSG) molecules of a given antigenic type, making up Ϸ10% of total cellular protein (2, 3). Eventually the host mounts an antibody response against a given VSG variant. However, as parasites can switch between hundreds of antigenically diverse VSG coats during the course of a chronic infection, trypanosomes expressing a new VSG variant can escape the host antibody response against the old one (4, 5). This highly sophisticated strategy of antigenic variation makes African trypanosomes a paradigm for antigenic variation in general.Although a great deal is known about mechanisms mediating VSG switching, we know relatively little about the role of VSG itself in other aspects of immune evasion and pathogenicity. To investigate this, we inhibited VSG synthesis by performing inducible RNA interference (RNAi) both in vitro and in vivo. Inhibition of VSG synthesis in vitro triggers a rapid and specifi...

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“…Indeed, though this switch reaction does not involve DNA rearrangements, it remains possible that it shares repair-related or replication-related functions that act in recombination-based switching (see below). Such a link would explain why mutation of core homologous recombination (HR) factors not only impairs VSG switching by recombination (see below), but also VSG switching by transcription (108)(109)(110), and why multiple treatments that cause DNA damage or replication arrest in T. brucei can elevate silent BES expression (111).…”

Section: The Genomic Components Of Antigenic Variation In Trypanosomamentioning

confidence: 99%

DNA Recombination Strategies During Antigenic Variation in the African Trypanosome

2015

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Survival of the African trypanosome in its mammalian hosts has led to the evolution of antigenic variation, a process for evasion of adaptive immunity that has independently evolved in many other viral, bacterial and eukaryotic pathogens. The essential features of trypanosome antigenic variation have been understood for many years and comprise a dense, protective Variant Surface Glycoprotein (VSG) coat, which can be changed by recombination-based and transcription-based processes that focus on telomeric VSG gene transcription sites. However, it is only recently that the scale of this process has been truly appreciated. Genome sequencing of Trypanosoma brucei has revealed a massive archive of >1000 VSG genes, the huge majority of which are functionally impaired but are used to generate far greater numbers of VSG coats through segmental gene conversion. This chapter will discuss the implications of such VSG diversity for immune evasion by antigenic variation, and will consider how this expressed diversity can arise, drawing on a growing body of work that has begun to examine the proteins and sequences through which VSG switching is catalyzed. Most studies of trypanosome antigenic variation have focused on T. brucei , the causative agent of human sleeping sickness. Other work has begun to look at antigenic variation in animal-infective trypanosomes, and we will compare the findings that are emerging, as well as consider how antigenic variation relates to the dynamics of host–trypanosome interaction.

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Bloodstream Form-specific Up-regulation of Silent VSG Expression Sites and Procyclin in Trypanosoma brucei after Inhibition of DNA Synthesis or DNA Damage

Cited by 49 publications

References 81 publications

TbISWI Regulates Multiple Polymerase I (Pol I)-Transcribed Loci and Is Present at Pol II Transcription Boundaries in Trypanosoma brucei

TbISWI Regulates Multiple Polymerase I (Pol I)-Transcribed Loci and Is Present at Pol II Transcription Boundaries in Trypanosoma brucei

Variant surface glycoprotein RNA interference triggers a precytokinesis cell cycle arrest in African trypanosomes

DNA Recombination Strategies During Antigenic Variation in the African Trypanosome

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