Variant Surface Glycoprotein Genes Research Articles

The role of genomic location and flanking 3'UTR in the generation of functional levels of variant surface glycoprotein in Trypanosoma brucei.

Summary Trypanosoma brucei faces relentless immune attack in the mammalian bloodstream, where it is protected by an essential coat of Variant Surface Glycoprotein (VSG) comprising ∼10% total protein. The active VSG gene is in a Pol I‐transcribed telomeric expression site (ES). We investigated factors mediating these extremely high levels of VSG expression by inserting ectopic VSG117 into VSG221 expressing T. brucei. Mutational analysis of the ectopic VSG 3′UTR demonstrated the essentiality of a conserved 16‐mer for mRNA stability. Expressing ectopic VSG117 from different genomic locations showed that functional VSG levels could be produced from a gene 60 kb upstream of its normal telomeric location. High, but very heterogeneous levels of VSG117 were obtained from the Pol I‐transcribed rDNA. Blocking VSG synthesis normally triggers a precise precytokinesis cell‐cycle checkpoint. VSG117 expression from the rDNA was not adequate for functional complementation, and the stalled cells arrested prior to cytokinesis. However, VSG levels were not consistently low enough to trigger a characteristic ‘VSG synthesis block’ cell‐cycle checkpoint, as some cells reinitiated S phase. This demonstrates the essentiality of a Pol I‐transcribed ES, as well as conserved VSG 3′UTR 16‐mer sequences for the generation of functional levels of VSG expression in bloodstream form T. brucei.

Variant surface glycoprotein density defines an immune evasion threshold for African trypanosomes undergoing antigenic variation

Trypanosoma brucei is a protozoan parasite that evades its host’s adaptive immune response by repeatedly replacing its dense variant surface glycoprotein (VSG) coat from its large genomic VSG repertoire. While the mechanisms regulating VSG gene expression and diversification have been examined extensively, the dynamics of VSG coat replacement at the protein level, and the impact of this process on successful immune evasion, remain unclear. Here we evaluate the rate of VSG replacement at the trypanosome surface following a genetic VSG switch, and show that full coat replacement requires several days to complete. Using in vivo infection assays, we demonstrate that parasites undergoing coat replacement are only vulnerable to clearance via early IgM antibodies for a limited time. Finally, we show that IgM loses its ability to mediate trypanosome clearance at unexpectedly early stages of coat replacement based on a critical density threshold of its cognate VSGs on the parasite surface.

Metacyclic VSG expression site promoters are recognized by the same general transcription factor that is required for RNA polymerase I transcription of bloodstream expression sites

Infectious metacyclic Trypanosoma brucei cells develop in the salivary glands of tsetse flies. A critical aspect of the developmental program leading to acquisition of infectivity is the synthesis of a variant surface glycoprotein (VSG) coat. Metacyclic VSG genes are transcribed from a set of specialized VSG expression sites (ESs) that differ from bloodstream VSG ESs by being monocistronic, being significantly shorter, lacking long stretches of 70-bp repeats, and having distinct promoter sequences. Both metacyclic and bloodstream VSG ESs are transcribed by the multifunctional T. brucei RNA polymerase I (Pol I), however the factor that recognizes the divergent metacyclic VSG ES promoters and recruits Pol I during the development to infectious cells remains unknown. We used an in vitro assay to show that the promoters for both metacyclic and bloodstream VSG ESs are recognized by the same class I transcription factor A (CITFA). This general Pol I transcription initiation factor was previously shown to be essential for the transcription of bloodstream VSG genes, procyclin genes and rRNA genes, and was demonstrated to have distinct binding affinities for these three types of promoters. We now show that differences in the sequence of individual metacyclic VSG ESs promoters determine different affinities for CITFA.

Well-positioned nucleosomes punctuate polycistronic pol II transcription units and flank silent VSG gene arrays in Trypanosoma brucei

BackgroundThe compaction of DNA in chromatin in eukaryotes allowed the expansion of genome size and coincided with significant evolutionary diversification. However, chromatin generally represses DNA function, and mechanisms coevolved to regulate chromatin structure and its impact on DNA. This included the selection of specific nucleosome positions to modulate accessibility to the DNA molecule. Trypanosoma brucei, a member of the Excavates supergroup, falls in an ancient evolutionary branch of eukaryotes and provides valuable insight into the organization of chromatin in early genomes.ResultsWe have mapped nucleosome positions in T. brucei and identified important differences compared to other eukaryotes: The RNA polymerase II initiation regions in T. brucei do not exhibit pronounced nucleosome depletion, and show little evidence for defined −1 and +1 nucleosomes. In contrast, a well-positioned nucleosome is present directly on the splice acceptor sites within the polycistronic transcription units. The RNA polyadenylation sites were depleted of nucleosomes, with a single well-positioned nucleosome present immediately downstream of the predicted sites. The regions flanking the silent variant surface glycoprotein (VSG) gene cassettes showed extensive arrays of well-positioned nucleosomes, which may repress cryptic transcription initiation. The silent VSG genes themselves exhibited a less regular nucleosomal pattern in both bloodstream and procyclic form trypanosomes. The DNA replication origins, when present within silent VSG gene cassettes, displayed a defined nucleosomal organization compared with replication origins in other chromosomal core regions.ConclusionsOur results indicate that some organizational features of chromatin are evolutionarily ancient, and may already have been present in the last eukaryotic common ancestor.

High-resolution analysis of multi-copy variant surface glycoprotein gene expression sites in African trypanosomes.

BackgroundAfrican trypanosomes cause lethal diseases in humans and animals and escape host immune attack by switching the expression of Variant Surface Glycoprotein (VSG) genes. The expressed VSGs are located at the ends of telomeric, polycistronic transcription units known as VSG expression sites (VSG-ESs). Each cell has many VSG-ESs but only one is transcribed in bloodstream-form parasites and all of them are inactive upon transmission to the insect vector mid-gut; a subset of monocistronic metacyclic VSG-ESs are then activated in the insect salivary gland. Deep-sequence analyses have been informative but assigning sequences to individual VSG-ESs has been challenging because they each contain closely related expression-site associated genes, or ESAGs, thought to contribute to virulence.ResultsWe utilised ART, an in silico short read simulator to demonstrate the feasibility of accurately aligning reads to VSG-ESs. Then, using high-resolution transcriptomes from isogenic bloodstream and insect-stage Lister 427 Trypanosoma brucei, we uncover increased abundance in the insect mid-gut stage of mRNAs from metacyclic VSG-ESs and of mRNAs from the unusual ESAG, ESAG10. Further, we show that the silencing associated with allelic exclusion involves repression focussed at the ends of the VSG-ESs. We also use the approach to report relative fitness costs following ESAG RNAi from a genome-scale screen.ConclusionsBy assigning sequences to individual VSG-ESs we provide new insights into VSG-ES transcription control, allelic exclusion and impacts on fitness. Thus, deeper insights into the expression and function of regulated multi-gene families are more accessible than previously anticipated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3154-8) contains supplementary material, which is available to authorized users.

VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes

Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for defects in telomere-exclusive expression. VEX1 was sequestered by the active VSG and silencing of other VSGs failed when VEX1 was either ectopically expressed or depleted, indicating positive and negative regulation, respectively. Positive regulation affected VSGs and nontelomeric pol-I-transcribed genes, whereas negative regulation primarily affected VSGs. Negative regulation by VEX1 also affected telomeric pol-I-transcribed reporter constructs, but only when they contained blocks of sequence sharing homology with a pol-I-transcribed locus. We conclude that restricted positive regulation due to VEX1 sequestration, combined with VEX1-dependent, possibly homology-dependent silencing, drives a "winner-takes-all" mechanism of allelic exclusion.

A Conserved DNA Repeat Promotes Selection of a Diverse Repertoire of Trypanosoma brucei Surface Antigens from the Genomic Archive

African trypanosomes are mammalian pathogens that must regularly change their protein coat to survive in the host bloodstream. Chronic trypanosome infections are potentiated by their ability to access a deep genomic repertoire of Variant Surface Glycoprotein (VSG) genes and switch from the expression of one VSG to another. Switching VSG expression is largely based in DNA recombination events that result in chromosome translocations between an acceptor site, which houses the actively transcribed VSG, and a donor gene, drawn from an archive of more than 2,000 silent VSGs. One element implicated in these duplicative gene conversion events is a DNA repeat of approximately 70 bp that is found in long regions within each BES and short iterations proximal to VSGs within the silent archive. Early observations showing that 70-bp repeats can be recombination boundaries during VSG switching led to the prediction that VSG-proximal 70-bp repeats provide recombinatorial homology. Yet, this long held assumption had not been tested and no specific function for the conserved 70-bp repeats had been demonstrated. In the present study, the 70-bp repeats were genetically manipulated under conditions that induce gene conversion. In this manner, we demonstrated that 70-bp repeats promote access to archival VSGs. Synthetic repeat DNA sequences were then employed to identify the length, sequence, and directionality of repeat regions required for this activity. In addition, manipulation of the 70-bp repeats allowed us to observe a link between VSG switching and the cell cycle that had not been appreciated. Together these data provide definitive support for the long-standing hypothesis that 70-bp repeats provide recombinatorial homology during switching. Yet, the fact that silent archival VSGs are selected under these conditions suggests the 70-bp repeats also direct DNA pairing and recombination machinery away from the closest homologs (silent BESs) and toward the rest of the archive.

New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels.

BackgroundTrypanosoma (T.) evansi is a dyskinetoplastic variant of T. brucei that has gained the ability to be transmitted by all sorts of biting flies. T. evansi can be divided into type A, which is the most abundant and found in Africa, Asia and Latin America and type B, which has so far been isolated only from Kenyan dromedary camels. This study aimed at the isolation and the genetic and phenotypic characterisation of type A and B T. evansi stocks from camels in Northern Ethiopia.Methodology/principal findingsT. evansi was isolated in mice by inoculation with the cryopreserved buffy coat of parasitologically confirmed animals. Fourteen stocks were thus isolated and subject to genotyping with PCRs targeting type-specific variant surface glycoprotein genes, mitochondrial minicircles and maxicircles, minisatellite markers and the F1-ATP synthase γ subunit gene. Nine stocks corresponded to type A, two stocks were type B and three stocks represented mixed infections between A and B, but not hybrids. One T. evansi type A stock was completely akinetoplastic. Five stocks were adapted to in vitro culture and subjected to a drug sensitivity assay with melarsomine dihydrochloride, diminazene diaceturate, isometamidium chloride and suramin. In vitro adaptation induced some loss of kinetoplasts within 60 days. No correlation between drug sensitivity and absence of the kinetoplast was observed. Sequencing the full coding sequence of the F1-ATP synthase γ subunit revealed new type-specific single nucleotide polymorphisms and deletions.Conclusions/significanceThis study addresses some limitations of current molecular markers for T. evansi genotyping. Polymorphism within the F1-ATP synthase γ subunit gene may provide new markers to identify the T. evansi type that do not rely on variant surface glycoprotein genes or kinetoplast DNA.

Classico-molecular targeting of oligopeptidase B, cysteine protease and variable surface glycoprotein (VSG) genes of Trypanosoma evansi.

Trypanosomosis or Surra can rightly be attributed as the most economically important vector-borne haemoprotozoan disease encountering India. Surra infected chronic cases show almost similar types of signs and symptoms often confusing it with other haemoprotozoan infections, thereby, making it prerequisite for the development of aspecific and sensitive technique for its detection in susceptible animals. Blood microscopy and serology suffers from the hands of lack of sensitivity and specificity thereby leaving molecular detection techniques as one of the promising alternative. Alongside, there is utmost need for exploring of new molecular gene targets for the development of a putative alternative for diagnosis and immunoprophylaxsis. The present communication describes the identification and amplification of oligopeptidase B, cysteine protease and variable surface glycoprotein genes of T. evansi so as to exploit them in future as potential candidates for immune protection and/or molecular detection.

Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei.

Base J, β-D-glucosyl-hydroxymethyluracil, is a chromatin modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. In Trypanosoma brucei, J is enriched, along with histone H3 variant (H3.V), at sites involved in RNA Polymerase (RNAP) II termination and telomeric sites involved in regulating variant surface glycoprotein gene (VSG) transcription by RNAP I. Reduction of J in T. brucei indicated a role of J in the regulation of RNAP II termination, where the loss of J at specific sites within polycistronic gene clusters led to read-through transcription and increased expression of downstream genes. We now demonstrate that the loss of H3.V leads to similar defects in RNAP II termination within gene clusters and increased expression of downstream genes. Gene derepression is intensified upon the subsequent loss of J in the H3.V knockout. mRNA-seq indicates gene derepression includes VSG genes within the silent RNAP I transcribed telomeric gene clusters, suggesting an important role for H3.V in telomeric gene repression and antigenic variation. Furthermore, the loss of H3.V at regions of overlapping transcription at the end of convergent gene clusters leads to increased nascent RNA and siRNA production. Our results suggest base J and H3.V can act independently as well as synergistically to regulate transcription termination and expression of coding and non-coding RNAs in T. brucei, depending on chromatin context (and transcribing polymerase). As such these studies provide the first direct evidence for histone H3.V negatively influencing transcription elongation to promote termination.

Base J and H3.V Regulate Transcriptional Termination in Trypanosoma brucei

Trypanosoma brucei is a protozoan parasite that lacks many transcription factors found in other eukaryotes, such as those whose binding demarcates enhancers. T. brucei retains histone variants and modifications, however, and it is hypothesized that it relies on epigenetic marks to define transcription-related boundaries. The histone H3 variant (H3.V) and an alternate nucleotide, base J (ß-D-glucosyl-hydroxymethyluracil), are two chromatin marks found at both transcription termination sites (TTSs) and telomeres. Here, we report that the absence of both base J and H3.V result in transcription readthrough and the appearance of antisense transcripts near TTSs. Additionally, we find that maintaining the transcriptional silencing of pol I-transcribed telomeric Variant Surface Glycoprotein (VSG) genes appears to be dependent on deposition of H3.V alone. Our study reveals that gene expression depends on different epigenetic cues depending on chromosomal location and on the transcribing polymerase. This work provides insight into how these signals may have evolved into the more nuanced and fine-tuned gene regulatory mechanisms observed in other model systems.

Bromodomain Proteins Contribute to Maintenance of Bloodstream Form Stage Identity in the African Trypanosome

Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite’s surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design.

Identification of the ISWI Chromatin Remodeling Complex of the Early Branching Eukaryote Trypanosoma brucei

ISWI chromatin remodelers are highly conserved in eukaryotes and are important for the assembly and spacing of nucleosomes, thereby controlling transcription initiation and elongation. ISWI is typically associated with different subunits, forming specialized complexes with discrete functions. In the unicellular parasite Trypanosoma brucei, which causes African sleeping sickness, TbISWI down-regulates RNA polymerase I (Pol I)-transcribed variant surface glycoprotein (VSG) gene expression sites (ESs), which are monoallelically expressed. Here, we use tandem affinity purification to determine the interacting partners of TbISWI. We identify three proteins that do not show significant homology with known ISWI-associated partners. Surprisingly, one of these is nucleoplasmin-like protein (NLP), which we had previously shown to play a role in ES control. In addition, we identify two novel ISWI partners, regulator of chromosome condensation 1-like protein (RCCP) and phenylalanine/tyrosine-rich protein (FYRP), both containing protein motifs typically found on chromatin proteins. Knockdown of RCCP or FYRP in bloodstream form T. brucei results in derepression of silent variant surface glycoprotein ESs, as had previously been shown for TbISWI and NLP. All four proteins are expressed and interact with each other in both major life cycle stages and show similar distributions at Pol I-transcribed loci. They are also found at Pol II strand switch regions as determined with ChIP. ISWI, NLP, RCCP, and FYRP therefore appear to form a single major ISWI complex in T. brucei (TbIC). This reduced complexity of ISWI regulation and the presence of novel ISWI partners highlights the early divergence of trypanosomes in evolution.

Hidden Markov models for gene sequence classification

The article presents an application of Hidden Markov Models (HMMs) for pattern recognition on genome sequences. We apply HMM for identifying genes encoding the Variant Surface Glycoprotein (VSG) in the genomes of Trypanosoma brucei (T. brucei) and other African trypanosomes. These are parasitic protozoa causative agents of sleeping sickness and several diseases in domestic and wild animals. These parasites have a peculiar strategy to evade the host's immune system that consists in periodically changing their predominant cellular surface protein (VSG). The motivation for using patterns recognition methods to identify these genes, instead of traditional homology based ones, is that the levels of sequence identity (amino acid and DNA sequence) amongst these genes is often below of what is considered reliable in these methods. Among pattern recognition approaches, HMM are particularly suitable to tackle this problem because they can handle more naturally the determination of gene edges. We evaluate the performance of the model using different number of states in the Markov model, as well as several performance metrics. The model is applied using public genomic data. Our empirical results show that the VSG genes on T. brucei can be safely identified (high sensitivity and low rate of false positives) using HMM.

Localization of serum resistance-associated protein in Trypanosoma brucei rhodesiense and transgenic Trypanosoma brucei brucei.

African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human-infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance-associated protein (SRA protein), protects against ApoL1-mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA-mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well-characterized endosomal markers. By three-dimensional deconvolved immunofluorescence single-cell analysis, combined with double-labelling immunoelectron microscopy, we found that ≈ 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency.

Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes

African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing.

Synchronous expression of individual metacyclic variant surface glycoprotein genes in Trypanosoma brucei

One distinctive feature of the Trypanosoma brucei life cycle is the presence of two discrete populations that are based on differential expression of variant surface glycoproteins (VSGs). Both are adapted to the environmental pressures they face and more importantly, both contribute directly to transmission. Metacyclics in the tsetse fly enable transmission to a new mammalian host, whereas bloodstream trypanosomes must avoid immune destruction to the extent that sufficient numbers are available for transmission, when the insect vector takes a blood meal. At present, there are few investigations on the molecular aspects of parasite biology in the tsetse vector and specifically about the activation of metacyclic VSG gene expression. Here we used an established in vitro differentiation system based on the overexpression of the RNA-binding protein 6 (RBP6), to monitor two metacyclic VSGs (VSG 397 and VSG 653) during development from procyclics to infectious metacyclic forms. We observed that activation of these two mVSGs was simultaneous both at the transcript and protein level, and manifested by the appearance of only one of the mVSGs in individual cells.

Variant surface glycoproteins from Venezuelan trypanosome isolates are recognized by sera from animals infected with either Trypanosoma evansi or Trypanosoma vivax

Salivarian trypanosomes sequentially express only one variant surface glycoprotein (VSG) on their cell surface from a large repertoire of VSG genes. Seven cryopreserved animal trypanosome isolates known as TeAp-ElFrio01, TEVA1 (or TeAp-N/D1), TeGu-N/D1, TeAp-Mantecal01, TeGu-TerecayTrino, TeGu-Terecay03 and TeGu-Terecay323, which had been isolated from different hosts identified in several geographical areas of Venezuela were expanded using adult albino rats. Soluble forms of predominant VSGs expressed during the early infection stages were purified and corresponded to concanavalin A-binding proteins with molecular masses of 48–67kDa by sodium dodecyl sulfate-polyacrylamide gel electropohoresis, and pI values between 6.1 and 7.5. The biochemical characterization of all purified soluble VSGs revealed that they were dimers in their native form and represented different gene products. Sequencing of some of these proteins yielded peptides homologous to VSGs from Trypanosoma (Trypanozoon) brucei and Trypanosoma (Trypanozoon) evansi and established that they most likely are mosaics generated by homologous recombination. Western blot analysis showed that all purified VSGs were cross-reacting antigens that were recognized by sera from animals infected with either T. evansi or Trypanosoma (Dutonella) vivax. The VSG glycosyl-phosphatidylinositol cross-reacting determinant epitope was only partially responsible for the cross-reactivity of the purified proteins, and antibodies appeared to recognize cross-reacting conformational epitopes from the various soluble VSGs. ELISA experiments were performed using infected bovine sera collected from cattle in a Venezuelan trypanosome-endemic area. In particular, soluble VSGs from two trypanosome isolates, TeGu-N/D1 and TeGu-TeracayTrino, were recognized by 93.38% and 73.55% of naturally T. vivax-infected bovine sera, respectively. However, approximately 70% of the sera samples did not recognize all seven purified proteins. Hence, the use of a combination of various VSGs for the diagnosis of animal trypanosomosis is recommended.

Locus-specific control of DNA resection and suppression of subtelomeric VSG recombination by HAT3 in the African trypanosome.

The African trypanosome, Trypanosoma brucei, is a parasitic protozoan that achieves antigenic variation through DNA-repair processes involving Variant Surface Glycoprotein (VSG) gene rearrangements at subtelomeres. Subtelomeric suppression of DNA repair operates in eukaryotes but little is known about these controls in trypanosomes. Here, we identify a trypanosome histone acetyltransferase (HAT3) and a deacetylase (SIR2rp1) required for efficient RAD51-dependent homologous recombination. HAT3 and SIR2rp1 were required for RAD51-focus assembly and disassembly, respectively, at a chromosome-internal locus and a synthetic defect indicated distinct contributions to DNA repair. Although HAT3 promoted chromosome-internal recombination, it suppressed subtelomeric VSG recombination, and these locus-specific effects were mediated through differential production of ssDNA by DNA resection; HAT3 promoted chromosome-internal resection but suppressed subtelomeric resection. Consistent with the resection defect, HAT3 was specifically required for the G2-checkpoint response at a chromosome-internal locus. HAT3 also promoted resection at a second chromosome-internal locus comprising tandem-duplicated genes. We conclude that HAT3 and SIR2rp1 can facilitate temporally distinct steps in DNA repair. HAT3 promotes ssDNA formation and recombination at chromosome-internal sites but has the opposite effect at a subtelomeric VSG. These locus-specific controls reveal compartmentalization of the T. brucei genome in terms of the DNA-damage response and suppression of antigenic variation by HAT3.

Mono-allelic VSG expression by RNA polymerase I in Trypanosoma brucei: Expression site control from both ends?

Trypanosoma brucei is a vector borne, lethal protistan parasite of humans and livestock in sub-Saharan Africa. Antigenic variation of its cell surface coat enables the parasite to evade adaptive immune responses and to live freely in the blood of its mammalian hosts. The coat consists of ten million copies of variant surface glycoprotein (VSG) that is expressed from a single VSG gene, drawn from a large repertoire and located near the telomere at one of fifteen so-called bloodstream expression sites (BESs). Thus, antigenic variation is achieved by switching to the expression of a different VSG gene. A BES is a tandem array of expression site-associated genes and a terminal VSG gene. It is polycistronically transcribed by a multifunctional RNA polymerase I (RNAPI) from a short promoter that is located 45–60kb upstream of the VSG gene. The mechanism(s) restricting VSG expression to a single BES are not well understood. There is convincing evidence that epigenetic silencing and transcription attenuation play important roles. Furthermore, recent data indicated that there is regulation at the level of transcription initiation and that, surprisingly, the VSG mRNA appears to have a role in restricting VSG expression to a single gene. Here, we review BES expression regulation and propose a model in which telomere-directed, epigenetic BES silencing is opposed by BES promoter-directed, activated RNAPI transcription.