DNA double strand break position leads to distinct gene expression changes and regulates VSG switching pathway choice

Antigenic variation is an immune evasion strategy used by Trypanosoma brucei that results in the periodic exchange of the surface protein coat. Underlying this process is the movement of variant surface glycoprotein genes in or out of a specialized locus known as bloodstream form expression site by homologous recombination, facilitated by blocks of repetitive sequence known as the 70-bp repeats, that provide homology for gene conversion events. DNA double strand breaks are potent drivers of antigenic variation, however where these breaks must fall to elicit a switch is not well understood. To understand how the position of a break influences antigenic variation we established a series of cell lines to study the effect of an I-SceI meganuclease break in the active expression site. We found that a DNA break within repetitive regions is not productive for VSG switching, and show that the break position leads to a distinct gene expression profile and DNA repair response which dictates how antigenic variation proceeds in African trypanosomes.

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Escaping the immune system by DNA repair and recombination in African trypanosomes

Open Biology ◽

10.1098/rsob.190182 ◽

2019 ◽

Vol 9 (11) ◽

pp. 190182 ◽

Cited By ~ 3

Author(s):

Núria Sima ◽

Emilia Jane McLaughlin ◽

Sebastian Hutchinson ◽

Lucy Glover

Keyword(s):

Immune Response ◽

Dna Repair ◽

Trypanosoma Brucei ◽

Antigenic Variation ◽

Surface Glycoprotein ◽

Variant Surface Glycoprotein ◽

Surface Coat ◽

African Trypanosomes ◽

Active Expression ◽

Expression Site

African trypanosomes escape the mammalian immune response by antigenic variation—the periodic exchange of one surface coat protein, in Trypanosoma brucei the variant surface glycoprotein (VSG), for an immunologically distinct one. VSG transcription is monoallelic, with only one VSG being expressed at a time from a specialized locus, known as an expression site. VSG switching is a predominantly recombination-driven process that allows VSG sequences to be recombined into the active expression site either replacing the currently active VSG or generating a ‘new’ VSG by segmental gene conversion. In this review, we describe what is known about the factors that influence this process, focusing specifically on DNA repair and recombination.

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Gene conversions mediating antigenic variation in Trypanosoma brucei can occur in variant surface glycoprotein expression sites lacking 70-base-pair repeat sequences.

Molecular and Cellular Biology ◽

10.1128/mcb.17.2.833 ◽

1997 ◽

Vol 17 (2) ◽

pp. 833-843 ◽

Cited By ~ 58

Author(s):

R McCulloch ◽

G Rudenko ◽

P Borst

Keyword(s):

Gene Conversion ◽

Antigenic Variation ◽

Surface Glycoprotein ◽

Variant Surface Glycoprotein ◽

Mammalian Host ◽

Recognition Sequence ◽

African Trypanosomes ◽

Repeat Sequences ◽

Active Expression ◽

Expression Site

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid immune system-mediated killing by their mammalian host. An important mechanism for switching the expressed VSG gene is the duplicative transposition of a silent VSG gene into one of the telomeric VSG expression sites of the trypanosome, resulting in the replacement of the previously expressed VSG gene. This process appears to be a gene conversion reaction, and it has been postulated that sequences within the expression site may act to initiate and direct the reaction. All bloodstream form expression sites contain huge arrays (many kilobase pairs) of 70-bp repeat sequences that act as the 5' boundary of gene conversion reactions involving most silent VSG genes. For this reason, the 70-bp repeats seemed a likely candidate to be involved in the initiation of switching. Here, we show that deletion of the 70-bp repeats from the active expression site does not affect duplicative transposition of VSG genes from silent expression sites. We conclude that the 70-bp repeats do not appear to function as indispensable initiation sites for duplicative transposition and are unlikely to be the recognition sequence for a sequence-specific enzyme which initiates recombination-based VSG switching.

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Expression of a retroposon-like sequence upstream of the putative Trypanosoma brucei variant surface glycoprotein gene expression site promoter

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.7036-7044.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 7036-7044

Author(s):

M J Lodes ◽

B L Smiley ◽

A W Stadnyk ◽

J L Bennett ◽

P J Myler ◽

...

Keyword(s):

Gene Expression ◽

Trypanosoma Brucei ◽

Copy Number ◽

Surface Glycoprotein ◽

Variant Surface Glycoprotein ◽

Putative Promoter ◽

Glycoprotein Gene ◽

Active Expression ◽

Expression Site ◽

Total Copy Number

We have cloned the region spanning the putative promoter from two variant surface glycoprotein gene expression sites that are at each end of chromosome M4 of Trypanosoma brucei IsTat 7. Both expression sites contain a retroposon-like sequence (ESR) pseudogene whose 3' end is approximately 30 bp upstream of the putative expression site promoter. The ESRs from both expression sites share considerable sequence homology and are related to LINE-like elements, especially the T. brucei ingi retroposon. Other ESRs are located on large, but not intermediate or mini-, chromosomes in the IsTaR 1 serodeme, and the total copy number is 10 to 20, similar to that estimated for variant surface glycoprotein expression sites. No DNA rearrangements in the vicinity of the ESR and putative expression site promoter were detected following antigenic switches in the IsTaR 1 serodeme. ESR transcripts are present in bloodstream, but not procyclic, forms. Variation in transcript size and sequence between bloodstream variant antigenic types implies that only the ESR from the active expression site is transcribed. This pattern of expression reflects that of sequences downstream of the putative expression site promoter, suggesting that the region of coordinately controlled expression extends upstream of this promoter.

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Expression of a retroposon-like sequence upstream of the putative Trypanosoma brucei variant surface glycoprotein gene expression site promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.7036 ◽

1993 ◽

Vol 13 (11) ◽

pp. 7036-7044 ◽

Cited By ~ 11

Author(s):

M J Lodes ◽

B L Smiley ◽

A W Stadnyk ◽

J L Bennett ◽

P J Myler ◽

...

Keyword(s):

Gene Expression ◽

Trypanosoma Brucei ◽

Copy Number ◽

Surface Glycoprotein ◽

Variant Surface Glycoprotein ◽

Putative Promoter ◽

Glycoprotein Gene ◽

Active Expression ◽

Expression Site ◽

Total Copy Number

We have cloned the region spanning the putative promoter from two variant surface glycoprotein gene expression sites that are at each end of chromosome M4 of Trypanosoma brucei IsTat 7. Both expression sites contain a retroposon-like sequence (ESR) pseudogene whose 3' end is approximately 30 bp upstream of the putative expression site promoter. The ESRs from both expression sites share considerable sequence homology and are related to LINE-like elements, especially the T. brucei ingi retroposon. Other ESRs are located on large, but not intermediate or mini-, chromosomes in the IsTaR 1 serodeme, and the total copy number is 10 to 20, similar to that estimated for variant surface glycoprotein expression sites. No DNA rearrangements in the vicinity of the ESR and putative expression site promoter were detected following antigenic switches in the IsTaR 1 serodeme. ESR transcripts are present in bloodstream, but not procyclic, forms. Variation in transcript size and sequence between bloodstream variant antigenic types implies that only the ESR from the active expression site is transcribed. This pattern of expression reflects that of sequences downstream of the putative expression site promoter, suggesting that the region of coordinately controlled expression extends upstream of this promoter.

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Frequent Loss of the Active Site during Variant Surface Glycoprotein Expression Site Switching In Vitro inTrypanosoma brucei

Molecular and Cellular Biology ◽

10.1128/mcb.18.1.198 ◽

1998 ◽

Vol 18 (1) ◽

pp. 198-205 ◽

Cited By ~ 27

Author(s):

Mike Cross ◽

Martin C. Taylor ◽

Piet Borst

Keyword(s):

Active Site ◽

Antigenic Variation ◽

Surface Glycoprotein ◽

Variant Surface Glycoprotein ◽

Selection System ◽

African Trypanosomes ◽

Expression Site ◽

Gene Switching

ABSTRACT African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid being killed by their mammalian hosts. The active VSG gene is located in one of many telomeric expression sites. Replacement of the VSG gene in the active site or switching between expression sites can give rise to a new VSG coat. To study Trypanosoma brucei VSG expression site inactivation rather than VSG gene switching, it is useful to have an in vitro negative-selection system independent of the VSG. We have achieved this aim by using a viral thymidine kinase (TK) gene. Following integration of the TK gene downstream of the 221a VSG expression site promoter, transformant cell lines became sensitive to the nucleoside analog 1-(2-deoxy-2-fluoro-8-d-arabinofuranosyl)-5-iodouracil. These TK trypanosomes were able to revert to resistance at a rate approaching 10−5 per cell per generation. The majority of revertants expressed a new VSG gene even though there had been no selection against the VSG itself. Analysis of these switched variants showed that some had shut down TK expression via an in situ expression site switch. However, most variants had the complete 221 expression site deleted and another VSG expression site activated. We speculate that a new VSG expression site cannot switch on without inactivation of the old site.

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Expression site attenuation mechanistically links antigenic variation and development in Trypanosoma brucei

eLife ◽

10.7554/elife.02324 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 49

Author(s):

Christopher Batram ◽

Nicola G Jones ◽

Christian J Janzen ◽

Sebastian M Markert ◽

Markus Engstler

Keyword(s):

Trypanosoma Brucei ◽

Histone Methylation ◽

Antigenic Variation ◽

Tsetse Fly ◽

Developmental Competence ◽

Surface Glycoprotein ◽

G1 Phase ◽

African Trypanosomes ◽

Developmental Progression ◽

Expression Site

We have discovered a new mechanism of monoallelic gene expression that links antigenic variation, cell cycle, and development in the model parasite Trypanosoma brucei. African trypanosomes possess hundreds of variant surface glycoprotein (VSG) genes, but only one is expressed from a telomeric expression site (ES) at any given time. We found that the expression of a second VSG alone is sufficient to silence the active VSG gene and directionally attenuate the ES by disruptor of telomeric silencing-1B (DOT1B)-mediated histone methylation. Three conserved expression-site-associated genes (ESAGs) appear to serve as signal for ES attenuation. Their depletion causes G1-phase dormancy and reversible initiation of the slender-to-stumpy differentiation pathway. ES-attenuated slender bloodstream trypanosomes gain full developmental competence for transformation to the tsetse fly stage. This surprising connection between antigenic variation and developmental progression provides an unexpected point of attack against the deadly sleeping sickness.

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Cloning and characterization of a variant surface glycoprotein expression site from Trypanosoma equiperdum.

Molecular and Cellular Biology ◽

10.1128/mcb.6.8.2950 ◽

1986 ◽

Vol 6 (8) ◽

pp. 2950-2956 ◽

Cited By ~ 4

Author(s):

A Raibaud ◽

G Buck ◽

T Baltz ◽

H Eisen

Keyword(s):

Trypanosoma Brucei ◽

Southern Blot Analysis ◽

Surface Glycoprotein ◽

Variant Surface Glycoprotein ◽

African Trypanosomes ◽

Active Expression ◽

Expression Site ◽

Trypanosoma Equiperdum ◽

Dna Fragment

Variant surface glycoprotein (VSG) genes of African trypanosomes are expressed when they are inserted into one of several telomere-linked expression sites. We cloned and characterized an 11-kilobase (kb) DNA fragment located upstream of an expressed VSG gene. A DNA sequence of 1.8 kb that is located immediately upstream of the inserted VSG gene contains sequences homologous to the 76-base-pair repeats described as being upstream of VSG genes in Trypanosoma brucei (D. A. Campbell, M. P. Van Bree, and J. C. Boothroyd, Nucleic Acids Res. 12:2759-2774). There are no such sequences elsewhere in the 11-kb cloned region. Southern blot analysis using probes from the cloned region revealed multiple unlinked copies of the same or very similar regions. At least three of these are located near telomeres, and two have been shown to be used for the expression of known Trypanosoma equiperdum VSG genes. Like VSG genes, the upstream sequences themselves can be duplicated and deleted. The choice of expression site to be used by a duplicated VSG gene is nonrandom; the site used for expression of the parental VSG gene is strongly favored for use in the daughter variant. Furthermore, even when the parental expression site is not used, the VSG gene occupying it is replaced. Thus, an active expression site is a preferential target for gene conversion in the next variation event.

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RAD50 promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

10.1101/2020.03.17.994905 ◽

2020 ◽

Author(s):

Ann-Kathrin Mehnert ◽

Marco Prorocic ◽

Annick Dujeancourt-Henry ◽

Sebastian Hutchinson ◽

Richard McCulloch ◽

...

Keyword(s):

Dna Repair ◽

Homologous Recombination ◽

Antigenic Variation ◽

Strand Break ◽

Surface Glycoprotein ◽

Histone H2a ◽

African Trypanosomes ◽

Mrn Complex ◽

Dna Double Strand Break ◽

Damage Response

ABSTRACTHomologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 is required for RAD51-dependent homologous recombination, phosphorylation of histone H2A and controlled resection following a DNA double strand break (DSB). Perhaps surprisingly, DSB resection in the rad50 nulls was not impaired and appeared to peak earlier than in the parental strains. Finally, we show that RAD50 suppresses DNA repair using donors with short stretches of homology at a subtelomeric locus, with null strains producing a greater diversity of expressed VSG variants following DSB repair. We conclude that RAD50 promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation.

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Diversity and complexity of the large surface protein family in the compacted genomes of various Pneumocystis species

10.1101/814236 ◽

2019 ◽

Author(s):

Liang Ma ◽

Zehua Chen ◽

Da Wei Huang ◽

Ousmane H. Cissé ◽

Jamie L. Rothenburger ◽

...

Keyword(s):

Antigenic Variation ◽

Life Stage ◽

Surface Protein ◽

Opportunistic Pathogen ◽

Surface Proteins ◽

Surface Glycoprotein ◽

Mammalian Host ◽

Expression Site ◽

Phylogeny And Evolution ◽

Major Surface

AbstractPneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multi-copy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins, and provide detailed description of the classification, unique characteristics and phylogenetic relatedness of five Msg families. We further describe the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests a wide variety of functions for this superfamily, not only conferring antigenic variation to allow immune evasion but also mediating life-stage development, optimizing cell mobility and adhesion, and adapting to specific host niches or environmental conditions. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.

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