Variant Surface Glycoprotein Expression Research Articles

Repression of polymerase I‐mediated gene expression at Trypanosoma brucei telomeres

The African trypanosome, Trypanosoma brucei, is a flagellated pathogenic protozoan that branched early from the eukaryotic lineage. Unusually, it uses RNA polymerase I (Pol I) for mono-telomeric expression of variant surface glycoprotein (VSG) genes in bloodstream-form cells. Many other subtelomeric VSG genes are reversibly repressed, but no repressive DNA sequence has been identified in any trypanosomatid. Here, we show that artificially seeded de novo telomeres repress Pol I-dependent gene expression in mammalian bloodstream and insect life-cycle stages of T. brucei. In a telomeric VSG expression site, repression spreads further along the chromosome and this effect is specific to the bloodstream stage. We also show that de novo telomere extension is telomerase dependent and that the rate of extension correlates with the expression level of the adjacent gene. Our results show constitutive telomeric repression in T. brucei and indicate that an enhanced, developmental stage-specific repression mechanism controls antigenic variation.

Regulation of antigen gene expression in

The trypanosome genome is organized into long polycistronic units that seem to be permanently transcribed in proliferative stages of the parasite. Cellular differentiation is controlled primarily at the level of individual mRNA maturation and stability. The transcription units of the two major stage-specific antigens, the variant surface glycoprotein (VSG) of the bloodstream form and procyclin of the procyclic form, are subject to an additional layer of control: the mutually exclusive activation of RNA elongation and processing. The high recombination frequency prevailing in the telomere that harbours the active VSG expression site has been exploited by the parasite to both drive antigenic variation and generate VSG-based adaptive proteins.

VSG switching in Trypanosoma brucei: antigenic variation analysed using RNAi in the absence of immune selection.

Trypanosoma brucei relies on antigenic variation of its variant surface glycoprotein (VSG) coat for survival. We show that VSG switching can be efficiently studied in vitro using VSG RNAi in place of an immune system to select for switch variants. Contrary to models predicting an instant switch after inhibition of VSG synthesis, switching was not induced by VSG RNAi and occurred at a rate of 10(-4) per division. We find a highly reproducible hierarchy of VSG activation, which appears to be capable of resetting, whereby more than half of the switch events over 12 experiments were to one of two VSGs. We characterized switched clones according to switch mechanism using marker genes in the active VSG expression site (ES). Transcriptional switches between ESs were the preferred switching mechanism, whereby at least 10 of the 17 ESs identified in T. brucei 427 can be functionally active in vitro. We could specifically select for switches mediated by DNA rearrangements by inducing VSG RNAi in the presence of drug selection for the active ES. Most of the preferentially activated VSGs could be activated by multiple mechanisms. This VSG RNAi-based procedure provides a rapid and powerful means for analysing VSG switching in African trypanosomes entirely in vitro.

DNA metabolism and genetic diversity in Trypanosomes

Trypanosomes are protozoan parasites that cause major diseases in humans and other animals. Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of African and American Trypanosomiasis, respectively. In spite of large amounts of information regarding various aspects of their biology, including the essentially complete sequences of their genomes, studies directed towards an understanding of mechanisms related to DNA metabolism have been very limited. Recent reports, however, describing genes involved with DNA recombination and repair in T. brucei and T. cruzi, indicated the importance of these processes in the generation of genetic variability, which is crucial to the success of these parasites. Here, we review these data and discuss how the DNA repair and recombination machineries may contribute to strikingly different strategies evolved by the two Trypanosomes to create genetic variability that is needed for survival in their hosts. In T. brucei, two genetic components are critical to the success of antigenic variation, a strategy that allows the parasite to evade the host immune system by periodically changing the expression of a group of variant surface glycoproteins (VSGs). One component is a mechanism that provides for the exclusive expression of a single VSG at any one time, and the second is a large repository of antigenically distinct VSGs. Work from various groups showing the importance of recombination reactions in T. brucei, primarily to move a silent VSG into an active VSG expression site, is discussed. T. cruzi does not use the strategy of antigenic variation for host immune evasion but counts on the extreme heterogeneity of their population for parasite adaptation to different hosts. We discuss recent evidence indicating the existence of major differences in the levels of genomic heterogeneity among T. cruzi strains, and suggest that metabolic changes in the mismatch repair pathway could be an important source of antigenic diversity found within the T. cruzi population.

The central roles of telomeres and subtelomeres in antigenic variation in African trypanosomes

Telomeres and subtelomeres are important to the virulence of a number of pathogens, as they harbour large diverse gene families associated with the maintenance of infection. Evasion of immunity by African trypanosomes involves the differential expression of variant surface glycoproteins (VSGs), which are encoded by a family of >1500 genes and pseudogenes. This silent archive is located subtelomerically and is activated by gene conversion into specialized transcription units, which themselves are subject to silencing by allelic exclusion. Current research addresses the role of telomeres in the conversion and silencing mechanisms and in the diversification of the VSG archive.

A TFIIH Homologue Controls the Mutually Exclusive Expression of the Stage‐Specific Antigens of Trypanosoma brucei

Antigenic variation in Trypanosoma brucei relies on a mono‐allelic control of the multiple variant surface glycoprotein (VSG) expression sites (ESs). A single ES is active in bloodstream forms (BF) and none are in insect‐specific procyclic forms (PF), where procyclin replaces the VSG. In PF, disrupting TbTFIIH by conditional RNAi of any of the Tbp44, TbXPD or TbXPB subunits, or by over‐expression of a TbXPD mutant, led to strong transcriptional stimulation in the beginning of the ESs. Both RNA elongation and mRNA production were up‐regulated. This effect was linked to inhibition of procyclin, and occurred irrespective of the various cellular phenotypes, some of which were lethal due to cell cycle arrest. In BF, stimulation of procyclin was linked to inhibition of the active ES. Thus, TbTFIIH is involved in the mutually exclusive expression of the VSG and procyclin transcription units, both of which are transcribed by RNA Pol I.

Telomere length regulation and transcriptional silencing in KU80-deficient Trypanosoma brucei

KU is a heterodimer, consisting of approximately 70 and approximately 80 kDa subunits (KU70 and KU80, respectively), which is involved in a variety of nuclear functions. We generated tbKU80-deficient trypanosomes to explore the potential role of the tbKU complex in telomere maintenance and transcriptional regulation of variant surface glycoprotein (VSG) genes in Trypanosoma brucei. Using real-time PCR, we demonstrated that the expression of several different VSG genes remains tightly regulated in tbKU80-deficient bloodstream-form cell lines, suggesting that VSG transcription profiles do not change in these cells. Owing to developmental silencing of the VSG Expression Sites (ES), no VSG is transcribed in the insect procyclic stage. With a green fluorescent protein reporter system, we showed that tbKU80-deficient mutants are fully capable of ES silencing after differentiation into procyclic forms. Using T7 RNA polymerase to explore the transcriptional accessibility of ES chromatin in vivo, we demonstrated that tbKU80-deficient bloodstream-form cells were able to generate transcriptionally repressed ES chromatin after differentiation into procyclic cells. Finally, we demonstrated progressive telomere shortening in tbKU80-deficient mutants. The possible function of tbKU80 in telomere maintenance and regulation of telomerase is discussed.

Isolation of the repertoire of VSG expression site containing telomeres of Trypanosoma brucei 427 using transformation-associated recombination in yeast.

Trypanosoma brucei switches between variant surface glycoproteins (VSGs) allowing immune escape. The active VSG is in one of many telomeric bloodstream form VSG expression sites (BESs), also containing expression site-associated genes (ESAGs) involved in host adaptation. The role of BES sequence diversity in parasite virulence can best be understood through analysis of the full repertoire of BESs from a given T. brucei strain. However, few BESs have been cloned, as telomeres are highly underrepresented in standard libraries. We devised a strategy for isolating the repertoire of T. brucei 427 BES-containing telomeres in Saccaromyces cerevisiae by using transformation-associated recombination (TAR). We isolated 182 T. brucei 427 BES TAR clones, 167 of which could be subdivided into minimally 17 BES groups. This set gives us the first view of the breadth and diversity of BESs from one T. brucei strain. Most BESs ranged between 40 and 70 kb (average, 57 +/- 17 kb) and contained most identified ESAGs. Phylogenetic comparison of the cohort of BES promoter and ESAG6 sequences did not show similar trees, indicating rapid evolution most likely mediated by sequence exchange between BESs. This cloning strategy could be used for any T. brucei strain, facilitating research on the biodiversity of telomeric gene families and host-pathogen interactions.

Genetic variability in ESAG6 genes among Trypanosoma evansi isolates and in comparison to other Trypanozoon members

Bloodstream trypanosomes take up iron needed for their propagation through the transferrin receptor that, in Trypanosoma brucei, is encoded by expression-site-associated genes (ESAGs), ESAG6 and 7 genes located in variant surface glycoprotein expression sites. ESAG6 and 7 genes in different expression sites have been shown to encode transferrin receptors with varying affinities for polymorphic transferrins. T. brucei could cope with the different host transferrins by switching between expression sites. ESAG6- and 7-encoded transferrin receptor appear to be present in Trypanosoma evansi but the genes have not yet been characterized. In this study, we cloned and sequenced different members of ESAG6 genes in seven isolates of T. evansi from geographically distinct localities in Thailand. We assessed the intra- and inter-species genetic variability in the transferrin receptor gene regions involved in transferrin binding and established that T. evansi, like T. brucei, has widely diverse ESAG6 genes. In addition, T. evansi possess a clade of ESAG6 variants not observed in T. brucei and different T. evansi strains share at least two conserved variants. We further noted that T. evansi possesses all the reported T. equiperdum ESAG6 variants as a subset. Our findings depict a correlation between the genetic diversity in the transferrin-binding regions of ESAG6 genes with the broad host range of T. evansi and T. brucei compared to the narrow host range of Trypanosoma equiperdum.

Factors Affecting the Level and Localization of the Transferrin Receptor in Trypanosoma brucei

Transfer of bloodstream-form Trypanosoma brucei variant 221a from calf serum to dog serum-based medium induces acute iron starvation, as the transferrin receptor (Tf-R) of variant 221a binds dog Tf poorly. We show here that transfer to dog serum induces a 3-5-fold increase in Tf-R mRNA and protein within one doubling time (8 h). Because iron stores are still high 8 h after transfer, we infer that the signal for Tf-R overproduction is the decreased availability of cytosolic iron when cellular iron import drops. Up to 30% of the extra Tf-R spills out of the flagellar pocket onto the pellicular surface. Because the 5-fold increase in Tf-R is accompanied by a 5-fold increase in bovine Tf uptake, the up-regulation of Tf-R levels in response to Tf starvation helps the trypanosome to compete for limiting amounts of Tf. We noted that Tf-R levels also vary in calf serum medium. Cells in dense cultures contain up to 5-fold more Tf-R mRNA and protein than in dilute cultures. Only one-tenth of the extra Tf-R reaches the pellicular surface. The increase cannot be explained by a lack of Tf or to cell density sensing but is due to pericellular hypoxia. Our results show that bloodstream-form trypanosomes can regulate the expression of the two Tf-R subunit genes and the localization of their gene products in a flexible manner. This flexibility is made possible by the promoter-proximal position of the two genes in the variant surface glycoprotein expression site.

The Trypanosoma brucei reference strain TREU927/4 contains T. brucei rhodesiense-specific SRA sequences, but displays a distinct phenotype of relative resistance to human serum

The Trypanosoma brucei reference strain TREU927/4 exhibits some resistance to lysis by normal human serum (NHS), but this resistance is never complete even after selection. The genome of this strain contains a minimum of eight sequences related to the T. brucei rhodesiense-specific serum resistance-associated gene (SRA), which encodes a truncated variant surface glycoprotein (VSG) conferring full resistance to lysis by NHS. We selected two sequences showing the highest similarity to SRA and also preceded by a region (“cotransposed region”) present immediately upstream from SRA in the VSG expression site termed R-ES, where SRA is expressed in T. brucei rhodesiense. Whereas one of these sequences appears to be a pseudogene, the other, which is contained within a cluster of VSG basic copies (BCs), encodes a VSG truncated in the C-terminal domain. In the latter gene, an inserted region encoding surface-exposed loops similar to those of the BoTat 1.20 VSG interrupts the full SRA sequence. Therefore, this gene was termed SRA-BC, for the putative VSG basic copy from which SRA was derived. Neither this gene nor other SRA-like sequences appeared to be responsible for the relative resistance of TREU927/4 to NHS, since (i) transfection of SRA-BC in T. brucei brucei did not confer increased resistance; (ii) SRA transcripts could not be detected in this strain, even when focusing the search on the limited SRA sequence necessary to confer resistance and when using strain-specific SRA probes on RNA from cells selected in the presence of NHS.

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

The African trypanosome Trypanosoma brucei transcribes the active variant surface glycoprotein (VSG) gene from one of about 20 VSG expression sites (ESs). In order to study ES control, we made reporter lines with a green fluorescent protein gene inserted behind the promoter of different ESs. We attempted to disrupt the silencing machinery, and we used fluorescence-activated cell sorter analysis for the rapid and sensitive detection of ES up-regulation. We find that a range of treatments that either block nuclear DNA synthesis, like aphidicolin, or modify DNA-like cisplatin and 1-methyl-3-nitro-1-nitrosoguanidine results in up-regulation of silent ESs. Aphidicolin treatment was the most effective, with almost 80% of the cells expressing green fluorescent protein from a silent ES. All of these treatments blocked the cells in S phase. In contrast, a range of toxic chemicals had little or no effect on expression. These included berenil and pentamidine, which selectively cleave the mitochondrial kinetoplast DNA, the metabolic inhibitors suramin and difluoromethylornithine, and the mitotic inhibitor rhizoxin. Up-regulation also affected other RNA polymerase I (pol I) transcription units, as procyclin genes were also up-regulated after cells were treated with either aphidicolin or DNA-modifying agents. Strikingly, this up-regulation of silent pol I transcription units was bloodstream form-specific and was not observed in insect form T. brucei. We postulate that the redistribution of a limiting bloodstream form-specific factor involved in both silencing and DNA repair results in the derepression of normally silenced pol I transcription units after DNA damage.

The Trypanosoma brucei reference strain TREU927/4 contains T. brucei rhodesiense-specific SRA sequences, but displays a distinct phenotype of relative resistance to human serum.

The Trypanosoma brucei reference strain TREU927/4 exhibits some resistance to lysis by normal human serum (NHS), but this resistance is never complete even after selection. The genome of this strain contains a minimum of eight sequences related to the T. brucei rhodesiense-specific serum resistance-associated gene (SRA), which encodes a truncated variant surface glycoprotein (VSG) conferring full resistance to lysis by NHS. We selected two sequences showing the highest similarity to SRA and also preceded by a region ("cotransposed region") present immediately upstream from SRA in the VSG expression site termed R-ES, where SRA is expressed in T. brucei rhodesiense. Whereas one of these sequences appears to be a pseudogene, the other, which is contained within a cluster of VSG basic copies (BCs), encodes a VSG truncated in the C-terminal domain. In the latter gene, an inserted region encoding surface-exposed loops similar to those of the BoTat 1.20 VSG interrupts the full SRA sequence. Therefore, this gene was termed SRA-BC, for the putative VSG basic copy from which SRA was derived. Neither this gene nor other SRA-like sequences appeared to be responsible for the relative resistance of TREU927/4 to NHS, since (i) transfection of SRA-BC in T. brucei brucei did not confer increased resistance; (ii) SRA transcripts could not be detected in this strain, even when focusing the search on the limited SRA sequence necessary to confer resistance and when using strain-specific SRA probes on RNA from cells selected in the presence of NHS.

RNA polymerase I transcribes procyclin genes and variant surface glycoprotein gene expression sites in Trypanosoma brucei.

In eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to alpha-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. brucei cell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, alphabeta-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

Structural features affecting variant surface glycoprotein expression in Trypanosoma brucei.

The glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) of Trypanosoma brucei is the most abundant GPI-anchored protein expressed on any cell, and is an essential virulence factor. To determine what structural features affect efficient expression of VSG, we made a series of mutations in two VSGs. Inserting 18 amino acids, between the amino- and carboxy-terminal domains, reduced the expression of VSG 221 to about 3% of the wild-type level. When this insertion was combined with deletion of the single carboxy-terminal subdomain, expression was reduced a further three-fold. In VSG 117, which contains two carboxy-terminal subdomains, point mutation of the intervening N-glycosylation site reduced expression about 15-fold. Deleting the most carboxy-terminal subdomain and intervening region, including the N-glycosylation site, reduced expression to 15-20% of wild type VSG, and deletion of both subdomains reduced expression to <1%. Despite their low abundance, all VSG mutants were GPI anchored on the cell surface. Our results suggest that, for a protein to be efficiently displayed on the surface of bloodstream-form T. brucei, it is essential that it contains the conserved structural motifs of a T. brucei VSG. Serum resistance-associated protein (SRA), which confers human infectivity on T. brucei, strongly resembles a VSG deletion mutant. Expression of three epitope-tagged versions of SRA in T. brucei conferred total resistance to human serum. SRA possesses a canonical GPI signal sequence, but we were unable to obtain unequivocal evidence for the presence of a GPI anchor. SRA was not released during osmotic lysis, indicating that it is not GPI anchored on the cell surface.

The transferrin receptor genes of Trypanosoma equiperdum are less diverse in their transferrin binding site than those of the broad-host range Trypanosoma brucei.

Trypanosoma brucei and T. equiperdum infect the mammalian bloodstream and tissues. T. brucei is transmitted by tsetse flies between an extremely large range of mammals in sub-Saharan Africa. In contrast, T. equiperdum is restricted to equines, where it is transmitted as a venereal disease. Both species evade immune destruction by changing their variant surface glycoprotein (VSG), encoded in a telomeric VSG expression site. T. brucei has about 20 VSG expression sites, and it has been proposed that their genetic diversity plays a role in host adaptation. Two expression site-associated genes ESAG6 and ESAG7, encode variable transferrin receptor subunits allowing trypanosomes to internalize polymorphic transferrin molecules from different mammals. We investigated if there was a correlation between the size of the trypanosome host range and the degree of ESAG6 genetic diversity. Both T. equiperdum and T. brucei appear to have approximately similar numbers of ESAG6, however, the genetic diversity of the ESAG6 family varies in the two species. We sequenced 114 T. equiperdum ESAG6 genomic clones, resulting in the isolation of 10 T. equiperdum ESAG6 variants. The T. equiperdum ESAG6 genes were less genetically diverse than those of T. brucei in regions known to play a role in transferrin binding. This indicates that ESAG6 genetic diversity playing a role in host adaptation could have been lost in the absence of selection pressure. There was also evidence of positive selection ( d(N) /d(S) = approximately 5) acting on other ESAG6 regions not involved in transferrin binding, perhaps due to antigenic variation of these surface molecules.

Delineation of the regulated Variant Surface Glycoprotein gene expression site domain of Trypanosoma brucei.

The African trypanosome Trypanosoma brucei is protected in the bloodstream of the mammalian host by a dense Variant Surface Glycoprotein (VSG) coat. Although an individual cell has hundreds of VSG genes, the active VSG is transcribed in a mutually exclusive fashion from one of about twenty telomeric VSG expression sites. Expression sites are regulated domains flanked by 50 bp repeat arrays and extensive tracts of repetitive elements. We have integrated exogenous rDNA and expression site promoters upstream of the 50 bp repeats of the VO2 VSG expression site. Transcription from both types of exogenous promoter is downregulated and comparable to promoters targeted into the VSG Basic Copy arrays. We show that the upstream exogenous rDNA promoter escapes VSG expression site control, as switching the downstream VO2 VSG expression site on and off does not affect its activity. Therefore, the 50 bp repeat arrays appear to be the boundary of the regulated expression site domain.

Ku Is Important for Telomere Maintenance, but Not for Differential Expression of Telomeric VSG Genes, in African Trypanosomes

Trypanosome antigenic variation, involving differential expression of variant surface glycoprotein (VSG) genes, has a strong association with telomeres and with DNA recombination. All expressed VSGs are telomeric, and differential activation involves recombination into the telomeric environment or silencing/activation of subtelomeric promoters. A number of pathogen contingency gene systems associated with immune evasion involve telomeric loci, which has prompted speculation that chromosome ends provide conditions conducive for the operation of rapid gene switching mechanisms. Ku is a protein associated with eukaryotic telomeres that is directly involved in DNA recombination and in gene silencing. We have tested the hypothesis that Ku in trypanosomes is centrally involved in differential VSG expression. We show, via the generation of null mutants, that trypanosome Ku is closely involved in telomere length maintenance, more so for a transcriptionally active than an inactive telomere, but exhibits no detectable influence on DNA double strand break repair. The absence of Ku and the consequent great shortening of telomeres had no detectable influence either on the rate of VSG switching or on the silencing of the telomeric promoters of the VSG subset that is expressed in the tsetse fly.

Mutational analysis of the variant surface glycoprotein GPI-anchor signal sequence in Trypanosoma brucei.

The variant surface glycoproteins (VSG) of Trypanosoma brucei are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) anchor. All GPI-anchored proteins are synthesized with a C-terminal signal sequence, which is replaced by a GPI-anchor in a rapid post-translational transamidation reaction. VSG GPI signal sequences are extraordinarily conserved. They contain either 23 or 17 amino acids, a difference that distinguishes the two major VSG classes, and consist of a spacer sequence followed by a more hydrophobic region. The omega amino acid, to which GPI is transferred, is either Ser, Asp or Asn, the omega+2 amino acid is always Ser, and the omega+7 amino acid is almost always Lys. In order to determine whether this high conservation is necessary for GPI anchoring, we introduced several mutations into the signal peptide. Surprisingly, changing the most conserved amino acids, at positions omega+1, omega+2 and omega+7, had no detectable effect on the efficiency of GPI-anchoring or on protein abundance. Several more extensive changes also had no discernable impact on GPI-anchoring. Deleting the entire 23 amino-acid signal sequence or the 15 amino-acid hydrophobic region generated proteins that were not anchored. Instead of being secreted, these truncated proteins accumulated in the endoplasmic reticulum prior to lysosomal degradation. Replacing the GPI signal sequence with a proven cell-surface membrane-spanning domain reduced expression by about 99% and resulted not in cell surface expression but in accumulation close to the flagellar pocket and in non-lysosomal compartments. These results indicate that the high conservation of the VSG GPI signal sequence is not necessary for efficient expression and GPI attachment. Instead, the GPI anchor is essential for surface expression of VSG. However, because the VSG is a major virulence factor, it is possible that small changes in the efficiency of GPI anchoring, undetectable in our experiments, might have influenced the evolution of VSG GPI signal sequences.

A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei.

In the mammalian host, African trypanosomes generate consecutive waves of parasitaemia by changing their antigenic coat. Because this coat consists of a single type of variant surface glycoprotein (VSG), the question arises of how a trypanosome accomplishes the transcription of only one of a multi-allelic family of VSG expression site loci to display a single VSG type on the surface at any one time. No major differences have been detected between the single active expression site and the cohort of inactive expression sites. Here we identify an extranucleolar body containing RNA polymerase I (pol I) that is transcriptionally active and present only in the bloodstream form of the parasite. Visualization of the active expression site locus by tagging with green fluorescent protein shows that it is specifically located at this unique pol I transcriptional factory. The presence of this transcriptional body in postmitotic nuclei and its stability in the nucleus after DNA digestion provide evidence for a coherent structure. We propose that the recruitment of a single expression site and the concomitant exclusion of inactive loci from a discrete transcriptional body define the mechanism responsible for VSG mono-allelic expression.