Anita Dirks-Mulder

A ribosomal DNA promoter replacing the promoter of a telomeric VSG gene expression site can be efficiently switched on and off in T. brucei

Trypanosoma brucei survives in the mammalian blood-stream by regularly changing its variant surface glycoprotein (VSG) coat. The active VSG gene is located in a telomeric expression site, and coat switching occurs either by replacing the transcribed VSG gene or by changing the expression site that is active. To determine whether VSG expression site control requires promoter-specific sequences, we replaced the active VSG expression site promoter in bloodstream-form T. brucei with a ribosomal DNA (rDNA) promoter. These transformants were fully infective in laboratory animals, and the rDNA promoter, which is normally constitutively active, was efficiently inactivated and reactivated in the context of the VSG gene expression site. As there is no sequence similarity between the VSG expression site promoter and the rDNA promoter, VSG expression site control does not involve sequences specific to the VSG expression site promoter. We conclude that an epigenetic mechanism, such as telomeric silencing, is involved in VSG expression site control in bloodstream-form T. brucei.

Control of variant surface glycoprotein gene-expression sites in Trypanosoma brucei

Trypanosoma brucei has 20 similar telomeric‐expression sites for variant surface glycoprotein genes. Expression sites appear to be controlled at the level of transcription initiation, resulting in only one site being active at any time. Switching between expression sites occurs at a low rate. To analyse the switching mechanism, we used trypanosomes with two expression sites tagged with two different drug‐resistance genes and selected these on agarose plates containing both drugs. Double‐resistant clones arose at a low frequency of 10−7 per cell, but these behaved as if they rapidly switched between the two tagged expression sites and lost double resistance in the absence of selection. Using in situ hybridization we found that only 10% of the double‐resistant cells had two fluorescent spots corresponding to transcribed expression sites. Our results suggest that: (i) a double expressor is not a stable intermediate in expression site switching; (ii) expression sites are not independently switched on and off; and (iii) expression sites can be in a ‘pre‐active’ silent state from which they can be readily activated.

Telomere exchange can be an important mechanism of Variant Surface Glycoprotein gene switching in Trypanosoma brucei

Trypanosoma brucei undergoes antigenic variation by changing its Variant Surface Glycoprotein (VSG) coat. Although there are up to a thousand VSG genes, only one is transcribed at a time from a telomeric VSG expression site. Switching can involve DNA rearrangements exchanging the active VSG gene, or transcriptional activation of a new expression site and transcriptional silencing of the old one. Determining the mechanism mediating a switch has not always been easy, as the many virtually identical copies of VSG gene expression sites complicate transcriptional analysis. To overcome this problem, we have used bloodstream form T. brucei with a single copy VSG gene in an active expression site marked with a hygromycin resistance gene. We allowed these transformants to undergo switching of the active VSG gene, via three different experimental methods. We were able to select large numbers of switched trypanosomes from a single infected mouse using a new microtitre-dish based procedure developed for this purpose. The drug sensitivity of the switched trypanosomes allowed us to determine the transcriptional state of the marked expression site, and polymerase chain reaction (PCR) amplification was used to determine whether the single copy drug resistance gene and VSG gene present in the marked expression site had been retained. These studies showed that telomere exchange, which has been considered rare, can in some cases be an important mechanism of VSG gene switching. We describe 4 telomere exchange events between the active VSG 221 expression site and 4 different chromosomes.

Selection for activation of a new variant surface glycoprotein gene expression site in Trypanosoma brucei can result in deletion of the old one.

The African trypanosome Trypanosoma brucei expresses the active variant surface glycoprotein (VSG) gene in a telomeric VSG gene expression site. We have generated trypanosomes with a neomycin resistance gene inserted behind an active VSG gene expression site promoter, and a hygromycin resistance gene behind a silent one. By alternating drug selection, we could select for trypanosomes that had switched between the two marked VSG gene expression sites. Surprisingly, trypanosomes that had activated a new VSG gene expression site had often lost the old one. Using polymerase chain reaction (PCR), we screened large numbers of switched trypanosomes and found that sequences lost invariably included the drug marker near the promoter, as well as the telomeric VSG gene many tens of kilobases away. We postulate that stable activation of a new expression site requires silencing of the old one. If silencing does not occur at a sufficient rate by normal switch-off, stable activation of the new site can only occur if the old site is lost in random deletion events. The fact that we pick up these normally infrequent deletions, indicates that inactivation of the old VSG expression site could be rate limiting during switching in our strain of T. brucei.

J‐binding protein increases the level and retention of the unusual base J in trypanosome DNA

The nuclear DNA of Trypanosoma brucei and other kinetoplastid flagellates contains the unusual base β‐d‐glucosyl‐hydroxymethyluracil, called J, replacing part of the thymine in repetitive sequences. We have described a 100 kDa protein that specifically binds to J in duplex DNA. We have now disrupted the genes for this J‐binding protein (JBP) in T. brucei. The disruption does not affect growth, gene expression or the stability of some repetitive DNA sequences. Unexpectedly, however, the JBP KO trypanosomes contain only about 5% of the wild‐type level of J in their DNA. Excess J, randomly introduced into T. brucei DNA by growing the cells in the presence of the J precursor 5‐hydroxymethyldeoxyuridine, is lost by simple dilution as the KO trypanosomes multiply, showing that JBP does not protect J against removal. In contrast, cells containing JBP lose excess J only sluggishly. We conclude that JBP is able to activate the thymine modification enzymes to introduce additional J in regions of DNA already containing a basal level of J. We propose that JBP is a novel DNA modification maintenance protein.