Activation, silencing and mutually exclusive expression within the var gene family of Plasmodium falciparum

International Journal for Parasitology 36 (2006) 975–985 www.elsevier.com/locate/ijpara

Invited review

Activation, silencing and mutually exclusive expression within the var gene family of Plasmodium falciparum Matthias Frank b

a,b

, Kirk Deitsch

a,*

a Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA Division of International Health and Infectious Diseases, Weill Medical College of Cornell University, New York, NY 10021, USA

Received 13 March 2006; received in revised form 3 May 2006; accepted 11 May 2006

Abstract The var gene family of the human malaria parasite Plasmodium falciparum remains a topic of intense research focus due to the key role these antigen-encoding genes play in the ability of parasites to cause disease and avoid the human immune response. In recent years, as molecular tools for investigating the mechanisms that coordinate var gene expression have become more sophisticated, numerous insights have been acquired into how parasites manage to regulate transcription of this large gene family such that only a single gene is expressed at a time. The results of different experimental approaches have implicated mechanisms of chromatin modification, subnuclear localisation, promoter/promoter interactions and sterile RNAs in the silencing and activation of individual var genes, however, the roles that each of these different aspects play remain ill defined. In addition, some conflicting data regarding silencing and monoallelic expression of recombinant var promoters have recently been published, thus adding to the difficulty of understanding this complex phenomenon. In this review, we hope to present some of the existing data regarding this controversial topic in a way that will be both informative and constructive in our efforts to understand the molecular aspects of antigenic variation by malaria parasites. Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Malaria; Antigenic variation; Virulence genes; Allelic exclusion; Transcription; var genes; Gene expression; Intron; Chromatin; Epigenetic

1. Introduction The human malaria parasite Plasmodium falciparum continues to be responsible for substantial morbidity and mortality in many regions of the developing world, in particular sub-Saharan Africa (Snow et al., 2005). Of the three species of Plasmodium that infect humans, P. falciparum is unique in its ability to cytoadhere to the surface of postcapillary endothelial cells, thus leading to sequestration of the infected red blood cells and obstruction of blood flow (Kyes et al., 2001). This phenomenon is responsible for the severe disease phenotypes, including cerebral and placental malaria, that are unique to P. falciparum infections. Cytoadherence and the resulting sequestration of infected cells away from the peripheral circulation are

*

Corresponding author. Tel.: +1 212 746 4976; fax: +1 212 746 4028. E-mail address: [email protected] (K. Deitsch).

thought to have evolved to enable parasitised cells to avoid passage through the spleen where they would be recognised and destroyed (Barnwell et al., 1983; Miller et al., 1994). Thus, the ability of parasite-infected red blood cells to cytoadhere to blood vessel walls is both necessary for parasite survival and also underlies many of the disease manifestations of P. falciparum malaria. The cytoadhesion of parasite-infected red blood cells is mediated by a large family of hypervariable cell surface antigens collectively referred to as PfEMP1 (Deitsch and Wellems, 1996; Kyes et al., 2001). Individual parasites only express a single form of PfEMP1 at a time, exporting this protein to the surface of the infected red blood cell, where it acts as a receptor and binds to specific host molecules on the surface of endothelial cells, thus resulting in cytoadhesion and sequestration (Berendt et al., 1990). The interactions of PfEMP1 with molecules on the surface of endothelial cells are specific, with different forms of PfEMP1 displaying different binding affinities (Robinson

0020-7519/$30.00 Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.05.007

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et al., 2003). This specificity underlies the variable severity of P. falciparum infections, for instance, adhesion to brain- or placental-specific receptors resulting in cerebral or pregnancy-associated malaria, respectively. In addition, parasites can change which PfEMP1 they express over time (Roberts et al., 1992; Staalsoe et al., 2002), resulting in sudden changes in virulence over the course of an infection. The placement of PfEMP1 on the infected red blood cell surface exposes it to the immune system of the host, which readily makes antibodies that specifically recognise individual PfEMP1 molecules (Bull et al., 1999; Jensen et al., 2004). These antibodies successfully lead to clearance of parasitised red cells from the circulation and a concordant reduction in the parasite load of the infected individual. However, before complete resolution of the infection can be achieved, sub-populations of the parasites arise that have switched to expressing a different form of PfEMP1 and consequently are not recognised by the antibody response. This process of antigenic variation leads to repeated waves of parasitemia (Miller et al., 1994), with each wave representing a distinct sub-population of parasites that expresses a different PfEMP1 molecule on the surface of infected cells, thus resulting in changes in antigenicity, binding specificity and virulence. PfEMP1 therefore is a molecule that plays a crucial role in virulence of P. falciparum infections as well as in the parasite’s ability to maintain a persistent infection by avoiding host immunity. 2. The var gene family A long effort to identify and characterise the genes that encode PfEMP1 culminated in the discovery of the var gene family in 1995 (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). The parasite genome was estimated to contain between 50 and 150 var genes, although their exact number and arrangement was uncertain. All var genes have

the same basic structure consisting of two exons, the first encoding the extracellular portion of the protein and a single transmembrane domain while the second encodes a highly charged region that is proposed to anchor the protein within a knob structure at the red blood cell membrane (Fig. 1A). Interestingly, although PfEMP1 is clearly secreted by the parasite, no obvious signal peptide is found in the encoded amino acid sequence. Some light has been shed on how the protein is targeted to the infected cell surface, with the recent discovery of a unique trafficking signal found near the amino terminus of the protein (Hiller et al., 2004; Marti et al., 2004). The extracellular portion of the protein encoded by exon 1 is hypervariable in sequence and consists largely of domains that have a low level of similarity to the Duffy antigen binding domain identified in Plasmodium knowlesi and Plasmodium vivax (Adams et al., 1992). For this reason they are referred to as Duffy binding-like (DBL) domains (Su et al., 1995). In addition, most PfEMP1 molecules also contain a cysteine-rich interdomain region (CIDR) and, together with the DBLs, these domains mediate the cytoadherence of infected cells to surface molecules found on the endothelial wall (Baruch, 1999). While exon 1 of each var gene is hypervariable to allow for variation in binding affinities and antigenicity of the encoded PfEMP1, exon 2 is highly conserved among all var genes (Su et al., 1995). With the release of the completed and annotated genome, a more complete picture of the var gene family emerged (Gardner et al., 2002). Within the genome of the reference strain 3D7 there exist 59 intact var genes as well as numerous var fragments and pseudogenes. The genes are organised into clusters of tandemly repeated genes within the internal regions of several chromosomes, or as one to three copies found in the subtelomeric regions near most chromosome ends (Fig. 1B). Close examination of the surrounding regulatory DNA sequences found that there are three basic promoter types, referred to as UpsA, B

Fig. 1. Schematic diagram showing var gene organisation and structure. (A) Typical structure of a var gene. Promoters are shown with bent arrows and transcripts are shown as lines drawn above the genes. The transcription start point for the translated mRNA is shown as well as a potential regulatory element identified through electromobility shift assays (SPE1, SPE2 or CPE). Exon 1 encodes multiple Duffy binding-like domains as well as a cysteine rich interdomain region, while exon 2 is conserved and codes for the acidic terminal segment of PfEMP1. (B) Organisation of var genes within the Plasmodium falciparum genome. Clusters of genes containing UpsC-type promoters are often found in the internal regions of the chromosomes while UpsA and B containing genes are typically found within subtelomeric regions. Arrows indicate the direction of transcription.

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and C, and these promoter types reflect the chromosomal location of the given var gene (Voss et al., 2000; Kraemer and Smith, 2003; Lavstsen et al., 2003). For example, genes containing UpsA-type promoters are exclusively found in subtelomeric domains, oriented such that they are transcribed towards the telomere while those containing UpsC promoters are found in internal clusters of genes. Genes containing UpsB types are found both subtelomerically and in internal clusters. While the significance of the different promoter types is not fully understood, it has been speculated they may play a role in controlling expression of different subclasses of var genes or that they may be the result of selective recombination between different subgroups of genes during the generation of diversity within the family. 3. var gene transcription, switching rates and phenotypic selection Over the course of an infection, P. falciparum parasites undergo antigenic variation by changing which PfEMP1 is expressed on the surface of the infected cells. To do this efficiently, and to achieve the maximum efficacy out of the 60 var genes found in the genome, it is necessary for the parasite to express only a single var gene at a time, a phenomenon referred to as mutually exclusive expression. It has been demonstrated by single cell RT-PCR (Chen et al., 1998), nuclear run-on (Scherf et al., 1998) and Northern blot (Kyes et al., 2003; Horrocks et al., 2004) that var genes are in fact exclusively expressed, with a single full-length var transcript detectable in the late ring/early trophozoite stage when PfEMP1 is being produced and transported to the red blood cell surface. These experiments indicate that PfEMP1 production is regulated at the level of var gene transcription initiation, rather than at the level of protein synthesis. This also implies that the transcription of individual genes within the var gene family must be coordinated such that activation of expression of one gene coincides with silencing of the previously active gene, thus maintaining expression of only a single gene at any given time. Analysis of var gene transcription has been complicated by the fact that there are three different transcripts that have been reported to come from individual var genes, only one of which is a full-length mRNA that is translated into PfEMP1 (Fig. 1A). The first of these non-mRNA transcripts to be reported were the ‘‘sterile’’ transcripts that initiate within var introns and include exon 2 (Su et al., 1995). Rather than being expressed from a single var gene, these mRNAs appear to come from many genes simultaneously. They generally contain no canonical start methionine and do not appear to be translated, hence the term ‘‘sterile’’ transcripts. In addition, they are transcribed late in the cell cycle as opposed to early, when the full-length, translated var mRNA is made (Kyes et al., 2003). The second non-mRNA transcript associated with var genes is detected very early after invasion of the red blood cell by

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the merozoite (Chen et al., 1998; Scherf et al., 1998). In the first several hours after invasion, transcription of numerous var genes has been reported, however, as the cell matures, transcription is limited to a single gene that produces the mRNA that is ultimately translated into PfEMP1. This early, ‘‘loose’’ transcription from multiple var genes has been detected both by single cell RT-PCR (Chen et al., 1998) and by nuclear run-on (Scherf et al., 1998), however whether these transcripts are full length has not been determined and they are not detectably translated. The role that either of these non-mRNA transcripts play in the process of antigenic variation remains unknown. Because PfEMP1 is a cell surface receptor that mediates binding of infected red blood cells to specific ligands, it is possible to select populations of parasites for specific binding phenotypes and thus for expression of the corresponding var gene. This has been particularly useful in identifying the var gene responsible for binding to the placental receptor chrondroitin sulphate A (CSA), the mechanism underlying pregnancy-associated malaria (Fried and Duffy, 1996; Beeson et al., 2000; Duffy and Fried, 2003). Selection of parasites for the ability to bind to CSA led to the selective upregulation of a particular var gene called var2csa (Salanti et al., 2003) and subsequent work has demonstrated that indeed this gene is expressed in parasites isolated from infected placentas (Salanti et al., 2004). The protein product encoded by this gene is a primary candidate for what could potentially be a syndrome-specific anti-malaria vaccine (Smith and Deitsch, 2004). The success of this work in identifying an individual var gene that is associated with a particular disease phenotype raises the possibility that a similar approach may be possible to identify other genes or subsets of genes that mediate additional disease phenotypes, in particular cerebral malaria. In a natural infection, switching of expression between different var genes is responsible for allowing the parasites to avoid the antibody response that the host generates against PfEMP1 and thus to perpetuate a long-term infection. The switching rate therefore needs to be rapid enough to prevent the infection from being cleared but not so rapid as to lead to premature expenditure of the complete var repertoire. This interplay between the production of PfEMP1-specific antibodies by the infected individual and the process of var gene expression switching ultimately leads to the distinctive waves of parasitemia typical of clinical infections (Miller et al., 1994). The rate at which parasites vary their surface antigens has been measured using cultured parasites and indicated a switch rate of 2–3% per generation (Roberts et al., 1992). A more precise measurement based on var gene transcription using Northern blots to measure the ‘‘on’’ and ‘‘off’’ rates of individual genes calculated rates between 0.25% and 0.025% per generation (Horrocks et al., 2004). Further work will determine if extracellular factors influence switching rates and if var genes switch on and off at random or if, rather, there is a programmed order to their expression.

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4. Chromatin structure and its possible function in regulating var gene expression Investigations into the mechanisms that regulate var gene expression have been pursued by several different laboratories but can be broadly grouped into three basic approaches: first, analysis of alterations in chromatin structure and their contribution to gene silencing and activation; second, the functional evaluation of conserved var gene non-coding regions using episomal and integrated reporter constructs; and third, the association of different transcriptional states with changes in localisation of chromosomal segments containing var loci into different subnuclear compartments. While the details of the mechanisms by which the parasite exerts such tight control over the multi-copy var gene family remain incompletely defined, many studies support a role of epigenetic regulation. Epigenetic control of gene expression has been defined as changes in the transcriptional state of a gene that are not the result of changes in transcription factors or DNA sequence but rather are mediated by alterations in DNA modifications (for instance, methylation patterns) or in chromatin structure (i.e., histone acetylation or methylation) (Henikoff and Matzke, 1997). Here, we will review some of the recent evidence regarding these ideas. After the initial description of the multi-copy nature of the var gene family, it was apparent that the parasite must possess a regulatory mechanism that coordinates the expression of this gene family, ensuring that only a single gene is expressed in each individual parasite. Switches in expression were not associated with sequence changes at var loci, providing evidence for a switching process not dependent on DNA sequence changes and consistent with a mechanism based on epigenetic regulation (Scherf et al., 1998; Deitsch et al., 1999). Further evidence for an epigenetic basis for var gene regulation came from studies of switching dynamics in parasites first selected for expression of a particular var gene, then followed for 40 generations during which time the parasites were allowed to switch var gene expression without selection (Horrocks et al., 2004). These experiments demonstrated that each var gene has intrinsic ‘‘on’’ and ‘‘off’’ switch rates but these rates can be influenced by the var gene expression history of the parasite. The authors proposed a model in which an individual var gene can exist in three different states: transcriptionally active, transcriptionally inactive but capable of being activated and highly silenced. This model is consistent with the idea that alterations in the local chromatin environment could potentially mediate the transition between these transcriptional states. Chromatin structure has been shown to be modulated by histone modifications in many organisms (Felsenfeld et al., 1996). Phosphorylation, methylation and acetylation of histone proteins have all been implicated in gene regulation, presumably by changing the accessibility of various DNA sequences to the proteins necessary for gene expression (Eissenberg and Wallrath, 2003). In Saccharomyces

cerevisiae the deacetylase Sir2 has been implicated in silencing of the mating loci HML and HMR by assembling an area of silent heterochromatin in the chromosomal region where these loci are located (Moazed, 2001). Two recent studies have investigated the role that the P. falciparum homologue PfSir2 plays in regulating var gene expression. In the first, chromatin immuno-precipitation (ChIP) was used to localise PfSir2 within various regions of the parasite chromosomes (Freitas-Junior et al., 2005). PfSir2 was found to bind to the ends of the chromosomes and up to 55 kb toward the centromeres, regions that include up to two-thirds of the var gene family. However, when parasites were selected for activation of a particular subtelomeric var gene, this gene was no longer bound by PfSir2, thus correlating PfSir2 binding with gene silencing. ChIP assays were also used to show that histone H4 acetylation was associated with active var loci, a trait found in many other organisms. Interestingly, transcriptionally silent internal var genes showed no evidence of PfSir2 binding, indicating that silencing of these genes, typically driven by UpsC-type promoters, may be PfSir2-independent. In the second study, it was shown that P. falciparum subtelomeric regions indeed display many features characteristic of condensed heterochromatin and this chromatin environment can reversibly silence a promoter artificially inserted into this region of a chromosome (Duraisingh et al., 2005). To specifically investigate the role of PfSir2 in this process, targeted gene disruption was used to knock out expression of PfSir2. The effect of the knockout on var gene expression was assessed using microarray analysis to determine if var gene silencing was in anyway disrupted. In the PfSir2 knockout line, several var genes were upregulated as predicted, indicating that PfSir2 plays a role in silencing of these genes. However, expression of the majority of the var gene family was not affected. Specifically, a number of UpsA-type var genes were upregulated in the PfSir2 knockout line, while the majority of UpsB and UpsC genes remained silenced. Thus, while PfSir2 probably plays a role in regulating chromatin assembly in subtelomeric regions of the chromosomes and expression of a subset of var genes, other factors must be involved in silencing the majority of the family and in coordinating mutually exclusive expression. One possibility is a second Sir2-like gene found in the P. falciparum genome that may also play a role in var gene silencing, extending the complexity of this regulatory pathway. 5. Conserved non-coding DNA elements and their role in transcriptional control of individual var genes Early investigations into transcription control of var genes focused on the promoters and upstream regulatory regions found at each gene. The transcription start site of one gene was mapped using primer extension and found to be approximately 1 kb upstream of the open reading frame, indicating that var mRNAs contain a rather lengthy 5 0 untranslated region (UTR) (Deitsch et al., 1999). Several

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other potential regulatory elements were identified using electromobility shift assays (EMSA) with isolated nuclear extracts (Voss et al., 2003). These experiments identified specific sequence elements in both UpsB and UpsC upstream sequences that are bound by nuclear protein complexes. Interestingly, the DNA elements were bound by protein complexes extracted from nuclei taken from late-stage parasites, a point in the cell cycle when var upstream promoters are not transcriptionally active. This raises the possibility that the binding activities identified by EMSA are not directly involved in active transcription

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but, potentially, in the epigenetic modifications that mark genes for activation or silencing in subsequent cell cycles. More direct assays of var promoter activity employed transiently transfected plasmid constructs in which the upstream regulatory regions of various var genes were used to drive the expression of the reporter genes firefly luciferase or chloramphenicol acetyl transferase (Fig. 2A) (Deitsch et al., 1999; Voss et al., 2000; Vazquez-Macias et al., 2002). In all of these studies, var promoters placed on transiently transfected episomes were always transcriptionally active, allowing deletion analysis of the upstream regulatory

Fig. 2. Plasmid constructs used in transfection experiments designed to examine mechanisms for var promoter silencing and mutually exclusive expression. Plasmid constructs that have been used in either transient (top) or stable (bottom) transfection of cultured malaria parasites. All promoters are shown with bent arrows. A barred-circle indicates a transcriptionally silent var promoter, while active promoters are shown transcribing an mRNA molecule (curved line). The construct drawn in (A) is shown with a luciferase reporter gene, however, Voss et al. used chloramphenicol acetyl transferase instead of luciferase. For each construct, the default state of the var promoter is indicated, as well as whether this var promoter is ‘‘counted’’ by the mechanism that controls mutually exclusive expression of the var gene family. In the constructs shown in J and K the default state of the var promoter is silent, however, drug selection of transformed parasites results in activation of this recombinant var promoter and consequent silencing of the rest of the var gene family.

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region and the identification of sequences necessary for transcriptional activity. However, the fact that these promoters were transcriptionally active was in itself noteworthy, considering that the typical state of var promoters in the genome is silent. Thus, removal of var promoters from their chromosomal context and their placement on episomes appeared to disrupt the silencing process, rendering them constitutively active and indicating that an important element of the silencing process had been lost. The original expression constructs that assayed var promoter activity seemed to be missing an important element necessary for silencing and were consequently constitutively active. All var genes carry a conserved intron that separates exons 1 and 2, encoding the extracellular and intracellular domains of PfEMP1, respectively. The intron is noteworthy in that it appears to be the source of the ‘‘sterile’’ transcripts mentioned earlier (Fig. 1A). To investigate the role of this non-coding genetic element in var gene silencing, a var intron was placed downstream of the luciferase reporter gene in a construct used in transient transfection experiments (Fig. 2B). This element had no effect on expression of the unrelated hrp3 promoter, however, it had a very strong silencing effect when placed on the same plasmid as a var promoter (Deitsch et al., 2001). The silencing effect of the intron was not dependent on its orientation but required the transfected parasites transition through at least one S-phase, a characteristic typical of gene silencing that involves chromatin modification (Kirchmaier and Rine, 2001; Li et al., 2001). Further examination of the sequences and silencing properties of var introns established that each intron can be divided into three distinct regions as determined by GC content and strand asymmetry (Calderwood et al., 2003). The central region was found to contain promoter activity and this promoter is most active at the point in the cell cycle when the ‘‘sterile’’ transcripts become abundant (Kyes et al., 2003), providing further evidence that the intron is the likely source of these non-coding RNAs. Further, creation of a disabled intron devoid of promoter activity also resulted in loss of its function as a silencer (Fig. 2C) (Calderwood et al., 2003; Gannoun-Zaki et al., 2005), indicating that interactions between the two promoters found in each var gene, one upstream of the coding region and the second in the intron, may play a role in var gene silencing. 6. Stable transformants confirm the role of non-coding DNA elements in var gene silencing While transient transfection experiments in P. falciparum generate very reproducible results, experiments that employ them have certain limitations. In particular they are quite inefficient, with only a small portion of the population of parasites being studied actually taking up DNA. Thus, it is difficult to determine the uniformity of promoter activity in all parasites that take up DNA and it is impossible to determine whether these constitutively active var promoters are recognised by the process that controls

mutually exclusive var gene expression. Several recent studies have begun to address the role of non-coding DNA elements in var gene regulation using stably transformed parasite lines. Such studies have the advantage of also allowing the investigation of mechanisms that control mutually exclusive expression as well as gene activation and silencing. How parasites coordinate expression of this large, multi-copy gene family is one of the most fascinating questions of malaria biology and is central to the parasite’s ability to maintain a persistent infection. Through the use of stably transformed parasites and drug-selectable markers, it is possible to manipulate the expression of recombinant var promoters within an entire population of transformed parasites and subsequently assess how this manipulation affects the rest of the gene family. These studies have added another level of sophistication to our ability to study the phenomenon of antigenic variation and have yielded important information regarding how this process is controlled at the molecular level. Two recent reports have been published that support a central role for the var gene intron in var promoter silencing (Gannoun-Zaki et al., 2005; Frank et al., 2006). In the first, using plasmid constructs in which a var promoter was used to drive expression of the human dhfr gene, GannounZaki et al. (2005) found that constructs that contained only a var upstream promoter (Fig. 2E) efficiently transformed transfected parasites, indicating that in these plasmids the var promoter readily assumed a transcriptionally active state. However, when a var intron was included in the constructs, the only transformed parasites that could be recovered from the transfection experiments contained a spontaneous deletion within the central region of the intron (Fig. 2F), a portion of the intron that had previously been shown to be necessary for silencing (Calderwood et al., 2003). Thus, the presence on the plasmid of an intron appeared to silence the var promoter driving hdhfr expression, making it extremely difficult or impossible to recover transformed parasites when the intron remained intact. These experiments were therefore consistent with the hypothesis that in the presence of the intron, a var promoter naturally assumes the silent state. However, the data should not be interpreted to imply that var genes cannot be activated in the presence of an intact intron, as endogenous var genes do in fact become transcriptionally active without any alterations to the intron sequence. Rather, it is more likely that the low rate of var promoter activation (i.e., var gene switching), combined with the poor efficiency of parasite transfection, simply made the likelihood of obtaining transfectants in which the recombinant var promoter was initially active extremely low. In a subsequent study, Frank et al. assessed the role of the intron in silencing using a more complex construct in which a var promoter is used to drive expression of a luciferase reporter gene (Calderwood et al., 2003; Frank et al., 2006). Since luciferase activity is not selected for in these experiments, it serves as an accurate measure of the default state of the var promoter within the construct. These

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plasmids also contain a var intron, which serves to silence the upstream var promoter. The promoter activity of the intron, previously observed in the production of the ‘‘sterile’’ transcripts, is utilised here to drive expression of the drug-selectable marker human dhfr, thus providing the transformed parasites with a drug-resistant phenotype (Fig. 2G). Surprisingly, rather than being uniformly silenced, approximately 50% of parasite lines stably transformed with this construct were actively expressing luciferase, suggesting that they had activated the upstream var gene promoter. Analysis of episomes recovered from the various transformed lines showed that intramolecular recombination within large multimeric plasmid concatamers had resulted in the generation of several different arrangements of var upstream promoters and introns and the different arrangements resulted in either an active or silent state of the upstream var promoter. Specifically, rearrangements that yielded equal numbers of var promoters and introns within the concatamers (Fig. 2H) always resulted in complete silencing of luciferase expression, while in plasmids in which the recombination event deleted an intron from the plasmid concatamer (Fig. 2I), the resulting ‘‘unpaired’’ var promoter expressed high levels of luciferase, indicating that it had assumed an active transcriptional state. Retransfection of rescued plasmids and integration of the concatemers into the chromosome confirmed this relationship. Analysis of the various transformed parasite lines supported a model in which there is a strict one-toone pairing requirement for promoters and introns in order for silencing to occur, however, it was not necessary that the intron be in its natural position downstream of the var promoter. Moreover, within a concatameric array of expression cassettes, an unpaired var promoter could be transcriptionally active while the immediately adjacent paired promoter was silent, indicating that each promoter is regulated independently. In a similar approach, Voss et al. recently utilised transfection constructs in which var promoters were used to drive expression of the hdhfr drug-selectable marker (Voss et al., 2006). In addition, the constructs employed in these experiments used an hsp86 promoter driving blasticidin S deaminase (bsd) to generate stable transformants (Fig. 2J). By initially selecting with blasticidin, the authors were able to determine the default transcriptional state of the transfected var promoter phenotypically by assaying for resistance to WR99210 conferred by expression of the hdhfr gene. Drug resistance assays and Northern blots indicated that the var promoter in these constructs naturally assumed the silent state, even in the absence of the intron, and that the presence of the intron only stabilised this silent state. While the papers by Voss et al. and Frank et al. both identify the var gene intron as a central component in the silencing process, the data are in clear disagreement regarding the default transcriptional activity of a var promoter and whether the intron is truly required for silencing. The constructs employed by Voss et al. demonstrated silencing of a var promoter in the absence of an intron but in the

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context of an additional heterologous promoter (hsp86). Previous work on the intron indicated that its silencing properties were dependent on its own promoter activity, suggesting that promoter/promoter interactions may be responsible for the silencing phenomenon (Figs. 2C and F) (Calderwood et al., 2003; Gannoun-Zaki et al., 2005). This raised the possibility that similar interactions may be taking place between the var and hsp86 promoters within the constructs employed by Voss et al., thus rendering the intron redundant and unnecessary for silencing. To investigate this possibility, we have recently created a series of constructs to determine if promoter/promoter interactions similar to those seen between the var promoter and intron can also occur between var promoters and other unrelated promoter sequences when they are artificially placed in close proximity to one another (Fig. 2D; Eisberg et al., unpublished data). Indeed, we find that many heterologous promoters, including the hsp86 promoter employed in the constructs of Voss et al., do in fact interact with var promoters resulting in very strong repression of var promoter activity. These results therefore suggest that it might be possible to reconcile the conflicting data reported in the Voss et al. and Frank et al. papers. Future studies employing the cell lines and constructs described in these two papers and additional cell lines in which endogenous var gene regulatory elements have been mutated or deleted will undoubtedly lead to further dissection of the DNA elements involved in var gene silencing. 7. Monoallelic expression of the var gene family As previously mentioned, one of the great advantages of strategies that employ stable transfection to assess the activity of recombinant var promoters is the additional attribute of being able to investigate mechanisms that control mutually exclusive expression. By selecting for activation of the recombinant var promoter in a stably transformed population of parasites, it is possible to then assess expression of the endogenous var genes and thus determine if the recombinant promoter is recognised and ‘‘counted’’ as a member of the family. In this way, it is possible to begin to gain insights into the elusive mechanisms that coordinate expression of this large family of genes and lead to the elegant waves of parasitemia typical of P. falciparum infections. Investigating mutually exclusive var gene expression was greatly aided by the development by Salanti et al. of a primer set that allows simultaneous measurement of the levels of expression of every var gene in the 3D7 parasite genome using quantitative real-time RT-PCR (Salanti et al., 2003). This provides a very rapid, detailed and accurate way to determine if experimental approaches affect coordinated var gene expression. The three papers previously mentioned that used stably transfected plasmid constructs to investigate var promoter silencing also considered the effect of the recombinant var promoters on expression of the rest of the gene family. Gannoun-Zaki et al. found that parasites carrying

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episomal constructs containing a mutated intron and thus a constitutively active var promoter continued to express endogenous, chromosomal var genes, thus concluding that the recombinant var promoters in this configuration were not recognised and were ‘‘uncounted’’(Gannoun-Zaki et al., 2005). Similar conclusions were drawn from the experiments of Frank et al. who found that ‘‘unpaired’’, constitutively active var promoters that were integrated into the subtelomeric hrp2 locus did not affect expression of endogenous var genes, once again indicating that these promoters were unrecognised (Frank et al., 2006). Voss et al., however, were apparently able to infiltrate the pathway that coordinates mutually exclusive expression with recombinant var promoters on both episomal and integrated recombinant constructs (Voss et al., 2006). In these experiments, selection for activation of the recombinant var promoter using drug pressure resulted in strong repression of expression of the entire var gene family as measured by Northern blot hybridisation, indicating that the recombinant promoter was indeed ‘‘counted’’. The one caveat of these experiments is that the transfected episomes are found as multi-copy concatamers containing numerous copies of the recombinant var promoter, thus it is formally impossible to determine if only one of these promoters is transcriptionally active. Indeed, selection for expression of the recombinant var promoters using drug pressure also led to a substantial increase in the copy number of the concatamer, implying that many of these var promoters may in fact be simultaneously active. This does not detract from the fact that all chromosomal var genes were silenced but does have implications for understanding the more subtle point as to how individual var promoters are recognised and counted. Recently, Dzikowski et al. avoided this potential complication by using a strategy in which targeted integration into the genome was used to selectively modify individual var genes, thus creating single copy, recombinant var genes that can be selected for activation through the application of drug pressure (Dzikowski et al., 2006). In all of the genetic modifications generated, the recombinant var promoters were kept paired with introns, thus approximating the native structure of var genes (Fig. 2K). In contrast to the study of Gannoun-Zaki et al., the constructs employed by Dzikowski et al. employed two selectable markers, thus allowing the generation of a large population of transformed parasites prior to selecting for activation of the initially silenced recombinant var promoter. We hypothesise that this strategy provided a large enough pool of transformed parasites to enable selection of the very small number that had spontaneously activated the var promoter while in the presence of an intact intron. Similar to the results of Voss et al., selection for activation of this var promoter effectively silenced all other members of the gene family, again indicating that the recombinant promoters used in these constructs are recognised by the mechanism that controls mutually exclusive expression and are indeed ‘‘counted’’. In addition, these experiments

demonstrate the production of a functional PfEMP1 protein is not necessary for an active var gene to be counted, a result that is different from other models of allelic exclusion described in mammalian systems (Serizawa et al., 2004; Corcoran, 2005). Taken together, certain conclusions can be drawn from the results of these four published studies. In all cases, recombinant var promoters that were defective in the silencing process and that were therefore constitutively active were also unrecognised by the mechanism controlling mutually exclusive expression. Hence active transcription from the var promoter described by Gannoun-Zaki et al. (Figs. 2E and F) and the ‘‘unpaired’’ promoters of Frank et al. (Fig. 2I), both of which were impaired in silencing and constitutively active, did not affect transcription from the rest of the var gene family. However, in the transgenic parasite lines described by Voss et al. (Fig. 2J) and Dzikowski et al. (Fig. 2K) the recombinant var promoters were able to be silenced, indicating that they were more properly regulated. Selection for activation of these silenced promoters also resulted in silencing of all other var promoters in the genome, thus demonstrating that these promoters are now recognised and ‘‘counted’’. These experiments therefore indicate that the processes of silencing and the control of mutually exclusive expression may potentially be somehow linked. Two additional examples provide similar results. Disruption of the coding region of the var2csa gene through the insertion of an expression cassette into exon 1 also disrupts silencing of this locus and thus renders the upstream promoter of this var gene constitutively active (Viebig et al., 2005). However, these parasites now no longer ‘‘count’’ this gene and therefore also express other members of the family. Similarly, the pseudogene var1csa (also called varcommon) has been shown to have a deletion of the region of the intron that is required for silencing and is continuously transcribed in many parasite lines (Kyes et al., 2003; Winter et al., 2003). Once again, this constitutively active var promoter that cannot be silenced is also unrecognised and has no impact on mutually exclusive expression of the rest of the family. 8. Perinuclear localisation and var gene activation The evidence that modifications in chromatin structure might play a role in the regulation of var gene expression has led to investigations into how chromatin is organised within the parasite nucleus. In yeast, the nucleus has been shown to be organised into regions of condensed heterochromatin near the nuclear periphery and regions of more loosely packed euchromatin near the centre. In addition, the perinuclear localisation of subtelomeric genes is associated with gene silencing, whereas the activation of subtelomeric genes is associated with their movement away from the nuclear periphery (Andrulis et al., 1998; Tham et al., 2001). While the var genes in P. falciparum are scattered in small groups of one to seven throughout the genome

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of the parasite, within the three-dimensional space of the nucleus they have been shown to gather into larger clusters that can be observed using fluorescent in situ hybridisation (FISH) (Freitas-Junior et al., 2000). This technique has shown that much like yeast, the telomeres and the var genes located in the adjacent subtelomeric regions cluster together into ‘‘bouquets’’ of six to eight chromosome ends near the periphery of the nucleus, within a region of condensed heterochromatin (Fig. 3). In addition, the tandem arrays of var genes located in the central regions of the chromosomes also are found near the nuclear periphery (Ralph et al., 2005) and recently Voss et al. (2006) have shown that they colocalise with telomeric clusters. Additional studies have employed FISH to follow the movement of transcriptionally active or silent var promoters in wild type and genetically modified cultured parasites. While activation of the subtelomeric var2csa locus appeared to be associated with movement away from a telomeric cluster (Ralph et al., 2005), an activated episomal central var promoter driving the human dhfr gene remained associated with a telomeric cluster (Voss et al., 2006), thus the exact role of the clustering of var genes within the nucleus remains unclear. To look more closely at nuclear organisation, plasmid constructs were employed that carried either var promoters or promoters from other unrelated genes on either episomes or integrated into subtelomeric regions of the genome. These constructs were then followed

Fig. 3. Model of the organisation of Plasmodium falciparum nucleus. The nucleus is divided into regions of condensed heterochromatin (dark blue, near the nuclear periphery) and transcriptionally active euchromatin (light blue, near the centre of the nucleus). Clusters of var genes found in the central regions of chromosomes (orange spot) as well as those positioned in subtelomeric regions (yellow spots) are localised near the nuclear periphery. Note that a specific region of euchromatin has been observed to extend from the centre of the nucleus into the nuclear periphery. var gene activation has been associated with re-location of a silent var gene into this less condensed and thus presumably more transcriptionally competent subnuclear compartment.

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to determine if localisation into different subnuclear compartments is associated with changes in gene expression (Duraisingh et al., 2005; Voss et al., 2006). Consistent with a role for subnuclear organisation, activation of these recombinant promoters coincided with their increased colocalisation with other active promoters within the nuclear periphery. These observations have led to the hypothesis that transcriptionally active var promoters are confined to a specific subnuclear region or expression site, a model also proposed for regulation of VSG expression in the antigenic variation of African trypanosomes (Navarro and Gull, 2001). Consistent with this hypothesis, electron microscopic studies identified a zone of relaxed euchromatin within the mostly condensed heterochromatin of the nuclear periphery (Ralph et al., 2005). However, in P. falciparum this likely constitutes only one layer of regulation, since many var genes are located in close proximity to each other along the chromosome, yet activation of one var gene is not associated with activation of the adjacent var gene. Thus, a mechanism must exist to silence individual var genes even when they are located within a region of the nucleus that is permissive for transcription. Frank et al. have recently provided evidence that the var intron may mediate this phenomenon by showing that in plasmid constructs, a var promoter that is paired with an intron is silent even if the neighbouring var promoter is active. The intron therefore appears to impose a transcriptional repression on a var promoter even when it is present in a nuclear region that is permissive for var gene transcription. The nature of the epigenetic mechanism that marks an individual var gene promoter to assume the active state remains elusive and continues to be a central question in antigenic variation of P. falciparum. The clustering of the telomeres into ‘‘bouquets’’ has also been suggested as a potential mechanism for bringing var genes into close proximity for recombination to generate diversity within the family (Freitas-Junior et al., 2000). In this context, it is noteworthy that while exon I coding regions of var genes from different parasite strains are extremely variable in sequence, the non-coding regions such as var promoters and introns are highly conserved, supporting a conserved functional role for these genetic elements. In addition, several other hypervariable, multi-copy gene families are also located in these chromosomal regions, in particular the rifin and stevor genes (Gardner et al., 2002). These chromosomal regions, therefore, may represent specialised areas or ‘‘islands’’ for a class of genes that is under special transcriptional regulatory control and that is also subject to continuous sequence alterations to generate diversity. Thus, the subnuclear localisation of these chromosomal segments and their clustering with the nucleus may play a significant role in both of these processes. 9. Conclusion The last couple of years have resulted in a remarkable progress in the understanding of the regulation of the var

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gene family. In this review, we have tried to summarise recent developments regarding the epigenetic factors that control var gene expression at the molecular level. While important, these factors are unlikely to be the sole determinants of host–pathogen interaction and disease development. Host factors such as the immune response (Bull et al., 2000), receptor adhesion (Salanti et al., 2003) and haemoglobin polymorphisms (Fairhurst et al., 2005) have all been shown to contribute to the final outcome of a P. falciparum infection. In addition, other recent studies have employed infection of human volunteers to investigate antigenic variation during natural infections (Peters et al., 2002; Lavstsen et al., 2005). It is our hope that recent advances in this field will eventually lead to a better understanding of the factors that determine disease outcome and immunity, thus contributing to future interventions against this devastating disease. References Adams, J.H., Sim, B.K., Dolan, S.A., Fang, X., Kaslow, D.C., Miller, L.H., 1992. A family of erythrocyte binding proteins in malaria parasites. Proc. Natl. Acad. Sci. USA 89, 7085–7089. Andrulis, E.D., Neiman, A.M., Zappulla, D.C., Sternglanz, R., 1998. Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 395, 525, [394 (1998) 592]. Barnwell, J.W., Howard, R.J., Coon, H.G., Miller, L.H., 1983. Splenic requirement for antigenic variation and expression of the variant antigen on the erythrocyte membrane in cloned Plasmodium knowlesi malaria. Infect. Immun. 40, 985–994. Baruch, D.I., 1999. Adhesive receptors on malaria-parasitized red cells. Baillieres Best Pract. Res. Clin. Haematol. 12, 747–761. Baruch, D.I., Pasloske, B.L., Singh, H.B., Bi, X., Ma, X.C., Feldman, M., Taraschi, T.F., Howard, R.J., 1995. Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 82, 77–87. Beeson, J.G., Rogerson, S.J., Cooke, B.M., Reeder, J.C., Chai, W., Lawson, A.M., Molyneux, M.E., Brown, G.V., 2000. Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria. Nat. Med. 6, 86–90. Berendt, A.R., Ferguson, D.J.P., Newbold, C.I., 1990. Sequestration in Plasmodium falciparum malaria: sticky cells and sticky problems. Parasitol. Today 6, 247–254. Bull, P.C., Kortok, M., Kai, O., 2000. Plasmodium falciparum-infected erythrocytes: agglutination by diverse Kenyan plasma is associated with severe disease and young host age. J. Infect. Dis. 182, 641, [182 (2000) 252]. Bull, P.C., Lowe, B.S., Kortok, M., Marsh, K., 1999. Antibody recognition of Plasmodium falciparum erythrocyte surface antigens in Kenya: evidence for rare and prevalent variants. Infect. Immun. 67, 733–739. Calderwood, M.S., Gannoun-Zaki, L., Wellems, T.E., Deitsch, K.W., 2003. Plasmodium falciparum var genes are regulated by two regions with separate promoters, one upstream of the coding region and a second within the intron. J. Biol. Chem. 278, 34125–34132. Chen, Q., Fernandez, V., Sundstrom, A., Schlichtherle, M., Datta, S., Hagblom, P., Wahlgren, M., 1998. Developmental selection of var gene expression in Plasmodium falciparum. Nature 394, 392–395. Corcoran, A.E., 2005. Immunoglobulin locus silencing and allelic exclusion. Semin. Immunol. 17, 141–154. Deitsch, K.W., Calderwood, M.S., Wellems, T.E., 2001. Malaria. Cooperative silencing elements in var genes. Nature 412, 875–876.

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