Variant Surface Glycoprotein Gene Expression Site Research Articles

Biological Variation Among African Trypanosomes: I. Clonal Expression of Virulence Is Not Linked to the Variant Surface Glycoprotein or the Variant Surface Glycoprotein Gene Telomeric Expression Site

The potential association of variant surface glycoprotein (VSG) gene expression with clonal expression of virulence in African trypanosomes was addressed. Two populations of clonally related trypanosomes, which differ dramatically in virulence for the infected host, but display the same apparent VSG surface coat phenotype, were characterized with respect to the VSG genes expressed as well as the chromosome telomeric expression sites (ES) utilized for VSG gene transcription. The VSG gene sequences expressed by clones LouTat 1 and LouTat 1A of Trypanosoma brucei rhodesiense were identical, and gene expression in both clones occurred precisely by the same gene conversion events (duplication and transposition), which generated an expression-linked copy (ELC) of the VSG gene. The ELC was present on the same genomic restriction fragments in both populations and resided in the telomere of a 330-kb chromosome; a single basic copy of the LouTat 1/1A VSG gene, present in all variants of the LouTat 1 serodeme, was located at an internal site of a 1.5-Mb chromosome. Restriction endonuclease mapping of the ES telomere revealed that the VSG ELC of clones LouTat 1 and 1A resides in the same site. Therefore, these findings provide evidence that the VSG gene ES and, potentially, any cotranscribed ES-associated genes do not play a role in the clonal regulation of virulence because trypanosome clones LouTat 1 and 1A, which differ markedly in their virulence properties, both express identical VSG genes from the same chromosome telomeric ES.

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.

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.

Biosynthesis and function of the modified DNA base beta-D-glucosyl-hydroxymethyluracil in Trypanosoma brucei.

beta-D-Glucosyl-hydroxymethyluracil, also called J, is a modified DNA base conserved among kinetoplastid flagellates. In Trypanosoma brucei, the majority of J is present in repetitive DNA but the partial replacement of thymine by J also correlates with transcriptional repression of the variant surface glycoprotein (VSG) genes in the telomeric VSG gene expression sites. To gain a better understanding of the function of J, we studied its biosynthesis in T. brucei and found that it is made in two steps. In the first step, thymine in DNA is converted into hydroxymethyluracil by an enzyme that recognizes specific DNA sequences and/or structures. In the second step, hydroxymethyluracil is glucosylated by an enzyme that shows no obvious sequence specificity. We identified analogs of thymidine that affect the J content of the T. brucei genome upon incorporation into DNA. These analogs were used to study the function of J in the control of VSG gene expression sites. We found that incorporation of bromodeoxyuridine resulted in a 12-fold decrease in J content and caused a partial derepression of silent VSG gene expression site promoters, suggesting that J might strengthen transcriptional repression. Incorporation of hydroxymethyldeoxyuridine, resulting in a 15-fold increase in the J content, caused a reduction in the occurrence of chromosome breakage events sometimes associated with transcriptional switching between VSG gene expression sites in vitro. We speculate that these effects are mediated by the packaging of J-containing DNA into a condensed chromatin structure.

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.

Characterization of the ligand-binding site of the transferrin receptor in Trypanosoma brucei demonstrates a structural relationship with the N-terminal domain of the variant surface glycoprotein.

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N-terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface-exposed loops and generate the ligand-binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG-like structure abolished the binding. Chimeric proteins containing the N-terminal dimerization domain of VSG and the C-terminal half of either pESAG7 or pESAG6, which contains the ligand-binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N-terminal domain of the VSG and that the ligand-binding site corresponds to the exposed surface loops of the protein.

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.

Families of adenylate cyclase genes in Trypanosoma brucei

Four genes for adenylate cyclase have been characterizzed in Trypanosoma brucei. One of them, esag 4 (for expression site associated gene 4) is present in different VSG (variant surface glycoprotein) gene expression sites and, thus, is only expressed in the bloodstream form of the parasite. The others, termed gresag 4.1, 4.2 and 4.3 (for genes related to esag 4) are expressed in both bloodstream and procyclic forms. In addition, we cloned a esag 4-related gene from T. congolense. Here we characterize the genomic organization of gresag 4.1 and 4.3. While gresag 4.3 is unique, gresag 4.1 exists as a multigenic family of at least nine members located on a 3-Mb chromosome. Six of them are clustered in a region of 300 kb, three copies being tandemly linked. The determination of the nucleotide sequence of a conserved 1.6 kb PstI fragment demonstrated the presence of two separate subgroups in this family. This gene arrangement is present in different isolates of T.b. brucei/rhodesiense/gambiense. Several gresag 4.1 copies are transcribed in both bloodstream and procyclic forms.

Analysis of a hybrid [formula omitted] promoter in procyclic trypanosomes

The parasite Trypanosoma brucei changes its variant surface glycoprotein (VSG) coat to escape the host immune system. At a chromosomal locus, we analyzed the promoter that controls expression of VSG genes, using a system developed in collaboration with Ürményi and Van der Ploeg (Ürményi, T.P. and Van der Ploeg, L.H.T. (1995) Nucleic Acids Res. 23, 1010–1016), and showed that the variant surface glycoprotein expression site (VSG ES) promoter directed <6% the CAT activity produced by the procyclic acidic repetitive protein (PARP) promoter at the same locus. We identified a fragment from the PARP promoter (bp −743 to −111) that contained no intrinsic promoter activity. However, when this fragment was cloned 5′ to 3′ upstream of the VSG ES promoter, and this hybrid PARP VSG ES promoter was stably integrated at the RNA polymerase (Pol) II largest subunit gene locus, expression from a CAT gene cassette increased 10-fold. Nascent RNA analysis independently showed that the relative efficiency of α-amanitin-resistant transcription directed by the hybrid PARP VSG ES promoter was more than 6-fold higher than that directed by the wild-type VSG ES promoter. Furthermore, using nascent RNA protection assays, we mapped the transcription start site of the hybrid PARP VSG ES promoter to the same initiation site as that of the wild-type VSG ES promoter. Finally, we evaluated the functional activity of the hybrid PARP VSG ES mutant promoter at the dominant VSG gene expression site on the 1.5-Mb chromosome. At this locus, as well, the hybrid PARP VSG ES promoter directed almost 3-times as much CAT activity as that of the wild-type VSG ES promoter.

Targeting of exogenous DNA into Trypanosoma brucei requires a high degree of homology between donor and target DNA

Integration of exogenous DNA into the trypanosome genome occurs by homologous recombination only. To test whether a high degree of homology between donor and target DNA is required, we have inserted marker genes for drug resistance into the promoter area of variant surface glycoprotein (VSG) gene expression sites of Trypanosoma brucei, using targeting fragments from two expression sites that are 92% identical. We observed integrations into expression sites that are known to be perfectly matched to the donor flanks, and into subsets of uncharacterized expression sites that are specific for each type of targeting fragment, and that could be similar or identical to the donor flanks. This requirement for very high homology was found in both procyclic and bloodstream-form trypanosomes. We speculate that trypanosomes have a mismatch repair system that suppresses recombination between divergent DNA sequences, and we discuss ways in which the trypanosome might circumvent the requirement for perfect DNA homology in the duplicative transposition of a VSG gene into a VSG gene expression site.

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 gene expression in trypanosomes.

Trypanosomes are protozoan agents of major parasitic diseases such as Chagas' disease in South America and sleeping sickness of humans and nagana disease of cattle in Africa. They are transmitted to mammalian hosts by specific insect vectors. Their life cycle consists of a succession of differentiation and growth phases requiring regulated gene expression to adapt to the changing extracellular environment. Typical of such stage-specific expression is that of the major surface antigens of Trypanosoma brucei, procyclin in the procyclic (insect) form and the variant surface glycoprotein (VSG) in the bloodstream (mammalian) form. In trypanosomes, the regulation of gene expression is effected mainly at posttranscriptional levels, since primary transcription of most of the genes occurs in long polycistronic units and is constitutive. The transcripts are processed by transsplicing and polyadenylation under the influence of intergenic polypyrimidine tracts. These events show some developmental regulation. Untranslated sequences of the mRNAs seem to play a prominent role in the stage-specific control of individual gene expression, through a modulation of mRNA abundance. The VSG and procyclin transcription units exhibit particular features that are probably related to the need for a high level of expression. The promoters and RNA polymerase driving the expression of these units resemble those of the ribosomal genes. Their mutually exclusive expression is ensured by controls operating at several levels, including RNA elongation. Antigenic variation in the bloodstream is achieved through DNA rearrangements or alternative activation of the telomeric VSG gene expression sites. Recent discoveries, such as the existence of a novel nucleotide in telomeric DNA and the generation of point mutations in VSG genes, have shed new light on the mechanisms and consequences of antigenic variation.

A foreign transcription unit in the inactivated VSG gene expression site of the procyclic form of Trypanosoma brucei and formation of large episomes in stably transformed trypanosomes

Transcription of the variant surface glycoprotein (VSG) gene expression site (ES) in Trypanosoma brucei is inactivated upon differentiation from the bloodstream form to the insect-adapted, procyclic form. This paper demonstrates that a foreign transcription unit, containing a procyclic acidic repetitive protein (PARP) gene promoter driving a neomycin phosphotransferase ( neo) gene, can be fully active once integrated at the lingering, transcriptionally inactive VSG ES of procyclic trypanosomes. Following targeting into the ES two types of transformants were identified. Type one transformants were generated by integration of the PARP- neo gene into the region downstream of the long 70-bp repeat array of the silent telomeric ES encoding VSG gene 118. α-amanitin-resistant transcription at the neo locus proceeded from the PARP promoter to approximately 2.5 kb downstream of the integration site and terminated in front of the VSG 118 gene. Type two transformants contained variously sized large episomes (ranging from 135 kb to 500 kb), consisting of tandemly linked input plasmids. Transcription of the neo gene in the episomes was also resistant to α-amanitin. The presence of large amounts of the active episomal PARP promoter did not significantly affect the transcription of most RNA polymerase II transcribed genes, but resulted in a significant and equal reduction of the transcriptional efficiency of the endogenous PARP genes and VSG gene promoter sequences. This observation suggests that transcription of the PARP gene and the VSG gene expression sites in insect form trypanosomes may share a common transcriptional machinery.

Control of gene expression in trypanosomes.

Trypanosomes are protozoan agents of major parasitic diseases such as Chagas' disease in South America and sleeping sickness of humans and nagana disease of cattle in Africa. They are transmitted to mammalian hosts by specific insect vectors. Their life cycle consists of a succession of differentiation and growth phases requiring regulated gene expression to adapt to the changing extracellular environment. Typical of such stage-specific expression is that of the major surface antigens of Trypanosoma brucei, procyclin in the procyclic (insect) form and the variant surface glycoprotein (VSG) in the bloodstream (mammalian) form. In trypanosomes, the regulation of gene expression is effected mainly at posttranscriptional levels, since primary transcription of most of the genes occurs in long polycistronic units and is constitutive. The transcripts are processed by transsplicing and polyadenylation under the influence of intergenic polypyrimidine tracts. These events show some developmental regulation. Untranslated sequences of the mRNAs seem to play a prominent role in the stage-specific control of individual gene expression, through a modulation of mRNA abundance. The VSG and procyclin transcription units exhibit particular features that are probably related to the need for a high level of expression. The promoters and RNA polymerase driving the expression of these units resemble those of the ribosomal genes. Their mutually exclusive expression is ensured by controls operating at several levels, including RNA elongation. Antigenic variation in the bloodstream is achieved through DNA rearrangements or alternative activation of the telomeric VSG gene expression sites. Recent discoveries, such as the existence of a novel nucleotide in telomeric DNA and the generation of point mutations in VSG genes, have shed new light on the mechanisms and consequences of antigenic variation.

Expression site-associated genes transcribed independently of variant surface glycoprotein genes in Trypanosoma brucei

Expression site-associated genes (ESAGs) of Trypanosoma brucei are found upstream of variant surface glycoprotein (VSG) genes in bloodstream expression sites. There are at least 6 different ESAGs in each of these expression sites, and each ESAG is repetitive in the genome. ESAGs are believed to reside only in VSG expression sites and to be co-transcribed with the VSG gene from a common α-amanitin-insensitive promoter. Our results show that this is not always true. The transcriptionally active 1.22 metacyclic expression site contains no ESAGs, but ESAGs are highly transcribed in these cells. The level of transcription indicates that more than one copy of each of these genes is active. Furthermore, some of these genes are transcribed, to produce steady state RNA, in procyclic culture cells which do not express the VSG gene: there is differential expression of ESAGs between the bloodstream and procyclic phases of the trypanosome life cycle. Thus ESAGs can be transcribed outwith an active VSG gene expression site and in the absence of expression of the VSG.

A similar gene is shared by both the variant surface glycoprotein and procyclin gene transcription units of Trypanosoma brucei.

The genes for the variant surface glycoprotein (VSG) and procyclin are expressed in a mutually exclusive manner during the life cycle of Trypanosoma brucei and synthesize the most abundant mRNAs specific to the bloodstream and procyclic stages of the parasite, respectively. Genes belonging to the polycistronic transcription unit of the VSG gene (expression site-associated genes [ESAGs]) are uniquely expressed in the bloodstream form, but some members of ESAG families (genes related to ESAGs [GRESAGs]) are independently transcribed outside the VSG gene expression site. We report here that a gene related to ESAG 2, GRESAG 2.1, is present and expressed in a procyclin gene transcription unit (PARP A locus), which is polycistronic. Members of the ESAG 2 family are thus present in the two major differentially stage-regulated transcription units of this parasite.

A Similar Gene Is Shared by Both the Variant Surface Glycoprotein and Procyclin Gene Transcription Units of Trypanosoma brucei

The genes for the variant surface glycoprotein (VSG) and procyclin are expressed in a mutually exclusive manner during the life cycle of Trypanosoma brucei and synthesize the most abundant mRNAs specific to the bloodstream and procyclic stages of the parasite, respectively. Genes belonging to the polycistronic transcription unit of the VSG gene (expression site-associated genes [ESAGs]) are uniquely expressed in the bloodstream form, but some members of ESAG families (genes related to ESAGs [GRESAGs]) are independently transcribed outside the VSG gene expression site. We report here that a gene related to ESAG 2, GRESAG 2.1, is present and expressed in a procyclin gene transcription unit (PARP A locus), which is polycistronic. Members of the ESAG 2 family are thus present in the two major differentially stage-regulated transcription units of this parasite.

Genetics of antigenic variation in African trypanosomes

The major surface antigens of Trypanosoma brucei are the VSG (variant surface glycoprotein) at the bloodstream stage, and procyclin at the procyclic stage. Variation in the VSG allows the parasite to escape the antibody response of its mammalian host. This occurs through either DNA rearrangement in the telomeric VSG gene expression site, or alternate activation, without DNA rearrangement, of different telomeric expression sites. The VSG and procyclin genes each belong to large, polycistronic transcription units. Although the promoters of these units are both active at the two main stages of the parasite life cycle, stage-specific controls operating at the level of RNA elongation and processing lead to strictly differential expression of the end products of the two units. Despite their mutually exclusive control of expression, the VSG and procyclin transcription units share common characteristics. Both contain a similar gene, and both are transcribed by the same type of RNA polymerase, unusually resistant to α-amanitin. Among the eight genes present in the VSG transcription unit, two may be involved in the synthesis of cyclic AMP. The function of the other genes is unknown.

Expression of a minichromosomal variant surface glycoprotein gene in Trypanosoma brucei

African trypanosomes contain numerous variant surface glycoprotein (VSG) genes, but express only one at a time. When different VSG genes are activated by gene duplicative or non-duplicative mechanisms, antigenic variation occurs. Although transcriptionally inactive VSG genes can have either an intra-chromosomal or a telomeric location, all expressed VSG genes so far examined are telomeric. Some VSG genes are located on minichromosomes of approximately 100 kilobases (kb), but all those thus far described are transcriptionally inactive. We have found that the single copy of the IsTat 1.1 VSG gene in the IsTaR 1 serodeme has a telomeric location on a minichromosome when inactive. When it is activated, it is not duplicated and remains on a minichromosome. These data indicate that minichromosomes contain or can acquire all the DNA sequences necessary in cis for VSG gene expression and antigenic switching. Of several sequences associated with expressed VSG genes or their transcripts, only the 76-base pair (bp) repeat element was found in minichromosomal DNA. Variant antigenic types (VATs) which occurred immediately before and after VAT-1, expressed VSG genes located on larger chromosomes. Thus, different chromosomes can act as VSG gene expression sites.