Telomeric Expression Sites Research Articles

Histone deacetylases play distinct roles in telomeric VSG expression site silencing in African trypanosomes.

African trypanosomes evade the host immune response through antigenic variation, which is achieved by periodically expressing different variant surface glycoproteins (VSGs). VSG expression is monoallelic such that only one of approximately 15 telomeric VSG expression sites (ESs) is transcribed at a time. Epigenetic regulation is involved in VSG control but our understanding of the mechanisms involved remains incomplete. Histone deacetylases are potential drug targets for diseases caused by protozoan parasites. Here, using recombinant expression we show that the essential Trypanosoma brucei deacetylases, DAC1 (class I) and DAC3 (class II) display histone deacetylase activity. Both DAC1 and DAC3 are nuclear proteins in the bloodstream stage parasite, while only DAC3 remains concentrated in the nucleus in insect-stage cells. Consistent with developmentally regulated localization, DAC1 antagonizes SIR2rp1-dependent telomeric silencing only in the bloodstream form, indicating a conserved role in the control of silent chromatin domains. In contrast, DAC3 is specifically required for silencing at VSG ES promoters in both bloodstream and insect-stage cells. We conclude that DAC1 and DAC3 play distinct roles in subtelomeric gene silencing and that DAC3 represents the first readily druggable target linked to VSG ES control in the African trypanosome.

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.

Telomeric Expression Sites Are Highly Conserved in Trypanosoma brucei

Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs) of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T. brucei were independently tagged and sequenced. BESs are polymorphic in size and structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs do exist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs). The consequences of duplicated or missing ESAGs, including ESAG9, a newly named ESAG12, and additional variant surface glycoprotein genes (VSGs) were evaluated by functional assays after BESs were tagged with a drug-resistance gene. Phylogenetic analysis of constituent ESAG families suggests that BESs are sequence mosaics and that extensive recombination has shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecular mechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.

Bioinformatic insights to the ESAG5 and GRESAG5 gene families in kinetoplastid parasites

Trypanosoma brucei, the causative agent of African sleeping sickness, evades the immune response by expressing a coat of variant surface glycoprotein (VSG). VSG is expressed from a single telomeric expression site (ES), along with a number of expression site associated genes ( ESAGs). Thus far, the function of most ESAGs is unknown. One ES contains the serum resistance associated gene ( SRA), which confers resistance to trypanosome lytic factor in T. b. rhodesiense. Only three other ESAGs – 5, 6 and 7 – are present in this ES. ESAGs 6 and 7 encode a heterodimeric transferrin receptor, but the function of ESAG5 has not been identified. We present here a bioinformatic analysis of ESAG5 and distinguish between T. brucei-specific ESAGs and Genes Related to ESAG5 ( GRESAGs), which occur outside of ESs in chromosomal-internal contexts. Further, a genome-wide survey of these genes across kinetoplastids identifies a family of GRESAG5s in a number of species. Analysis of phylogenetic relationships indicates that this family may have evolved from a single ancestral copy. Predicted properties of (GR)ESAG5 proteins indicate a glycosylated protein containing either a signal peptide or transmembrane domain. Further analysis indicates a possible relationship to the lipid transfer/lipopolysaccharide-binding family which includes the bactericidal/permeability increasing (BPI) protein. Together, these results provide insights into the structure and evolution of an important extended gene family, and present a number of testable hypotheses which will aid in elucidating the function of ESAG5.

Telomere length in Trypanosoma brucei

Trypanosoma brucei thwarts the host immune response by replacing its variant surface glycoprotein (VSG). The actively transcribed VSG is located in one of ∼20 telomeric expression sites (ES). Antigenic variation can occur by transcriptional switching, reciprocal translocations, or duplicative gene conversion events among ES or with the large repertoire of telomeric and non-telomeric VSG. In recently isolated strains, duplicative gene conversion occurs at a high frequency and predominates, but the switching frequency decreases dramatically upon laboratory-adaptation. Uniquely, T. brucei telomeres grow—apparently indefinitely—at a steady rate of 6–12 base pairs (bp) per population doubling (PD), but the telomere adjacent to an active ES undergoes frequent truncations. Using two-dimensional gel electrophoresis, we demonstrate that all of the chromosome classes of fast-switching and minimally propagated T. brucei have shorter telomeres than extensively propagated Lister 427 clones, suggesting a link between laboratory adaptation, telomere growth, and VSG switching rates.

The variant surface glycoprotein as a tool for adaptation in African trypanosomes

African trypanosomes (prototype: Trypanosoma brucei) are flagellated protozoan parasites that infect a wide variety of mammals, causing nagana in cattle and sleeping sickness in humans. These organisms can cause prolonged chronic infections due to their ability to successively expose different antigenic variants of the variant surface glycoprotein (VSG). The genomic loci where the VSG genes are expressed are telomeric and contain polycistronic transcription units with several genes that are involved in adaptation of the parasite to the host. At least three of these genes, which respectively encode the two subunits of the heterodimeric receptor for transferrin and a protein conferring resistance to the human trypanolytic factor apolipoprotein L-I, share the same origin as the VSG. The high recombination potential of the telomeric VSG expression sites, coupled to their dynamic mono-allelic expression control, provides trypanosomes with a powerful capacity for adaptation to their hosts.

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.

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.

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.

The architecture of variant surface glycoprotein gene expression sites in Trypanosoma brucei

Trypanosoma brucei evades the immune system by switching between Variant Surface Glycoprotein (VSG) genes. The active VSG gene is transcribed in one of approximately 20 telomeric expression sites (ESs). It has been postulated that ES polymorphism plays a role in host adaptation. To gain more insight into ES architecture, we have determined the complete sequence of Bacterial Artificial Chromosomes (BACs) containing DNA from three ESs and their flanking regions. There was variation in the order and number of ES-associated genes ( ESAGs). ESAGs 6 and 7, encoding transferrin receptor subunits, are the only ESAGs with functional copies in every ES that has been sequenced until now. A BAC clone containing the VO2 ES sequences comprised approximately half of a 330 kb ‘intermediate’ chromosome. The extensive similarity between this intermediate chromosome and the left telomere of T. brucei 927 chromosome I, suggests that this previously uncharacterised intermediate size class of chromosomes could have arisen from breakage of megabase chromosomes. Unexpected conservation of sequences, including pseudogenes, indicates that the multiple ESs could have arisen through a relatively recent amplification of a single ES.

Expression site activation in Trypanosoma brucei with three marked variant surface glycoprotein gene expression sites

The genes for the Variant Surface Glycoprotein (VSG) of Trypanosoma brucei are transcribed in telomeric expression sites (ESs). There are about 20 different ESs per trypanosome nucleus. Usually, only one is active at a time, but trypanosomes can switch the ES that is active at a low rate (<10 −5 per cell per generation). To study activation and silencing of ESs, we have generated a line of T. brucei 427 with three ESs marked with a different drug resistance gene. We show that a selection with any combination of two of these drugs leads to an unstable double-resistant phenotype in which the two ESs containing the corresponding marker genes switch backward and forward at a very high rate (>10 −1 per cell per generation). Unstable triple-resistant trypanosomes were not obtained. We conclude that the unstable rapid-switching state is a natural intermediate in ES switching. It only involves two ESs, whereas the other ESs are not expressed. Furthermore, we show that ‘inactive’ ESs can exist at several different stable levels of activation. Whereas, a ‘silent’ ES shows a low level of expression of promoter proximal sequences, the level of activation can be reversibly increased, leading to partially activated ESs.

An update on antigenic variation in African trypanosomes.

African trypanosomes can spend a long time in the blood of their mammalian host, where they are exposed to the immune system and are thought to take advantage of it to modulate their own numbers. Their major immunogenic protein is the variant surface glycoprotein (VSG), the gene for which must be in one of the 20--40 specialized telomeric expression sites in order to be transcribed. Trypanosomes escape antibody-mediated destruction through periodic changes of the expressed VSG gene from a repertoire of approximately 1000. How do trypanosomes exclusively express only one VSG and how do they switch between them?

Organization of telomeres during the cell and life cycles of Trypanosoma brucei.

The genome of Trypanosoma brucei contains about 120 chromosomes, which do not visibly condense during mitosis. We have analyzed the organization and segregation of these chromosomes by in situ hybridization using fluorescent telomere probes. At the onset of mitosis, telomeres migrate from their nuclear peripheral location and congregate into a central zone. This dense group of telomeres then splits into two entities that migrate to opposite nuclear poles. Segregation continues until the double-sized nucleus divides and, before cytokinesis occurs, the telomeres reorganize into the discrete foci observed at interphase. During migration, the telomeres are located at the free end of the mitotic spindle. Treatment with the microtubule polymerization inhibitor rhizoxin prevents telomere clustering and chromosomal segregation. In the insect-specific procyclic form as well as in the non-dividing bloodstream stumpy form, telomeres tend to cluster close to the nuclear periphery at interphase. In contrast, in the proliferative bloodstream slender form the telomeres preferentially locate in the central zone of the nucleus. Thus, telomeres are closer to the nuclear periphery during those life cycle stages where the telomeric expression sites for the variant surface glycoprotein are all inactive, suggesting that transcriptional inactivation of these sites is related to their subnuclear localization.

Differential RNA elongation controls the variant surface glycoprotein gene expression sites of Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei develops antigenic variation to escape the immune response of its host. To this end, the trypanosome genome contains multiple telomeric expression sites competent for transcription of variant surface glycoprotein genes, but as a rule only a single antigen is expressed at any time. We used reverse transcription-PCR (RT-PCR) to analyse transcription of different segments of the expression sites in different variant clones of two independent strains of T. brucei. The results indicated that RNA polymerase is installed and active at the beginning of many, if not all, expression sites simultaneously, but that a progressive arrest of RNA elongation occurs in all but one site. This defect is linked to inefficient RNA processing and RNA release from the nucleus. Therefore, functional transcription in the active site appears to depend on the selective recruitment of a RNA elongation/processing machinery.

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.

In situ analysis of a variant surface glycoprotein expression-site promoter region in Trypanosoma brucei

In Trypanosoma brucei, the active variant surface glycoprotein genes ( vsg) are located at telomeric expression sites (ES), whose expression is highly regulated during the life cycle. In the procyclic form, all ESs are repressed. In the bloodstream form, where antigenic variation occurs, only one of approximately 20 ESs is active at a given time. We have investigated chromatin structure and DNA sequence around the ES promoter to identify cis-acting regulatory regions. A marker gene, inserted 1 kb downstream of the ES promoter, was used as a specific probe to map the position of nuclease hypersensitive sites. A prominent hypersensitive site was detected within the core promoter. This site was present in both active and inactive ES promoters, suggesting that a protein complex is bound to the promoter irrespective of its transcriptional state. However, none of the regions showed differential nuclease sensitivity between active and inactive transcriptional states. A systematic deletion analysis of the sequences surrounding the active ES promoter in situ confirmed the absence of cis-regulatory elements. We find that only 70 bp within the ES promoter are necessary to support ES regulation. Analysis of the reporter activities in an inactive bloodstream-form ES revealed the existence of an intermediate promoter activity in some clones, but we never observed full activation of more than one ES. The vsg mRNA from this intermediate ES was expressed less efficiently.

Analysis of a variant surface glycoprotein gene expression site promoter of Trypanosoma brucei by remodelling the promoter region

Trypanosoma brucei survives in the mammalian bloodstream by antigenic variation of its variant surface glycoprotein (VSG) coat. VSG genes are found in telomeric expression sites (ESs), and only one ES is fully transcribed at a time. The parasite changes its coat by either bringing another VSG gene into the active ES, or by switching on another ES and silencing the first. It has previously been shown that the promoter of an active ES can be replaced by a ribosomal promoter without affecting the function of the ES. This study has now analysed the conserved sequences flanking the ES promoter by deletion or replacement of these sequences in intact trypanosomes. The results show that the sequences 3′ of the promoter and extending down to the first protein-coding gene, ESAG 7, are not required in the bloodstream-form parasite either for high-level transcription or for switching of the ES. Transformants in which the sequences 5′ of the promoter extending up to simple-sequence 50-bp repeats had been removed were not obtained unless the 5' ES sequences were replaced with exogenous DNA, or unless the ES promoter was replaced by a ribosomal promoter, and even these transformants were rare. Transformants lacking the 5′ ES sequences displayed a less complete transcriptional repression of silent ESs. These results indicate that the area 5′ of an ES promoter is required for optimal functioning of an ES.

Frequent loss of the active site during variant surface glycoprotein expression site switching in vitro in Trypanosoma brucei.

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid being killed by their mammalian hosts. The active VSG gene is located in one of many telomeric expression sites. Replacement of the VSG gene in the active site or switching between expression sites can give rise to a new VSG coat. To study Trypanosoma brucei VSG expression site inactivation rather than VSG gene switching, it is useful to have an in vitro negative-selection system independent of the VSG. We have achieved this aim by using a viral thymidine kinase (TK) gene. Following integration of the TK gene downstream of the 221a VSG expression site promoter, transformant cell lines became sensitive to the nucleoside analog 1-(2-deoxy-2-fluoro-8-D-arabinofuranosyl)-5-iodouracil. These TK trypanosomes were able to revert to resistance at a rate approaching 10(-5) per cell per generation. The majority of revertants expressed a new VSG gene even though there had been no selection against the VSG itself. Analysis of these switched variants showed that some had shut down TK expression via an in situ expression site switch. However, most variants had the complete 221 expression site deleted and another VSG expression site activated. We speculate that a new VSG expression site cannot switch on without inactivation of the old site.