Molecular and Biochemical Parasitology 91 (1998) 67–76

Control of VSG gene expression sites in Trypanosoma brucei

Piet Borst *, Wilbert Bitter 1, Patricia A. Blundell, Ines Chaves, Mike Cross,Herlinde Gerrits, Fred van Leeuwen, Richard McCulloch 2, Martin Taylor 3,

Gloria Rudenko

Di6ision of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

Abstract

Antigenic variation in African trypanosomes continues to be one of the most elaborate and intriguing strategiesever devised by a protozoan parasite to avoid complete destruction by the immune defense of its mammalian host.Here we review some of the recent advances in our understanding of this strategy, concentrating on (unpublished)work from our laboratory. © 1998 Francqui Foundation. Published by Elsevier Science B.V. All rights reserved.

Keywords: Expression site; Trypanosoma brucei ; Modified base; J; Transferrin receptor; Antigenic variation

1. Basic features of antigenic variation in Africantrypanosomes

Fig. 1 specifies what is required to make asuccess of antigenic variation. We briefly discusssome of these points where new evidence is avail-able.

The basic features of surface coat switching inT. brucei have been amply reviewed in recentyears [1–10] and are summarized in Fig. 2. Asmost of the genes for Variant Surface Glyco-proteins (VSGs) are located in a chromosome-in-ternal position [11], switching mechanism A [12] isthe main route for switching the VSG gene in theactively transcribed telomeric VSG gene expres-sion site (ES). The presence of some 100 mini-chromosomes [13], which carry a VSG gene atleast at one end, in addition to some 25 largerchromosomes [14] also provides a substantialrepertoire of telomeric VSG genes that can enterthe active ES by mechanisms B or C. MechanismC, discovered by Etienne Pays and co-workers[15], initially appeared to be used only rarely, butrecent work shows that this is not always so [16].

* Corresponding author. Tel.: +31 20 5122880; fax: +3120 6691383.

1 Present address: Molecular Cell Biology, Utrecht Univer-sity, P.O. Box 80.056, 3508 TB Utrecht, The Netherlands.

2 Present address: Wellcome Unit of Molecular Parasitol-ogy, The Anderson College, 56 Dumbarton Road, GlasgowG11 6NU, UK.

3 Present address: Applied Molecular Biology Unit Dept. ofMedical Parasitology, London School of Hygiene and Tropi-cal Medicine, Keppel Street, London WC1E 7HT, UK.

0166-6851/98/$19.00 © 1998 Francqui Foundation. Published by Elsevier Science B.V. All rights reserved.

PII S 0166 -6851 (97 )00184 -9

P. Borst et al. / Molecular and Biochemical Parasitology 91 (1998) 67–7668

Fig. 1.

How chromosome-internal genes enter an ac-tive expression site has long been a matter ofspeculation [1,6,17]. It is clear that this processdepends on relatively short imperfect sequences.On the 5% side these sequences consist of a shortarray of 70 bp repeats. Long arrays of theserepeats are present in the expression site in frontof the active VSG gene (Fig. 3). At the 3% end thecross-over between incoming gene and the generesiding in the ES may occur anywhere in the3%-UTR of the VSG gene or behind this [18]. Onewould expect the 70 bp repeats to be essential forthe entry of most chromosome-internal genes intothe ES. It has also been speculated that the 70 bprepeats are required as a target for a nuclease thatinitiates the duplicative transposition of VSGgenes into an active ES (reviewed in [1,6]). Mc-Culloch et al. [19] have shown, however, thattelomere gene conversion (mechanism B in Fig. 2)can still occur when the 70 bp repeats are deletedfrom the active ES.

In principle the trypanosome would be able toexpress its whole repertoire of VSG genes in asingle ES using mechanisms A–C in Fig. 2. Inpractice, there are multiple ESs, allowing in situswitches (mechanism D). Hybridization experi-

ments indicate that there are about 20 ESs, atleast six of which are functional (and the othersmay be as well).

2. Why multiple expression sites?

The multiplicity of ESs requires complex con-trols to ensure that only one out of 20 ESs isactive at a time. Initially, it was unclear why thetrypanosome would make the management of itscoat repertoire more complex than strictly neces-sary. In the past two years we have found, how-ever, that a plausible raison d’etre for multipleESs does not so much reside in the additionalopportunities these provide for switching coat,but in the ability to switch the expression siteassociated genes (ESAGs) co-expressed with theVSG gene in each ES (Fig. 3).

Pioneering work by the Overath laboratory,extended by several other laboratories (reviewedby [2,5,20]), has shown that ESAGs 6 and 7encode two sub-units for a heterodimeric transfer-rin-receptor (Tf-R), located in the flagellar pocket.Receptor sub-units encoded by the different ESsdiffer in an interesting fashion in their sequence,

P. Borst et al. / Molecular and Biochemical Parasitology 91 (1998) 67–76 69

Fig. 2. The major mechanisms of VSG gene switching in T. brucei (from Borst et al., 1997 [5]). The thick lines represent the so-called221 VSG expression site, with the flag indicating a promoter, the black filled box the 221 VSG gene, and the white box anothertelomeric VSG gene (VSG gene X). V, Y and Z are other chromosome-internal VSG genes. The hygromycin resistance geneintroduced behind the promoter of the 221 expression site (see Fig. 1) is indicated by a diagonally striped box. The dashed line withan arrow indicates transcription. The figure illustrates how the different mechanisms can be distinguished, by using an expressionsite marked with a resistance gene. In mechanisms A–C, the Hygro gene is actively transcribed following the switch, whereas it issilent in mechanism D. In mechanisms A and B the single copy 221 VSG gene is lost. Whether A or B occurred can be derived fromknowing the original location of the new VSG gene. In C, the 221 VSG gene moves to another chromosome.

suggesting that multiple ESs exist to allow anti-genic variation of receptors [21]. More recentwork has shown, however, that receptor variabil-ity may primarily be required to allow T. brucei tocope with the diversity of Tfs in a range ofmammalian hosts [2].

The sequence of Tf has evolved rapidly inmammalian evolution and, for instance, humanand bovine Tfs differ by 30% in amino acidsequence [22]. One cannot expect a single receptorto tightly bind ligands that vary so much instructure and, indeed, we have found that Tf-Rsencoded by different ESs markedly differ in theiraffinity for Tfs from different mammals [2]. Astriking example is the very low affinity of theTf-R encoded in the 221 ES for canine Tf. Vari-ants expressing this ES grow fine in bovine andmurine serum (and bind the corresponding Tfswith high affinity), but hardly at all in canineserum. Prolonged growth of trypanosomes in ca-nine serum leads to selection of normally growingtrypanosomes that have activated the V02 ES,

which encodes a Tf-R with substantial affinity forcanine Tf. Sluggish growth of VAT 221a in canineserum (and switching to the VO2 ES) can becompletely prevented by adding 0.1 mg bovine Tfml−1, which is less than 0.01% of the normal Tfconcentration in serum [22a].

We think that our results on the Tf-R stronglysupport the idea that the multiplicity of ESs servesto allow T. brucei to colonize a large range ofmammals, each with its unique plasma composi-tion in which the trypanosome has to survive. Theancestral trypanosome may have had a single ESin which ESAGs were gradually collected to allowan efficient activation of a series of genes requiredfor survival in a small set of mammalian hosts.Duplication of this ES would have allowed thesecond set of ESAGs to evolve and adapt to newhosts. The implication of this hypothesis is thatthe other ESAGs might also be selected for inclu-sion in the ES because they are involved in func-tions that need to be varied depending on thehost. This remains to be verified.

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Fig. 3. Schematic representation of a VSG gene expression site, modeled after [55,56]. The flag indicates the promoter, the numbersin the boxes indicate the ESAGs. The enlargements show features of ESAG 6 and 7, which encode subunits of the transferrinreceptor, and the area of the expression site replaced during gene conversion of a basic copy VSG gene. Positions of marker insertionor DNA replacements are indicated below the expression site.

3. Control of expression sites; generalconsiderations

The increase in host range, provided by themultiplicity of ESs, is bought at a price. Thetrypanosome now needs mechanisms to keep allbut one of the 20 or so ESs silent and to switchbetween ESs. Some of the mechanisms that havebeen considered are summarized in Fig. 4. Twomajor models can be distinguished: one in whichthere is cross-talk between sites and one in whichsites are activated and inactivated independently.Several arguments have led to a consensus thatcross-talk does not occur: The most importantargument is that experimental evidence was ob-tained for the simultaneous activity of two ESs ina single trypanosome [23,24]. Although this evi-dence seemed conclusive in 1985, we have secondthoughts in 1997 and we return to these doubts

below. A second argument was that the control ofthe ES promoter appears to have a major negativecomponent. In a plasmid in transient transfectionassays the promoter appears to be fully activated[25–27] and even when the promoter is stablyinserted in other locations of the genome than itsnormal position, the promoter may be active[28,29].

Hence, most efforts in the past 10 years wentinto the analysis of mechanisms of ES controlbased on independent (in)activation of ESs (seeFig. 4). Control by DNA rearrangements close tothe promoter, e.g. by promoter inversion, wasruled out at an early stage [25], but it has beenmore difficult to rule out that DNA rearrange-ments far upstream of the promoter contribute tocontrol [30–32]. With the introduction of se-lectable markers into ESs [28,33,34], it becameeasier to select for in situ switches (mechanism D

P. Borst et al. / Molecular and Biochemical Parasitology 91 (1998) 67–76 71

in Fig. 2) and check for DNA rearrangementsmore systematically. Navarro and Cross [35]found no systematic major alterations associatedwith switching in the large block of 50 bp re-peats [25,32] upstream of the ES promoter (seeFig. 3) and this is in agreement with our unpub-lished results. Although minor alterations in the50 bp repeats or major rearrangements even fur-ther upstream (an area not yet checked) cannotbe excluded as the cause of ES promoter(in)activation, we consider this option nowunattractive.

4. Does the expression site have to move to thenucleolus to be transcribed?

There is considerable evidence that the ES istranscribed by an RNA polymerase resemblingpolymerase I [36–38] (reviewed by Van derPloeg and Lee, 1997 [39]), the polymerase thatnormally restricts its activity to the large rRNAgenes. As transcribed rRNA genes are usuallylocated in the nucleolus of eukaryotes, it is con-ceivable that the ES has to move to the nucleo-lus to be transcribed, as suggested by Van derPloeg [40]. If there is only a single ES attach-ment site per nucleolus for which the multipleESs compete, this would also provide an expla-nation for mutually exclusive expression of dif-ferent ESs.

To test this idea, Zomerdijk [41] used fluores-cent in situ hybridization (FISH) to determinethe intra-nuclear location of nascent RNAderived from the active ES. He found no co-lo-calization, but some investigators were not con-vinced by his results, because these were basedon hybridization with repetitive probes. In thepast year we have reinvestigated this issue, usingan ES containing unique marker genes. This hasconfirmed that the active ES is not in the nucle-olus, whereas marker genes located in the rRNAgene array and driven by a ribosomal DNApromoter do localize to the nucleolus (unpub-lished results). Although this does not excludethat the active ES is in a specialized nuclearsub-compartment, we are confident that the ac-tive compartment is not the nucleolus.

5. Is the activity of the expression site promotercontrolled by DNA modification?

An unusual form of DNA modification in T.brucei was independently discovered by Pays etal. [42] and Bernards et al. [43]. This modifica-tion affected PstI and P6uII sites in and aroundtelomeric VSG genes in inactive ESs, but not inthe active one. Modification was absent in chro-mosome-internal VSG genes and undetectable inDNA from insect-form trypanosomes. This ap-peared to link modification to the inactivationof ESs and raised the possibility that the activ-ity of ESs might be controlled by a competitionbetween transcription initiation and modification[43].

The identification of a novel modified DNAbase in T. brucei, b-glucosyl-hydroxymethylu-racil or J for short [44,45], has opened the wayfor a critical test of the relation between modifi-cation and expression site control. We haveraised antibodies against J that allow immuno-precipitation of J-containing DNA fragments.These have made it possible to locate J moreprecisely in the T. brucei genome and to detectsmaller amounts of J in other organisms.

The anti-J antibodies precipitate silent VSGgene fragments known to contain modified PstIand P6uII sites. We have also shown, using spe-Fig. 4.

P. Borst et al. / Molecular and Biochemical Parasitology 91 (1998) 67–7672

Fig. 5. DNA modification due to J in and around telomeric VSG expression sites. Model of the distribution of J in active andinactive VSG expression sites (see Fig. 3). The ES promoter is indicated by a flag, transcription by a dashed line. The degree ofmodification was determined by anti-J immunoprecipitation of sonicated DNA fragments combined with dot-blot hybridizations.For ESAGs and 70 bp repeats the cumulative signal of all copies in the genome was analyzed. Telomeric repeats, 50 bp repeats, VSGgenes and promoter regions were also studied in individual expression sites by Southern-blot hybridizations. The absence of J inactively transcribed VSG genes suggests that J is most likely also absent from active ESAGs and transcribed 70 bp repeats, but thishas not been tested directly. No immunoprecipitation of the inactive 221 ES promoter itself was detected with restriction fragmentscontaining part of the HYG gene inserted directly downstream of the promoter.

cific oligonucleotides containing J, that substitu-tion of the T in one strand of a P6uII recognitionsite blocks cleavage by the enzyme. As no othermodified nucleotides have been detected in T.brucei, we think that the presence of J can fullyaccount for the modified restriction endonucleasesites detected in silent telomeric VSG genes byBernards et al. [43] and Pays et al. [42].

However, the distribution of J in telomericDNA is more complex than could have beenanticipated from modified restriction endonucle-ase sites, as illustrated by the summary of ourdata presented in Fig. 5. The bulk of J is insequences that cannot be probed with restrictionendonucleases, the (GGGTTA)n repeats present atthe ends of trypanosome telomeres [46], and the50 bp repeats upstream of the ES promoter area.An unexpected result is that these repeats retaintheir J when the ES is activated. When an ES issilenced, substitution of T by J spreads over mostof the ES. The highest concentrations of J arefound in and around the VSG gene and in the 70bp repeats upstream of the VSG gene, but signifi-cant amounts of J are also found in the ESAGsand in the promoter area. No J has been detectedat all in chromosome-internal silent VSG genes orin (transcribed) house-keeping genes.

Although these results are compatible with themodel of Bernards et al. [43] for control of telom-

eric silencing by DNA modification, other resultsmake us less enthusiastic about this model. First,we have found that J is not restricted to Kineto-plastida that undergo antigenic variation, but isalso present in Trypanosoma cruzi, Leishmania,Crithidia and even in the distant bodonid/crypo-tobiid Trypanoplasma borreli, a fish parasite. Sec-ondly, we have succeeded in increasing ordecreasing the level of J in the trypanosomegenome 10-fold (by growing trypanosomes in nu-cleoside analogues) and this did not grossly affectthe rate of expression site switching. Finally, ourrecent work has shown that the modifying enzymeresponsible for the first step in J biosynthesis canrecognize a remarkably diverse number of se-quences, but only if these occur in a certaincontext, i.e. in or near repetitive sequences thatare not transcribed [46]. This suggests that modifi-cation requires a specific chromatin structure orsub-nuclear location of the DNA, which must beimposed before DNA modification.

Although these results do not disprove themodel of Bernards et al. [43], they indicate that Jmay stabilize transcriptional repression, ratherthan initiate it, and that J may have wider func-tions than in antigenic variation. An obvious pos-sibility is that J helps to repress recombinationbetween repeated sequences located on differentchromosomes. A tight link between transcrip-

P. Borst et al. / Molecular and Biochemical Parasitology 91 (1998) 67–76 73

tional repression and absence of recombinationhas been demonstrated in fission yeast [47]. Ifmodified DNA acts as a less effective donor forgene conversion than unmodified DNA, thiscould also help chromosome-internal VSG genesto compete against telomeric VSG genes for entryinto the active ES. It should also be noted that thepresence of J in ribosomal repeats (our unpub-lished results) correlates with observations in Sac-charomyces, showing that proteins involved intelomeric silencing and in transcriptional, repres-sion of silent mating type loci are also involved inthe normal function of the nucleolus (seeKennedy et al., 1997 [48], for a recent discussion).Obviously, these speculations can only be criti-cally tested when the level of J in the genome canbe drastically modified. This will probably requireidentification and disruption of genes involved inJ biosynthesis.

6. Sequences required for the control ofexpression sites

The last mechanism mentioned in Fig. 4 istelomeric silencing, as first proposed byGottschling et al. [49] and invoked both by theCross group [34,50] and our group [5,21,33].Telomeric silencing in yeast depends on a specificchromatin state, initiated by RAP1 binding totelomeric repeats, which recruits the SIR2–4 geneproducts, which in turn bind hypoacetylated his-tones. Histones lacking acetyl groups in specificpositions are thought to be critical for repression,but the exact mechanism is still far from clear (seediscussions by Pazin and Kadonaga, 1997 [51];Roth and Allis, 1997 [52]). Competition betweentranscription initiation and repression governs thefrequency of switching between active and silentstates [53]. Characteristic of telomeric silencing isthat it involves a contiguous sequence of DNA,that it acts on a variety of PolII promoters, andeven on PolIII promoters (D. Gottschling, per-sonal communication). Some of these characteris-tics also apply to ES silencing. Horn and Cross[34] have shown that a heterologous promoter inthe downstream part of an ES is largely silencedwhen the ES is silenced. We have analyzed the ES

promoter area and found that the ES promotercan be replaced by a ribosomal DNA promoterwithout drastically affecting silencing, or the rateof switching [33]. This shows that the switchingmechanism is not dependent on the precise se-quence of the promoter. These results are compat-ible with telomeric silencing, but also with othermechanisms for the control of ESs.

The minimal ES promoter of 310 bp [26] re-placed by a ribosomal DNA promoter is embed-ded in 3 kb of sequence that is rather conservedbetween ESs. To test whether these sequences areessential, we have deleted them in bloodstreamform T. brucei. Replacement of the sequencesbetween the core promoter and ESAG7 (3% ESsequences) had no effect on transcription, on theability of the trypanosomes to switch off the 221ES, nor on the completeness of the repression ofthe silenced site (unpublished results). It provedmore difficult to obtain transformants in which allsequences between the core promoter and the 50bp repeats (the 5% ES sequences) had been deletedor replaced (unpublished results). Nevertheless,the fact that such clones are obtained at all showsthat even the 5% ES sequences are not indispens-able for high rates of promoter activity in the EScontext. It therefore appears that this high activityrequires no unique sequence elements either in thecore promoter area (where a ribosomal replace-ment promoter will do), or 5% or 3% of this core.

We also find, however, that the repression ofthe silenced promoter is less complete when theES core promoter is replaced by a ribosomal corepromoter [33] or when the 5% ES sequences aredeleted or replaced by lambda DNA (unpublishedresults). There are therefore some sequence-spe-cific elements recognized by the silencing machin-ery, and this may explain the high conservation ofthe 5% ES sequences closest to the core promoter.

7. Is there cross-talk between VSG geneexpression sites?

In the mid 1980’s two papers appeared report-ing that bloodstream form trypanosomes can existwith two simultaneously active VSG gene ESs(so-called double expressors) [23,24]. This sug-

P. Borst et al. / Molecular and Biochemical Parasitology 91 (1998) 67–7674

gested that ESs are independently activated andinactivated and that cross-talk between ESs doesnot occur. Absence of cross-talk, although notformally proven, became a generally acceptedconcept and the basis for mechanistic studies ofthe control of ESs. It was therefore a surprise thatno double expressors were observed in switchingexperiments using a marked expression site[34,50]. To test in a more rigorous fashion fordouble expressors as intermediates in in situswitches (mechanism D in Fig. 2), we generatedtrypanosomes with two marked ESs. Althoughthe selectable markers made it possible to rapidlyselect trypanosomes that had switched from oneES to the other, we did not obtain true doubleexpressors (unpublished results). Selection forgrowth on both drugs led to low level activationof one site and maximal activity of the other, butnever to two ESs with the same (high) activity.Our results so far indicate that two ESs cannot bemaximally active at the same time. Further sup-port for this idea has come from our observationthat switching is often associated with the com-plete loss of the previously active ES (unpublishedresults). We have no reason to think that only thetwo ESs studied are incompatible and our presentassumption is that the results with these ESs willhold for all ESs.

If this assumption is correct, how did Cornelis-sen et al. [23] and Baltz et al. [24] get their doubleexpressors? With hindsight, we must admit thatthe results of Cornelissen et al. are not completelyconclusive, as there is an alternative explanationfor the data (a complex telomere exchange) thatcould not be tested with the methods available in1985. Baltz et al. [24] obtained double expressorsproducing mixed coats and their results look un-ambiguous. However, their results were obtainedwith T. equiperdum and as this unfortunate para-site is doomed to stay in equines, it may not needmultiple ESs anymore [2] and control of ESs mayhave become more relaxed. Formally, it is alsodifficult to exclude that the double expressorsobtained by Baltz et al. [24] were rapidly switch-ing between two ESs, rather than stably express-ing two ESs simultaneously and at maximal rate.

We therefore think that mechanisms for EScontrol involving cross-talk between ESs cannot

be discounted. At present, we do not know atwhat level this cross-talk occurs. As trypanosomesseem happy with mixed coats [54], counter selec-tion probably does not occur at the level of VSGs.There might be counter selection, however, at thelevel of ESAG products, mixed products beingsub-optimal, or competition for limiting factorsrequired for transcription. These factors could bediffusible, they could represent limited access to asub-nuclear compartment, or combinations ofthese. An obvious way to start testing these alter-natives, is to create an abbreviated ES in whichthe sequences between ES promoter and telomereare deleted. We have found that the appropriatetargeting construct works in procyclic insect-formtrypanosomes (unpublished results) and we are inthe process of repeating these experiments withbloodstream trypanosomes. It should be clearfrom this discussion that none of the mechanismslisted in Fig. 4 can be ruled out, with the excep-tion of DNA rearrangements close to the pro-moter. The tools are available, however, to settlethis important issue in the coming years.

Acknowledgements

The work was supported by grants from theNetherlands Organization for Scientific Research(NWO/SON) to P.B., the Wellcome Trust toR.McC. and M.T., the European Molecular Biol-ogy Organization and the European Commissionto M.C., and the Gulbenkian PhD Program inBiology and Medicine (Portugal) to I.C.

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