# Plasmodium falciparum var Genes Are Regulated by Two Regions with Separate Promoters, One Upstream of the Coding Region and a Second within the Intron

Amy Jost, Jianbing Mu, Michael Calderwood, Laila Gannoun-Zaki, Thomas

Wellems, Kirk Deitsch

► To cite this version:

Amy Jost, Jianbing Mu, Michael Calderwood, Laila Gannoun-Zaki, Thomas Wellems, et al.. Plasmodium falciparum var Genes Are Regulated by Two Regions with Separate Promoters, One Upstream of the Coding Region and a Second within the Intron. Journal of Biological Chemistry, 2003, 278 (36), pp.34125-34132. 10.1074/jbc.M213065200. hal-02281911

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*Plasmodium falciparum var* Genes Are Regulated by Two Regions with Separate Promoters, One Upstream of the Coding Region and a Second within the Intron*

Received for publication, December 20, 2002, and in revised form, June 27, 2003
Published, JBC Papers in Press, June 27, 2003, DOI 10.1074/jbc.M213065200

Michael S. Calderwood‡, Laila Gannoun-Zaki‡, Thomas E. Wellems‡, and Kirk W. Deitsch§¶

From the ‡Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892 and §Weill Medical College of Cornell University, New York, New York 10021

Antigenic variation in *Plasmodium falciparum* malaria parasites results from switches in expression among members of the multicopy *var* gene family. This family is subject to allelic exclusion by which particular genes are expressed while the rest of the family remains transcriptionally silent. Evidence from reporter constructs indicates that *var* gene silencing involves a co-operative interaction between the *var* intron and an upstream element and requires transition of the para-sites through S-phase of the cell cycle. These findings implicate chromatin assembly in the process of regulating *var* gene expression and antigenic variation. Here we characterize the *var* intron and the elements within it that are necessary for *var* transcriptional silencing. Alignments of *var* introns show a highly conserved structure that consists of three discreet regions with distinct base pair compositions. The middle region is highly AT-rich and is sufficient to silence an associated *var* promoter. Constructs that include a typical *var* intron upstream of a reporter gene or drug-selectable marker reveal that the intron also possesses promoter activity, presumably providing an explanation for the origin of the previously described *var* "sterile" transcripts. Deletions that disable the promoter activity of the intron also eliminate its ability to function as a silencer. These findings suggest that interactions between the regions of these two promoters and the generation of the sterile transcripts play a significant role in regulating *var* gene expression.

The deadliest form of malaria is the result of infection by the protozoan parasite *Plasmodium falciparum*. The disease is characterized by recurrent waves of parasitemia in which each wave represents a population of infected red blood cells with distinct antigenic properties and cytoadherent phenotypes (1). The parasites in these waves use a mechanism of antigenic variation to switch among major variable parasite-produced proteins (PfEMP1) that are placed on the surface of the infected host red blood cells. These switches allow parasites to escape

clearance by the host antibody response, leading to persistent infections of variable severity. Antigenic variation thus ensures against the elimination of *P. falciparum* by interfering with the development of complete anti-PfEMP1 immunity.

PfEMP1 is encoded by a multicopy gene family termed *var* (2–4). About 60 different *var* genes are present within the haploid genome of each parasite (3, 5), encoding forms of PfEMP1 with unique antigenic and cytoadherent properties. Switches in expression between individual members of the *var* gene family account for the antigenic variation in a *P. falciparum* population over the course of an infection (4). A mechanism of allelic exclusion appears to control expression of the individual genes (6, 7), thus enabling the parasite population to survive. Without such exclusion, expression of all copies simultaneously would result in premature expenditure of the antigenic repertoire, whereas incomplete silencing of previously expressed copies would lead to clearance by antibodies generated against them. The molecular mechanisms responsible for activating and silencing the genes within this family, and thus coordinating the process of antigenic variation, therefore represent a process imperative for parasite survival.

Although gene duplications, deletions, and recombinations within the family are not uncommon, switches in expression were not found to be accompanied by promoter DNA sequence alterations (8). Silent promoters were shown to be rendered transcriptionally active when removed from the chromosome and placed on transfected episomes (8). These results indicated that switches in expression were probably not the result of changes in transcription factors. Silencing of episomal *var* promoters was restored, however, when a *var* intron was also incorporated into the transfected plasmid (9). Silencing because of the presence of the intron was specific to *var* promoters and depended upon transition of the transfected parasite through S-phase of the cell cycle. These findings associate control of *var* expression with changes in chromatin structure and epigenetic mechanisms of regulation that may have some features in common with chromatin-mediated gene regulation in other organisms.

Members of the *var* gene family are found in tandem arrayed clusters within the internal regions of chromosomes 4, 7, 8, and 12 and as individual genes within subtelomeric regions that also contain genes of the *rifin* and *stevor* families (1, 10). In both internal and subtelomeric regions, transcriptionally active *var* genes have been found adjacent to silent copies, indicating that chromosomal position does not dictate expression. Gene clusters appear to be partitioned into transcriptionally active and silent regions. Chromatin "boundaries" or "insulators" typically separate such regions to prevent the spread of silent and active chromatin structure into adjacent regions and to prevent inappropriate "cross-talk" between the regulatory elements of

<footnote>* This work was supported by National Institutes of Health Grant AI 52390. The Department of Microbiology and Immunology at Weill Medical College of Cornell University acknowledges the support of the William Randolph Hearst Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote>¶ A Stavros S. Niarchos Scholar. To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Ave., W-704, Box 62, New York, NY 10021. Tel.: 212-746-4976; Fax: 212-746-4028; E-mail: kwd2001@med.cornell.edu.</footnote>
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<figure><img src="image_8.png" /><figcaption>FIG. 1. Sequence alignments and nucleotide composition analysis of var introns from the subtelomeric and internal regions of different *P. falciparum* chromosomes. A, the intron sequences align into three distinct regions with prominent asymmetry in strand composition. Conserved repeats 5'-TGT(G/A)T(G/A)TG-3' are found only in region 1, whereas copies of the complementary repeat 5'-ACA(C/T)A(C/T)AC-3' are found only in region 3 (shaded). Conserved GT and AG dinucleotides at the intron splice boundaries are highlighted in reverse. Conserved repeat elements containing a putative initiator element are *boxed*, and the Inr-like initiator sequence (TCATA) is *shaded*. A similar conserved potential initiator element found on the opposite strand is *underlined*. GenBank™ sequence accession numbers (leading number of each label indicates the assigned chromosome): 1a, AL031747; 2a, AE001366; 2b, AE001434; 3a, AL034560; 3b, AL034559; 4a, AL035477; 7a, L40608; 7b and 7c, L40609; 12a, AC006279; 12b, AC006280. B, histograms showing base pair compositions of the forward strand of the three regions of the intron. Region 1 contains ≈20% G and 1% C content in the forward strand, whereas region 3 contains 2% G and 18% C. Contrasts are evident also in the T and A composition of these regions. Region 2 of the forward strand contains >70% A content.</figcaption></figure>
out region 1 of the intron from pVLH/int by restriction digestion with BamHI/DraI and cloning this fragment directly into BamHI/SmaI-digested pVLH-1. pVLH/intΔ2 was the result of a spontaneous deletion in pVLH/int that occurred while propagating the plasmid in E. coli. This plasmid was sequenced and found to be missing bp 161–686 of the intron. The plasmid pILH-1 was made by digesting pVLH/int with SstI and SmaI. The intron fragment was then cloned upstream of the luciferase coding region of similarly digested pGL2-basic (Promega). The simian virus 40 polyadenylation site of pGL2-basic was then replaced with a BamHI/ClaI fragment containing the hrp2 3' sequence from the plasmid pHLH-1. The various intron fragments were also cloned into BamHI/Not1-digested pILH-1 to generate plasmids in which the individual portions of the intron replaced the full-length intron to drive luciferase expression. Site-directed mutagenesis was performed by using the QuikChange mutagenesis kit (Stratagene) and the manufacturer's protocols.

**Parasite Transfection and Luciferase Assays**—Parasites were transfected as described (20). Briefly, uninfected human erythrocytes were electroporated in the presence of 50 µg of plasmid DNA in incomplete cytomix using a Bio-Rad gene pulser, 0.2-cm cuvettes, and conditions of 0.31 kV and 960 microfarads. Erythrocytes from two such electroporations were combined for each 5-ml culture and inoculated with late stage parasites purified using a Percoll/sorbitol gradient. Luciferase assays were performed on 5-ml cultures at selected times after transfection (9). Error bars in graphs represent standard deviations of experiments done in triplicate.

Previous work has shown that plasmids containing an intron require passage through S-phase to silence an upstream *var* promoter (9). However, because *var* promoters are only active during the ring stage (prior to S-phase), silencing is not detected after transfection until the ring stage of the following cycle, after which silencing is maintained through subsequent cell cycles. All transfection experiments described in this manuscript were performed by allowing parasites to invade DNA-loaded erythrocytes (20) and then separating the parasites from the DNA-loaded erythrocytes by Percoll/sorbitol gradients. Return of the purified parasites to culture with fresh, unloaded erythrocytes thus ensured that all transfected plasmids had been through at least one S-phase. Activity of the *var7b* promoter was always measured in ring-stage parasites unless otherwise stated.

## RESULTS

**Analysis of var Intron Structure**—Typical *var* genes contain two exons separated by a 0.8–1.2 kb intron (3). Fig. 1A shows an alignment of the sequences of introns from 11 different *var* sequences in the GenBank™ data base. These sequences are from several different parasite lines and include genes from both subtelomeric and internal regions of chromosomes. All show similar structure with three distinct regions that are distinguished by their sequence similarity and nucleotide composition (Fig. 1A). Several repeat sequence elements occur in these regions, including TGTATGTG in region 1 and the direct complement of this sequence (ACATACAC) in region 3.

Forward strand composition data, summarized by the histograms in Fig. 1B, show prominent asymmetry of the G *versus* C and A *versus* T contents of regions 1 and 3. The forward strand of region 2 consists of over 70% A with very little G or C. The differences among regions 1, 2, and 3 were found to be distinct without ambiguity of their borders in the alignments.

**Identification of an INR Element in a var Promoter**—Primer extension was previously performed by using mRNA from an expressed internal gene (*var7b*), and the transcription start site was mapped to a conserved A residue at the center position within the sequence TCATA (8). Interestingly, this 5-base pair sequence was recently identified as a promoter element of numerous genes in the parasite *T. vaginalis* (21). In all cases, the A at position 3 was the transcription start point, and certain specific point mutations within the sequence resulted in substantially reduced promoter activity. This element is very similar in function and sequence to a metazoan initiator element (Inr), suggesting that these elements were present in an ancestor early in eukaryotic evolution.

To test whether the TCATA sequence found at the transcription start site of the *var7b* promoter has a similar function to this sequence in Inr, we performed site-directed mutagenesis on the construct pVLH-1, which incorporates the *var7b* promoter driving expression of a luciferase reporter gene (8). Three specific mutations that reduce promoter activity in the *T. vaginalis* sequence were engineered into pVLH-1, and the resulting plasmids were transfected into *P. falciparum* parasites. These mutations produced reductions in promoter activity similar to those seen with experiments in *T. vaginalis*, suggesting the *P. falciparum* TCATA element has a function similar to that of the Inr in other organisms (Fig. 2B).

Inr Element from *T. vaginalis*: var transcription start site:

TCATA
TCATA

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**FIG. 2.** Effect on luciferase expression of nucleotide changes in an Inr-like element containing the *var7b* transcription start point. The transcription start point is located within the upstream *var7b* promoter region that resembles an Inr-like element also present in *T. vaginalis*. The transcription start point (A) is shown in bold. Luciferase expression levels from constructs containing site-specific mutations in the *var* initiator element are shown in the histogram. The sequence of the mutations is shown below the horizontal axis. Luciferase expression was detected from ring-stage parasites.

**Promoter Activity within the Var Intron**—Inspection of the intron sequence alignment shown in Fig. 1A identified several conserved copies of the putative Inr element described above. Therefore, we considered whether the *var* intron might also have promoter activity, thus providing a possible explanation for the origin of the “sterile” or “aberrant” exon 2 containing transcripts previously described (3, 16). These transcripts were shown to include a portion of the intron and exon II. Although a case has been made for low level translation of a few of these transcripts (22), the majority have no consistent start methionine or other features indicative of translation.

To determine whether a *var* intron can act as a functional promoter, we excised the intron previously used as a silencer in the plasmid pVLH/int (intron 2b in Fig. 1B) and placed it upstream of a luciferase reporter gene. The resulting constructs, pILH-1 and pVLH-1, were then transfected separately into *P. falciparum* parasites, which were then synchronized by Percoll/sorbitol gradient purifications (17). Luciferase expression levels were measured in both ring-stage parasites and late trophozoites (Fig. 3). As expected, the *var7b* promoter activity in the construct pVLH-1 was ring-specific, with expression levels falling substantially in late trophozoites. *var* genes were previously shown to be expressed in the ring stages of asexual parasites, between 3 and 18 h after merozoite invasion (23). By contrast, the construct that included intron 2b as a promoter showed very little activity in rings and much higher levels of activity in late trophozoites. This activity period is in agreement with the timing of the sterile transcripts detected by RNA blot analysis (16, 24) and coincides with S-phase of the cell cycle (25). Trophozoite stage activity of the intron promoter may thus relate to the dependence of *var* gene silencing on the transition of the parasite through S-phase (9) and assembly of silent chromatin that occurs during this period of the cell cycle (26–28).
<figure><img src="image_10.png" /><figcaption>FIG. 3. Luciferase expression levels showing activity of the *var7b* upstream and intron 2b promoters at different times in the cell cycle. Synchronized parasites were transfected with either pVLH-1 or pILH-1. Histograms indicate the luciferase levels of ring stages 12 h after invasion and of late trophozoite stages 36 h after invasion.</figcaption></figure>

To further characterize the intron promoter and to determine what portion or portions were necessary for promoter activity, we transfected parasites with constructs that included either the entire intron 2b in forward or reverse orientation, region 2 alone, or intron 2b with region 2 deleted. To determine the relative strength of the intron promoter, we compared the luciferase activity of these constructs to that of pVLH-1. Late trophozoites containing reporter constructs driven by intron 2b and ring-stage parasites transfected with pVLH-1 were assayed to allow a direct comparison of the peak activity of both promoters. The results (Fig. 4A) show that the intron can function as a promoter in either orientation, a characteristic previously described for several TATA-less promoters in other organisms (29–31). Further, the luciferase levels from the various constructs show that intron region 2, the region that includes putative Inr elements similar to that in the upstream *var* promoter, is necessary for intron promoter activity and is sufficient on its own for luciferase expression.

Constructs containing the intron 2b promoter gave significantly lower luciferase activity than those containing the *var7b* promoter (Fig. 4A). To determine whether this low level of promoter activity was indicative of a truly functional promoter and was not simply spurious, the plasmid pVLH/int was modified to include the coding region of the human *dhfr* gene downstream of intron 2b (Fig. 4B). In this construct (pVLH/IDH), the intron acts as a silencer of the upstream *var7b* promoter and as a promoter driving expression of the *dhfr* gene. As expected, parasites transfected with this construct demonstrated silencing of the luciferase gene similar to that seen with pVLH/int (data not shown). When grown in media containing pyrimethamine, drug resistant parasites were selected after 3 weeks of drug pressure, as is typical for parasites transfected with plasmids containing drug resistance markers. Plasmid rescue experiments showed that the resistant parasites were carrying the construct pVLH/IDH. The strong repression of luciferase expression indicates that *dhfr* expression is unlikely to be the result of “read-through” transcription initiating from the *var7b* promoter. In addition, control cultures of untransfected parasites or parasites transfected with a

A.

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B.

<figure><img src="image_12.png" /><figcaption>FIG. 4. Luciferase expression levels from parasites transfected with plasmids containing either the *var7b* upstream region or portions of intron 2b as a promoter. A, luciferase levels are indicated for parasites transfected with the following constructs: pVLH-1; pILH-1; pILH-1(+) in which intron 2b is placed upstream of the *luc* coding region in the reverse orientation; p2LH-1 in which region 2 of the intron is placed upstream of the *luc* coding region; and pILHintΔ2 in which region 2 has been deleted from the intron. Luciferase assays were done on ring-stage parasites for pVLH-1 and on late trophozoites for all other constructs, thus comparing peak promoter activity for all constructs. B, plasmid map for the construct pVLH/IDH.</figcaption></figure>

modified pVLH/IDH where the intron had been deleted failed to select any drug-resistant parasites. These results indicate that the promoter activity of the intron was sufficient to alter the phenotype of the parasite and, combined with previously published Northern blots that show significant transcription of the “sterile” transcripts (3, 16), strongly support the conclusion that *var* introns are functionally active promoters in *P. falciparum*.
*Relative Effects of Intron Regions 1, 2, and 3 on var Gene Silencing*—To explore the role of intron regions 1, 2, and 3 on *var* gene silencing, we constructed modified pVLH/int plasmids in which the full-length intron 2b was replaced by each individual region separately and in combination with one another. Luciferase expression levels from transfected parasites were then measured to determine the effects of the various regions of the intron on silencing of the associated *var7b* promoter. The expression assays were performed on ring stages after all the transfected parasites had passed through S-phase.

All constructs that included region 2, including constructs where region 2 was the only portion of the intron included, showed levels of *var* promoter silencing equivalent to that obtained from the complete intron (Fig. 5A). Constructs that included only regions 1 or 3, or both regions 1 and 3 but excluding region 2, showed partial repression of luciferase expression (50–70%) but were not able to fully silence *var7b* promoter activity. To further demonstrate association between the intron's promoter activity and its ability to function as a silencing element, each combination of intron 2b fragments was placed upstream of luciferase to test for promoter activity (Fig. 5B). These experiments show a direct association between the intron's promoter activity and its ability to silence the associated *var* promoter. In particular, these results highlight the importance of region 2 for both the silencing ability of the intron as well as its inherent promoter activity.

DISCUSSION

Silencing and selective expression of *var* genes have vital roles in the antigenic variation that is central to the pathogenesis of *P. falciparum* malaria. Models for *var* transcriptional control, however, are undeveloped, and only limited analogies can be drawn to the regulatory mechanisms of multigene families in other eukaryotes. Systems that show some analogies (for example, the *vsg* genes of African trypanosomes (32), the *vsp* genes of *Giardia* (33), and the odor receptor genes of mammals (34)) are controlled by mechanisms that remain unclear and appear to differ considerably from those in *P. falciparum*. A mechanism in which regions containing two separate promoters co-operate to control gene silencing has not been described for these other multicopy gene families and may be unique to *var* genes.

Reports differ on the number of *var* genes expressed at one time in a single parasite. Nuclear run-on experiments using parasites selected for their cytoadherence phenotype showed a single *var* gene transcribed in trophozoites (7). Single-cell reverse transcriptase-PCR experiments identified transcripts from multiple *var* genes in early ring-stage parasites, but only a single transcript remained in trophozoites (6). Detection of only a single transcript band on RNA blots is consistent with proposals that multiple transcripts in early rings are low level and transient, possibly because they are truncated or unstable (24). In contrast, other experiments using single-cell reverse transcriptase-PCR have described multiple *var* transcripts in trophozoites, suggesting that more than one *var* gene can be transcribed at this stage of the cell cycle (35). Nevertheless, it is clear that progression through the cell cycle has an important effect on the regulation and detection of *var* gene transcripts. One manifestation of this effect is the co-operative S-phase-dependent silencing of episomal constructs carrying *var* promoters in *cis* with *var* introns. The transcription from *var* introns occurs later in the cell cycle than the ring-stage peak of upstream *var* promoter activity and may relate to the S-phase dependence of silencing.

A function or explanation for the existence of the *var* sterile transcripts has been lacking since they were first described along with the discovery of the *var* gene family (3). Recent work

A.

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B.

<figure><img src="image_14.png" /><figcaption>FIG. 5. The silencing and promoter activities of intron 2b both require region 2. A, luciferase activity from parasites transfected with plasmids containing the *var7b* promoter driving *luc* expression along with different portions of the intron. Control plasmid pVLH-1 contains no intron and demonstrates full activity. Plasmid pVLH/int has the entire *var* intron 2b and demonstrates complete silencing. “Δ” in the names of the other plasmids indicates the region of the intron that has been deleted. B, luciferase activity from constructs that contain different portions of intron 2b driving luciferase expression. Control plasmid pXLH-1 contains the luciferase coding region with no promoter. Plasmid pILH-1 has the entire intron driving luciferase expression. The remaining plasmids are labeled as described for A, with Δ indicating the portion of the intron that has been deleted.</figcaption></figure>

on chromatin-mediated gene silencing in other organisms may provide some suggestions. The boundary elements *scs* and *scs'* in *Drosophila* (13) and several boundaries described in yeast (14) are actively transcribed. Promoter activities found in these boundaries are important for their ability to prevent the spread
of silent chromatin outside the silenced locus (36). It has also been shown that several yeast transcription factors can act as boundaries when bound to DNA, suggesting that certain promoters may behave as insulators and partition genomes into functionally independent domains (14, 15). This behavior is pronounced in the human β-globin locus where non-coding transcripts precisely delineate transcriptionally active and silent domains (37). Similar to the transcripts from the var intron, the β-globin non-coding transcripts are expressed during early S-phase at the time when the globin locus is replicated and chromatin assembly and modification occurs. Disruption of the production of these transcripts interferes with chromatin remodeling and proper gene regulation. Thus, the promoter activities of var introns could be functioning in a manner that likewise separates the transcriptionally active and silent chromatin domains of the chromosome neighborhoods in which var genes reside. Alternatively, the intron promoter could function directly in silencing of the upstream var promoter through promoter competition. This has also been proposed as a model for regulating expression of the β-globin locus in vertebrates (38–40). In this system, cis-linked promoters compete for a single shared enhancer, with a developmental switch in expression resulting from one promoter out- competing the other. An effect of promoter competition has recently been described for an insulator element in *Drosophila* (41). If such a mechanism applies to var genes, both promoters would be competing for a single enhancer element, and silencing of the upstream var promoter would be accompanied by the corresponding up-regulation of the intron promoter. This model would explain the apparent weakness of the intron promoter in the construct pILH-1 where it has been separated from any activator sequences that it may be competing for.

The luciferase assays described in Fig. 4A show that the intron has bi-directional promoter activity. This is in agreement with previous work showing that the intron can function as a silencer in either orientation (9). The bi-directionality of the silencing and promoter functions of the intron may be related to the symmetry displayed graphically in Fig. 1B, as inversion of the intron produces no overall change in the strand asymmetries of the DNA. We also note that a conserved sequence matching the Inr element is present on the opposite strand of the introns aligned in Fig. 1A and could function as a transcription start site for the inverted intron. Several other bi-directional promoters have been described previously in other organisms (29–31). Interestingly, the human *hGMFG* promoter is active in a single orientation; if the upstream regulatory regions are deleted, however, the core promoter functions bi-directionally (31). In the plasmid pILH-1, the intron may likewise be separated from the influence of upstream regulatory sequences, thus behaving as a bi-directional core promoter.

Although the non-coding regions of the *P. falciparum* genome are known for being extremely AT rich, the base composition of the intron is unique in that all three regions display distinct strand asymmetries (Fig. 1B). In region 2, the forward strand is over 70% A, with the remaining base pairs made up mostly of T residues, whereas the reverse strand of region 1 and the forward strand of region 3 contain A, T, and C but show an almost total absence of G. The function of this asymmetry is unknown; however, sequences with such asymmetric composition are easily unwound (42) and may function as binding sites for DNA or chromatin-binding proteins (43). DNA-binding proteins containing AT-hooks recognize the base pair composition of AT-rich target sites similar to region 2 (44) and can regulate gene expression through the modification of chromatin structure (45). AT-hook-containing proteins have been shown to play a role in the attachment of DNA to the nuclear matrix or

scaffold (46, 47) and are found encoded in the *P. falciparum* genome. Such attachment is predicted to lead to the formation of chromatin loops (48) with the attachment sites acting as boundaries between regions of the genome that are packaged into separate loops of chromatin representing different domains (49, 50). Such a role would be consistent with a model in which the intron serves as both a silencer and a boundary element that separates each var gene from the neighboring chromatin environment.

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