These authors contributed equally to this work.
Identification of a cis-acting DNA–protein interaction implicated in singular var gene choice in Plasmodium falciparum
Article first published online: 4 SEP 2012
© 2012 Blackwell Publishing Ltd
Volume 14, Issue 12, pages 1836–1848, December 2012
How to Cite
Brancucci, N. M. B., Witmer, K., Schmid, C. D., Flueck, C. and Voss, T. S. (2012), Identification of a cis-acting DNA–protein interaction implicated in singular var gene choice in Plasmodium falciparum. Cellular Microbiology, 14: 1836–1848. doi: 10.1111/cmi.12004
Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/onlineopen#OnlineOpen_Terms
- Issue published online: 19 NOV 2012
- Article first published online: 4 SEP 2012
- Accepted manuscript online: 14 AUG 2012 10:23AM EST
- Manuscript Accepted: 8 AUG 2012
- Manuscript Revised: 6 AUG 2012
- Manuscript Received: 27 JUL 2012
- Swiss National Science Foundation. Grant Numbers: PP00A-110835, PP00P3_130203
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- Experimental procedures
- Supporting Information
Plasmodium falciparum is responsible for the most severe form of malaria in humans. Antigenic variation of P. falciparum erythrocyte membrane protein 1 leads to immune evasion and occurs through switches in mutually exclusive var gene transcription. The recent progress in Plasmodium epigenetics notwithstanding, the mechanisms by which singularity of var activation is achieved are unknown. Here, we employed a functional approach to dissect the role of var gene upstream regions in mutually exclusive activation. Besides identifying sequence elements involved in activation and initiation of transcription, we mapped a region downstream of the transcriptional start site that is required to maintain singular var gene choice. Activation of promoters lacking this sequence occurs no longer in competition with endogenous var genes. Within this region we pinpointed a sequence-specific DNA–protein interaction involving a cis-acting sequence motif that is conserved in the majority of var loci. These results suggest an important role for this interaction in mutually exclusive locus recognition. Our findings are furthermore consistent with a novel mechanism for the control of singular gene choice in eukaryotes. In addition to their importance in P. falciparum antigenic variation, our results may also help to explain similar processes in other systems.
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- Experimental procedures
- Supporting Information
Many unicellular pathogens use antigenic variation to escape adaptive immune responses in the host. The widespread occurrence of this strategy in evolutionary distant species underscores its key role in pathogen survival and spreading. While the underlying control pathways are highly diverse in different systems, both mechanistically and in terms of complexity, antigenic variation is defined by two basic concepts. First, the antigens are encoded by gene families, the members of which are expressed in a mutually exclusive manner. Second, switches in the expression of individual members lead to antigenic variation of surface-exposed antigens. In several medically important pathogens such as Borrelia spp., Neisseria spp., Giardia lamblia, Plasmodium falciparum and Trypanosoma brucei, this paradigm of clonal phenotypic variation reaches a remarkable yet poorly understood level of sophistication (Deitsch et al., 2009; Dzikowski and Deitsch, 2009; Morrison et al., 2009; Prucca and Lujan, 2009).
The apicomplexan parasite P. falciparum causes several hundred million malaria cases and close to one million deaths annually (World Health Organization, 2010). Malaria-associated morbidity and mortality is a result of the intra-erythrocytic developmental cycle (IDC) where repeated rounds of parasite invasion into red blood cells (RBCs) are followed by intracellular maturation and replication. During this stage of infection parasites expose the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the RBC surface (Leech et al., 1984). This highly polymorphic antigen, encoded by the 60-member var gene family, undergoes antigenic variation to facilitate chronic infection and transmission (Biggs et al., 1991; Roberts et al., 1992; Smith et al., 1995; Su et al., 1995; Gardner et al., 2002). Furthermore, PfEMP1 mediates sequestration of infected RBC aggregates in the microvasculature of various organs and is thus directly responsible for severe outcomes, including cerebral and placental malaria (MacPherson et al., 1985; Pongponratn et al., 1991; Baruch et al., 1996; Gardner et al., 1996; Reeder et al., 1999; Beeson and Duffy, 2005).
var genes are transcribed by RNA polymerase II (RNA polII) in ring-stage parasites during the first half of the IDC (Scherf et al., 1998; Dzikowski et al., 2006; Kyes et al., 2007). Notably, only one var gene is transcribed at any time while all other members are silenced (Scherf et al., 1998). Switches in var gene transcription, and consequently antigenic variation of PfEMP1, are independent of detectable recombination events and occur by in situ var gene activation (Scherf et al., 1998). var gene silencing is explained by the fact that all var genes are positioned in subtelomeric and some chromosome-internal heterochromatic regions (Gardner et al., 2002; Flueck et al., 2009; Lopez-Rubio et al., 2009; Salcedo-Amaya et al., 2009). These chromosomal domains are uniformly enriched in histone 3 lysine 9 tri-methylation (H3K9me3) and P. falciparum heterochromatin protein 1 (PfHP1) (Flueck et al., 2009; Lopez-Rubio et al., 2009; Perez-Toledo et al., 2009; Salcedo-Amaya et al., 2009). The presence of these epigenetic marks is directly linked to var gene silencing (Chookajorn et al., 2007; Lopez-Rubio et al., 2007; Perez-Toledo et al., 2009). In contrast, the active var locus is associated with H3K9ac and H3K4me2/me3 instead (Lopez-Rubio et al., 2007). Interestingly, singular var gene activation is linked to locus repositioning into a dedicated perinuclear expression site (Duraisingh et al., 2005; Ralph et al., 2005; Marty et al., 2006; Voss et al., 2006; Dzikowski et al., 2007). While the mechanisms underlying this process are largely unknown, a recent study identified a critical role for nuclear actin in locus repositioning and mutually exclusive expression (Zhang et al., 2011). Moreover, Volz et al. identified a H3K4-specific methyltransferase (PfSET10) and demonstrated its exclusive localization to the active var locus suggesting a role for this enzyme in the transmission of epigenetic memory (Volz et al., 2012).
In recent years, var gene promoters emerged as key components in all layers of var gene regulation. Experiments where var gene promoters drive transcription of drug-selectable reporter genes have been particularly informative in studying var promoter function. In absence of drug selection var promoters are predominantly silenced, whereas drug challenge selects for parasites carrying active promoters (Voss et al., 2006; 2007). Importantly, this forced activation is sufficient to infiltrate a drug-selectable reporter into the mutual exclusion programme (Voss et al., 2006; 2007; Dzikowski et al., 2007). In addition to var promoters, the var intron acts as a cooperative partner in silencing and mutual exclusion (Deitsch et al., 2001; Calderwood et al., 2003; Gannoun-Zaki et al., 2005; Frank et al., 2006; Voss et al., 2006; Dzikowski et al., 2007).
We postulated that transcriptional control of var genes may be mediated by unknown sequence information contained within the promoter region. In this study, we developed a functional promoter mapping approach tailored to identify and characterize var gene-specific regulatory information. We mapped an autonomous upstream activating sequence (UAS) that is essential for var promoter activation. Notably, we also identified a region downstream of the transcriptional start site (TSS) and demonstrate an important role for this element in mutually exclusive promoter recognition. In absence of this sequence var promoters are fully active but, unlike wild-type promoters, do not compete with endogenous var gene transcription. Within this region we identified a 47 bp motif that interacts in a sequence-specific manner with an unknown nuclear protein. Together, our results show for the first time that the complex regulation of mutually exclusive var gene transcription involves functional cis-acting modules with intrinsic and position-dependent activities. They are furthermore consistent with a novel mechanism in sustaining singular gene choice in eukaryotes.
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- Experimental procedures
- Supporting Information
Functional var promoter mapping by bi-directional deletion analysis
To identify regulatory var promoter elements we employed a system suitable to analyse promoter activity in stably transfected parasites. All reporter constructs are based on the parental plasmid pBC (Fig. 1A) where the blasticidin deaminase (bsd) resistance cassette selects for stable episomes. A 2.5 kb var upsC upstream sequence (PFL1960w) controls transcription of the dual reporter encoding human dihydrofolate reductase fused to green fluorescent protein (hdhfr-gfp). A var gene intron element is located downstream of the hdhfr-gfp cassette to account for its role in var gene regulation. A telomere-associated repeat element 6 sequence (TARE6/rep20) is included for improved plasmid segregation (O'Donnell et al., 2002). In such a context, homogenous populations carrying active upsC promoters are obtained via selection with the antifolate drug WR99210 (WR) (Voss et al., 2006; 2007).
To identify elements involved in promoter activation and mutual exclusion we sequentially truncated the upsC upstream sequence from either the 5′ or 3′ end (Fig. 1B). We chose this bi-directional approach to identify possible functional regions both up- and downstream of the putative TSS. Based on a multiple upsC sequence alignment and the previous experimental mapping of an upsC TSS we expected the TSS of PFL1960w at position −1167 (Deitsch et al., 1999; Voss et al., 2000). Transfected parasites were challenged with WR and resistant populations were obtained for all but one cell line, 3D7/pBC3 (Fig. 1B). Several attempts to select for WR-resistant 3D7/pBC3 parasites failed showing that the region between −1656 to −1217 comprises an important UAS and/or the core promoter. To test if any of the deletions affected promoter strength we determined relative hdhfr-gfp transcript levels in ring-stage parasites by quantitative reverse transcriptase PCR (qRT-PCR). As shown in Fig. 1B, transcript levels in 3D7/pBC1 and 3D7/pBC2 were similar to those in 3D7/pBC indicating that the sequence upstream of −1656 does not contribute to var promoter activity. The promoter in pBC5, lacking 491 bp of the 5′ UTR, was also fully active. In contrast, the truncation encompassing bps −1057 to −1 in pBC4 caused a significant reduction in steady-state transcript levels. Hence, this approach identified two regulatory regions, located upstream and downstream of the putative TSS, respectively, which fulfil important roles in var promoter function.
Functional identification of an autonomous upsC upstream activating sequence
To learn more about the nature of the putative UAS we set out to analyse its function in the context of a minimal heterologous promoter. We decided to use the knob-associated histidine rich protein (kahrp) gene promoter for three reasons. First, the TSS of this gene has been mapped to 849 bp upstream of the ATG (Lanzer et al., 1992). Second, similar to var genes the timing of kahrp transcription peaks in ring-stage parasites. Lastly, the kahrp locus is not enriched in H3K9me3/PfHP1 (Flueck et al., 2009; Lopez-Rubio et al., 2009; Salcedo-Amaya et al., 2009), which is an important consideration in order to avoid heterochromatin-mediated masking of autonomous cis-acting activities. Hence, we generated plasmid pBKmin-RI where bps −1115 to −1 of the kahrp upstream sequence control transcription of the hdhfr-gfp reporter (Fig. 2A). Parasites carrying pBKmin-RI episomes were readily obtained after transfection. Notably, the disposition of this plasmid to integrate into the endogenous kahrp locus allowed us to measure Kmin activity also in a chromosomal environment. This integration event essentially causes a promoter swap where Kmin drives expression of the endogenous kahrp gene and the endogenous kahrp promoter controls transcription of the hdhfr-gfp reporter (Figs 2B and S1). Compared with the endogenous full-length kahrp promoter, the episomal and chromosomal minimal promoters displayed a 300-fold and 1000-fold reduced activity respectively (Fig. 2C). Hence, Kmin clearly fulfilled the requirements for a minimal promoter.
We cloned two overlapping fragments containing the putative upsC UAS upstream of Kmin to create upsC-Kmin hybrid promoters (pBC1Kmin and pBC2Kmin) (Figs 2D and S1). The region downstream of the upsC TSS encompassing bps −463 to −20, which has no effect on upsC promoter activity (Fig. 1B), was used as negative control (pBC3Kmin). qRT-PCR analysis revealed that upsC fragments C1 (−1679 to −1200) and C2 (−1401 to −727) consistently activated Kmin to a similar extent in both the episomal and chromosomal context whereas fragment C3 had no effect. Furthermore, neither the var intron nor the rep20 element altered Kmin activity. Together, these findings corroborate the results obtained with the upsC deletion constructs and are consistent with the presence of a var UAS located between bps −1401 and −1217. The fact that this element activates transcription from a heterologous minimal promoter suggests an autonomous, context-independent function in activating RNA polII-mediated transcription.
Transcriptional initiation from an alternative TSS compensates for the loss of core promoter function
Here, we investigated the functional region downstream of the putative TSS that is defined by plasmids pBC4 and pBC5 (−1057 to −491). Deletion of this region caused a substantial reduction in steady-state transcripts (Fig. 1B), suggesting it may contain important activating sequences. Northern blot analysis confirmed the reduced abundance of steady-state transcripts in 3D7/pBC4 compared with 3D7/pBC and 3D7/pBC5 (Fig. 3). An independent time-course experiment confirmed these results and excluded the possibility of altered transcriptional timing and/or transcript accumulation in 3D7/pBC4 parasites (Fig. S2). However, these experiments also revealed that the size difference between pBC- and pBC4-derived transcripts was much smaller than expected. In spite of the 1057 bp deletion in the 5′ UTR, pBC4-derived transcripts were larger than those originating from pBC5 where only 491 bp of the 5′ UTR were deleted (Fig. 3). This shows that transcription from the truncated pBC4 upstream sequence initiated from an alternative upstream TSS. Consequently, the reduced steady-state transcript levels observed in 3D7/pBC4 were not related to the deletion of important activating sequences but rather to the loss of proper core promoter function and transcriptional initiation from the natural TSS.
A regulatory region downstream of the TSS is involved in mutually exclusive var gene expression
Transgenic parasites carrying activated full-length var promoters do not transcribe endogenous var genes and fail to express PfEMP1 (Dzikowski et al., 2006; 2007; Voss et al., 2006; 2007; Chookajorn et al., 2007; Howitt et al., 2009; Witmer et al., 2012). This implies that mutually exclusive locus recognition may be mediated by cis-acting regulatory sequence elements located in var gene upstream regions. To test this hypothesis and to identify such functional elements we investigated if any of the activated truncated promoters escaped mutually exclusive activation. The negative control line 3D7/pBM, in which the unrelated ring stage-specific mahrp1 promoter controls hdhfr-gfp transcription, expressed PfEMP1 at normal levels, whereas parasites of the positive control line 3D7/pBC exhibited the expected PfEMP1 knock-down phenotype (Fig. 4A). PfEMP1 expression was also abolished in 3D7/pBC2 showing that the region ranging from −2488 to −1656 bps upstream of the start codon is not important for mutually exclusive locus recognition. In contrast, 3D7/pBC4 and 3D7/pBC5 parasites expressed PfEMP1 at levels similar to the 3D7/pBM-negative control line. Interestingly, both truncated promoters lack the same 491 bp sequence downstream of the TSS suggesting that this region carries sequence information important for mutually exclusive locus recognition.
To map this region more precisely we cloned three additional truncated upsC sequences in pBC6, pBC7 and pBC8 (Fig. 4B). Similar to the full-length promoter in 3D7/pBC, 3D7/pBC8 parasites failed to express PfEMP1 demonstrating that the pBC8 promoter was activated in a mutually exclusive manner. In contrast, 3D7/pBC6 and 3D7/pBC7 expressed PfEMP1 at levels similar to two negative controls (WR-selected 3D7/pBM and unselected 3D7/pBC) showing that these truncated promoters were not subject to mutually exclusive recognition as already observed for 3D7/pBC5. Together, this series of experiments pinpointed a putative 101 bp mutual exclusion element (MEE) (bps −316 to −215) that drives the upsC promoter into mutually exclusive activation; in absence of the MEE promoters escape this restriction and are activated in parallel to endogenous var transcription.
The mutual exclusion element interacts specifically with an unknown nuclear factor
The proposed function of the MEE in mutually exclusive activation may be directly linked to the specific recruitment of an unknown regulatory factor. We therefore tested three overlapping fragments (MEE1–MEE3) in electromobility shift assays (EMSA) using parasite nuclear extracts. Whereas MEE1 and MEE3 showed no sign of specific binding (data not shown), the central 47 bp MEE2 fragment formed a DNA–protein complex that was specifically competed in a dose-dependent manner by an excess of homologous competitor only (Fig. 5A). To characterize this interaction in more detail we performed competition EMSAs using a set of mutated MEE2 sequences (Fig. 5B). As expected, scrambled MEE2 failed to compete underscoring the sequence-specificity of this interaction. Four out of six fragments carrying consecutively mutated 8mers (MEE2-mut2/-mut3/-mut5/-mut6) competed with similar efficiency as the MEE2 wild-type sequence (Figs 5B and S3A). In contrast, MEE2-mut4 failed to compete even at a 100-fold molar excess, and MEE2-mut1 competed with intermediate efficiency. Hence, we conclude that the 8 bp ATAGATTA sequence mutated in MEE2-mut4 represents a core motif necessary for this specific interaction, whereas the 8mer sequence at the 5′ end of MEE2 may have ancillary function in complex formation.
Next, we asked if the MEE2 element also occurs upstream of other var genes. We inspected all var upstream sequences (−600 to −1 relative to the start ATG) and identified a perfect or slightly deviated MEE2 core motif with the consensus sequence (A/T)(A/T)(A/T)GA(T/A)TA in 44 (73%) out of all 60 var genes. Strikingly, in all but four cases this motif (i) is conserved in terms of orientation and position relative to the ATG start codon, (ii) is embedded in an overall highly similar sequence context including a characteristic poly-dT stretch, and (iii) occurs in upsB-, upsC-, upsB/C- and upsB/A-type var genes (Fig. S4). The remaining four core motifs were found in one upsB/C and three upsA-type upstream sequences but they did not share these characteristics; they occurred in a different sequence context and relative position/orientation. In EMSA experiments, the MEE2-like motif derived from another upsC var gene (PF07_0048), in which six nucleotide positions are changed compared with MEE2 including one substitution in the core motif, competed as efficiently as the wild-type MEE2 motif (Fig. 5B). Similarly, the element found upstream of an upsB-type var gene (PFL0005w), in which 19 positions are altered including two in the core motif, competed albeit with lower efficiency (Fig. S3B). In contrast, competitors derived from a var upsA (PFD1235w) and a var-unrelated rif (PFB0035c) upstream region, in which an AT(A/T)GATTA core motif is present at the same relative position as in MEE2, failed to inhibit formation of the MEE2–protein complex (Fig. S3C).
Together, our results show that the MEE2-interacting factor (MIF) also binds to related motifs found in a large proportion of var upstream regions. Interestingly, however, MIF does not bind to unrelated sequences that contain a perfectly conserved 8 bp MEE2 core motif. Hence, this core motif is necessary but not sufficient for binding and the local var upstream sequence context plays an important role in mediating stable and sequence- specific complex formation.
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- Experimental procedures
- Supporting Information
The importance of mutually exclusive transcription of gene families is exemplified by antigenic variation in unicellular pathogens as a prime strategy to secure survival and transmission. In T. brucei, the causing agent of African sleeping sickness, mutually exclusive transcription of variant surface glycoprotein genes is carried out by an extranucleolar RNA polI-containing body (Navarro and Gull, 2001). Another paradigm of mutual exclusion is that of singular odorant receptor (OR) gene choice in individual olfactory neurones in mammals (McClintock, 2010). Here, exclusive transcription of one out of over a thousand OR genes involves regulatory DNA elements both upstream and in the coding regions (Qasba and Reed, 1998; Vassalli et al., 2002; Lomvardas et al., 2006; Fuss et al., 2007; Nguyen et al., 2007), and a negative protein feedback mechanism (Serizawa et al., 2003; Lewcock and Reed, 2004; Shykind et al., 2004). In addition, and in remarkable analogy to mutually exclusive var regulation, Lomvardas and colleagues recently described a functional association of H3K9me3 and H3K4me3 with silenced and active OR loci respectively (Magklara et al., 2011). These important discoveries notwithstanding, we still lack detailed knowledge as to how mutually exclusive transcription is achieved in any system. In this study, we developed and successfully applied a complementary functional approach to study mutual exclusion in P. falciparum var gene transcription. For the first time, we identified cis-acting entities as important mediators of var gene activation and singular gene choice.
var gene transcription is mediated by RNA polII and occurs stage-specifically by activation in ring-stage parasites and subsequent repression or poising during the rest of the IDC (Kyes et al., 2007; Lopez-Rubio et al., 2007). Here, we identified a UAS element essential for upsC promoter activation. The position of this element upstream of the natural TSS, and the competence to activate transcription from a heterologous promoter, are attributes inherently associated with the role of UAS elements in transcriptional activation (Levine and Tjian, 2003). Our results are therefore consistent with the sequence-specific recruitment of a transcriptional activator by the UAS to orchestrate the assembly of the pre-initiation complex (PIC) and/or to activate RNA polII-dependent transcription. Interestingly, the fact that this element functions autonomously in a euchromatic context implies a ubiquitous rather than spatially restricted distribution of the transcriptional activator involved, which somewhat precludes a restricted role for this factor in mutually exclusive var activation.
The current model of mutually exclusive var transcription postulates the existence of a physically restricted perinuclear zone dedicated to the expression of a single var gene (Duraisingh et al., 2005; Ralph et al., 2005; Voss et al., 2006; 2007; Dzikowski et al., 2007; Lopez-Rubio et al., 2009). Activation requires entry into this zone with concomitant substitution of the formerly active locus, linked to the removal of H3K9me3/PfHP1 and deposition of H3K9ac and H3K4me2/3 marks predominantly along the region downstream of the TSS (Lopez-Rubio et al., 2007; Perez-Toledo et al., 2009). We identified a deletion downstream of the TSS as the common denominator of all four promoter variants that escaped mutually exclusive activation. Unlike full-length promoters, activation of promoters lacking this region did not occur at the expense of, but in parallel to, the transcription of an endogenous var gene. Notably, this deletion did not alter the relative activity of the promoter showing that the processes of promoter activation and mutually exclusive recognition are uncoupled from each other. The specific binding of a nuclear factor or complex (MIF) to a cis-acting sequence motif present in this region (MEE2) corroborates this hypothesis and suggests an important role for this DNA–protein interaction in mutually exclusive promoter activation. The presence of MEE2-related motifs in a large subset of var genes provides circumstantial evidence for a conserved mechanism of singular var gene choice. Although the exact function of this interaction remains to be discovered, binding of MIF to the mutual exclusion element may earmark var loci for mutually exclusive activation. Additional experiments tailored towards identifying MIF and dissecting the exact function of this interaction in var regulation are now required to test this hypothesis. In this context it is worth mentioning that the 47 bp MEE2 sequence does not contain any obvious ApiAP2 transcription factor-binding motifs (Campbell et al., 2010).
Using promoter deletion analyses combined with ectopic insertion of var elements into a euchromatic locus we were able to systematically reconstruct some of the control steps of var gene activation and mutual exclusion. Based on these novel findings, and by integrating current knowledge, we propose a speculative mechanistic model for mutually exclusive var gene activation (Fig. 6). The position of var loci in heterochromatic perinuclear clusters prevents accessibility to specific and general transcription factors and this is probably the most important determinant of transcriptional inactivity (Freitas-Junior et al., 2000; 2005; Duraisingh et al., 2005; Ralph et al., 2005; Voss et al., 2006; Flueck et al., 2009; Lopez-Rubio et al., 2009; Perez-Toledo et al., 2009). The MEE2-interacting factor or complex MIF may bind downstream of the TSS to reinforce repression and/or to prevent or reduce leaky transcription from silenced loci. Such a function may be crucial in keeping var genes repressed that are positioned within euchromatic zones at the nuclear periphery (Ralph et al., 2005). Singular var gene choice may occur through the recognition of the MEE2/MIF complex, or an alternative var locus-specific sequence tag, by the unique var gene expression site (VES) (Duraisingh et al., 2005; Voss et al., 2006; Dzikowski et al., 2007; Lopez-Rubio et al., 2009). Once locked in, the VES may trigger the exchange of H3K9me3/PfHP1 with H3K4me2/3 and H3K9ac marks and the dissociation of the repressive MIF complex. Physical association of the active var locus with the VES may also play a crucial role in epigenetic memory, i.e. in keeping the var gene in place for re-activation in daughter cells (Lopez-Rubio et al., 2007). In this context, it is tempting to speculate that the recently identified histone methyltransferase PfSET10 (Volz et al., 2012) may be one component of the VES compartment.
This model proposes a novel logic in mutually exclusive gene expression and provides us with an informed working hypothesis for further functional dissection of the mechanisms orchestrating singular var gene choice. In particular, targeted identification of the proteins or protein complexes interacting with the regulatory elements characterized in this study will be a promising and exciting avenue to pursue. Detailed insight into this complex regulatory system is important for our understanding of immune evasion and virulence of P. falciparum and other pathogens. Furthermore, our results will also help to understand conceptually similar processes in other organisms.
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- Experimental procedures
- Supporting Information
Parasite culture and transfection
Plasmodium falciparum 3D7 parasites were cultured as described previously (Trager and Jenson, 1978). Growth synchronization was achieved by repeated sorbitol lysis (Lambros and Vanderberg, 1979). Transfections were performed as described (Voss et al., 2006). Parasites were selected on 2.5 μg ml−1 blasticidin-S-HCl and 4 nM WR99210. Transfection constructs are described in supporting experimental procedures.
Quantitative reverse transcription PCR
qPCR was performed on reverse transcribed total RNA and gDNA isolated from synchronous parasite cultures. A detailed protocol, relative transcript calculation and primer sequences are provided in supporting experimental procedures and Table S1.
Southern and Northern blot analysis
gDNA was digested with appropriate restriction enzymes overnight and separated in 0.5× TBE-buffered 0.7% agarose gels. Total RNA was isolated from saponin-released parasites using TriReagent (Ambion). RNA was glyoxylated for 1 h at 55°C in five volumes glyoxal reaction mixture and electrophoresis was performed using 1× BPTE-buffered 1.5% agarose gels (Sambrook and Russell, 2001). Blots were probed with 32P-dATP-labelled hdhfr, kahrp and hsp86 PCR fragments. Membranes were stripped by boiling in 0.1% SDS for 15 min in between hybridizations.
Western blot analysis
Detection of hDHFR-GFP and GAPDH (loading control) was performed on whole-cell lysates. Primary antibody dilutions were: mouse anti-GFP (Roche Diagnostics, 11814460001), 1:500; mouse anti-GAPDH 1-10B (kind gift of Claudia Daubenberger), 1:20 000. PfEMP1 was extracted from trophozoite-infected RBC pellets (Triton X-100-insoluble/SDS soluble fraction) as described (van Schravendijk et al., 1993). Extracts were separated by SDS-PAGE using 5% polyacrylamide gels using Tris-glycine or Tris-acetate buffers. PfEMP1 was detected using the monoclonal mouse anti-PfEMP1 antibody 1B/6H-1 (Duffy et al., 2002), 1:500.
Electromobility shift assay
High-salt nuclear extracts and EMSAs were prepared and carried out as described (Voss et al., 2002) with the following modifications. Proteins were extracted with 500 mM KCl and incubated with 20 fmol of radiolabelled probe in 1× EMSA buffer in presence of 200 ng of poly(dA-dT) as non-specific competitor. Complementary oligonucleotide sequences used to generate double-stranded probes and competitors are listed in Table S1.
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We are grateful to Dania Mueller for help in qRT-PCR experiments and Sandra Birrer for help in cloning and transfection. We thank Tim-Wolf Gilberger for critical reading of the manuscript. N.M.B.B. received a Boehringer Ingelheim PhD fellowship (http://www.bifonds.de). This work is supported by the Swiss National Science Foundation (PP00A-110835; PP00P3_130203; http://www.snf.ch).
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- Supporting Information
- 1996) Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1. Proc Natl Acad Sci USA 93: 3497–3502. , , , , and (
- 2005) The immunology and pathogenesis of malaria during pregnancy. Curr Top Microbiol Immunol 297: 187–227. , and (
- 1991) Antigenic variation in Plasmodium falciparum. Proc Natl Acad Sci USA 88: 9171–9174. , , , , , , et al. (
- 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. , , , and (
- 2010) Identification and genome-wide prediction of DNA binding specificities for the ApiAP2 family of regulators from the malaria parasite. PLoS Pathog 6: e1001165. , , , , and (
- 2007) Epigenetic memory at malaria virulence genes. Proc Natl Acad Sci USA 104: 899–902. , , , , , , et al. (
- 1999) Intra-cluster recombination and var transcription switches in the antigenic variation of Plasmodium falciparum. Mol Biochem Parasitol 101: 107–116. , , and (
- 2001) Malaria. Cooperative silencing elements in var genes. Nature 412: 875–876. , , and (
- 2009) Common strategies for antigenic variation by bacterial, fungal and protozoan pathogens. Nat Rev Microbiol 7: 493–503. , , and (
- 2002) Transcription of multiple var genes by individual, trophozoite-stage Plasmodium falciparum cells expressing a chondroitin sulphate A binding phenotype. Mol Microbiol 43: 1285–1293. , , , , , , et al. (
- 2005) Heterochromatin silencing and locus repositioning linked to regulation of virulence genes in Plasmodium falciparum. Cell 121: 13–24. , , , , , , et al. (
- 2009) Genetics of antigenic variation in Plasmodium falciparum. Curr Genet 55: 103–110. , and (
- 2006) Mutually exclusive expression of virulence genes by malaria parasites is regulated independently of antigen production. PLoS Pathog 2: e22. , , and (
- 2007) Mechanisms underlying mutually exclusive expression of virulence genes by malaria parasites. EMBO Rep 8: 959–965. , , , , , , et al. (
- 2009) Plasmodium falciparum heterochromatin protein 1 marks genomic loci linked to phenotypic variation of exported virulence factors. PLoS Pathog 5: e1000569. , , , , , , et al. (
- 2006) Strict pairing of var promoters and introns is required for var gene silencing in the malaria parasite Plasmodium falciparum. J Biol Chem 281: 9942–9952. , , , , , and (
- 2000) Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum. Nature 407: 1018–1022. , , , , , , et al. (
- 2005) Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites. Cell 121: 25–36. , , , , , , et al. (
- 2007) Local and cis effects of the H element on expression of odorant receptor genes in mouse. Cell 130: 373–384. , , and (
- 2005) A silenced Plasmodium falciparum var promoter can be activated in vivo through spontaneous deletion of a silencing element in the intron. Eukaryot Cell 4: 490–492. , , , , and (
- 1996) Variant antigens and endothelial receptor adhesion in Plasmodium falciparum. Proc Natl Acad Sci USA 93: 3503–3508. , , , and (
- 2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419: 498–511. , , , , , , et al. (
- 2009) Clonally variant gene families in Plasmodium falciparum share a common activation factor. Mol Microbiol 73: 1171–1185. , , , , , and (
- 2007) Plasmodium falciparum var gene expression is developmentally controlled at the level of RNA polymerase II-mediated transcription initiation. Mol Microbiol 63: 1237–1247. , , , , , and (
- 1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65: 418–420. , and (
- 1992) A sequence element associated with the Plasmodium falciparum KAHRP gene is the site of developmentally regulated protein–DNA interactions. Nucleic Acids Res 20: 3051–3056. , , and (
- 2003) Sub-grouping of Plasmodium falciparum 3D7 var genes based on sequence analysis of coding and non-coding regions. Malar J 2: 27. , , , , and (
- 1984) Identification of a strain-specific malarial antigen exposed on the surface of Plasmodium falciparum-infected erythrocytes. J Exp Med 159: 1567–1575. , , , and (
- 2003) Transcription regulation and animal diversity. Nature 424: 147–151. , and (
- 2004) A feedback mechanism regulates monoallelic odorant receptor expression. Proc Natl Acad Sci USA 101: 1069–1074. , and (
- 2006) Interchromosomal interactions and olfactory receptor choice. Cell 126: 403–413. , , , , , and (
- 2007) 5’ flanking region of var genes nucleate histone modification patterns linked to phenotypic inheritance of virulence traits in malaria parasites. Mol Microbiol 66: 1296–1305. , , , , , and (
- 2009) Genome-wide analysis of heterochromatin associates clonally variant gene regulation with perinuclear repressive centers in malaria parasites. Cell Host Microbe 5: 179–190. , , and (
- 2010) Achieving singularity in mammalian odorant receptor gene choice. Chem Senses 35: 447–457. (
- 1985) Human cerebral malaria. A quantitative ultrastructural analysis of parasitized erythrocyte sequestration. Am J Pathol 119: 385–401. , , , , and (
- 2011) An epigenetic signature for monoallelic olfactory receptor expression. Cell 145: 555–570. , , , , , , et al. (
- 2006) Evidence that Plasmodium falciparum chromosome end clusters are cross-linked by protein and are the sites of both virulence gene silencing and activation. Mol Microbiol 62: 72–83. , , , , , and (
- 2009) Antigenic variation in the African trypanosome: molecular mechanisms and phenotypic complexity. Cell Microbiol 11: 1724–1734. , , and (
- 2001) A pol I transcriptional body associated with VSG mono-allelic expression in Trypanosoma brucei. Nature 414: 759–763. , and (
- 2007) Prominent roles for odorant receptor coding sequences in allelic exclusion. Cell 131: 1009–1017. , , , , and (
- 2002) A genetic screen for improved plasmid segregation reveals a role for Rep20 in the interaction of Plasmodium falciparum chromosomes. EMBO J 21: 1231–1239. , , , , , , et al. (
- 2009) Plasmodium falciparum heterochromatin protein 1 binds to tri-methylated histone 3 lysine 9 and is linked to mutually exclusive expression of var genes. Nucleic Acids Res 37: 2596–2606. , , , , , , et al. (
- 1991) Microvascular sequestration of parasitized erythrocytes in human falciparum malaria: a pathological study. Am J Trop Med Hyg 44: 168–175. , , , and (
- 2009) Antigenic variation in Giardia lamblia. Cell Microbiol 11: 1706–1715. , and (
- 1998) Tissue and zonal-specific expression of an olfactory receptor transgene. J Neurosci 18: 227–236. , and (
- 2005) Antigenic variation in Plasmodium falciparum is associated with movement of var loci between subnuclear locations. Proc Natl Acad Sci USA 102: 5414–5419. , , and (
- 1999) The adhesion of Plasmodium falciparum-infected erythrocytes to chondroitin sulfate A is mediated by P. falciparum erythrocyte membrane protein 1. Proc Natl Acad Sci USA 96: 5198–5202. , , , , , , et al. (
- 1992) Rapid switching to multiple antigenic and adhesive phenotypes in malaria. Nature 357: 689–692. , , , , , , et al. (
- 2009) Dynamic histone H3 epigenome marking during the intraerythrocytic cycle of Plasmodium falciparum. Proc Natl Acad Sci USA 106: 9655–9660. , , , , , et al. (
- 2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: CSHL Press. , and (
- 1998) Antigenic variation in malaria: in situ switching, relaxed and mutually exclusive transcription of var genes during intra-erythrocytic development in Plasmodium falciparum. EMBO J 17: 5418–5426. , , , , , , et al. (
- 1993) Immunochemical characterization and differentiation of two approximately 300-kD erythrocyte membrane-associated proteins of Plasmodium falciparum, PfEMP1 and PfEMP3. Am J Trop Med Hyg 49: 552–565. , , , , and (
- 2003) Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse. Science 302: 2088–2094. , , , , , , et al. (
- 2004) Gene switching and the stability of odorant receptor gene choice. Cell 117: 801–815. , , , , , , et al. (
- 1995) Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82: 101–110. , , , , , , et al. (
- 1995) The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes. Cell 82: 89–100. , , , , , , et al. (
- 1978) Cultivation of malarial parasites. Nature 273: 621–622. , and (
- 2002) Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb. Neuron 35: 681–696. , , , , and (
- 2012) PfSET10, a Plasmodium falciparum methyltransferase, maintains the active var gene in a poised state during parasite division. Cell Host Microbe 11: 7–18. , , , , , , et al. (
- 2000) Genomic distribution and functional characterisation of two distinct and conserved Plasmodium falciparum var gene 5’ flanking sequences. Mol Biochem Parasitol 107: 103–115. , , , , , , et al. (
- 2002) Plasmodium falciparum possesses a cell cycle-regulated short type replication protein A large subunit encoded by an unusual transcript. J Biol Chem 277: 17493–17501. , , , and (
- 2006) A var gene promoter controls allelic exclusion of virulence genes in Plasmodium falciparum malaria. Nature 439: 1004–1008. , , , , , , et al. (
- 2007) Alterations in local chromatin environment are involved in silencing and activation of subtelomeric var genes in Plasmodium falciparum. Mol Microbiol 66: 139–150. , , , , , , et al. (
- 2012) Analysis of subtelomeric virulence gene families in Plasmodium falciparum by comparative transcriptional profiling. Mol Microbiol 84: 243–259. , , , , , , et al. (
- World Health Organization (2010) World Malaria Report 2010. Geneva: WHO Press.
- 2011) A critical role of perinuclear filamentous actin in spatial repositioning and mutually exclusive expression of virulence genes in malaria parasites. Cell Host Microbe 10: 451–463. , , , , , , et al. (
- Top of page
- Experimental procedures
- Supporting Information
Supporting experimental procedures
Fig. S1. Southern analysis of gDNA isolated from parasites presented in Fig. 2.
A. Autoradiographs of Southern blots showing episomal maintenance or plasmid integration into the endogenous kahrp locus in 3D7/pBKmin and 3D7/pBKmin-RI. gDNA was digested with BglII and HindIII. Blots were probed with a radiolabelled kahrp fragment. E, episomal; I, integrated.
B. Autoradiographs of Southern blots showing episomal maintenance or plasmid integration into the endogenous kahrp locus in 3D7/pBC1Kmin, 3D7/pBC2Kmin and 3D7/pBC3Kmin. gDNA was digested with BglII and HindIII. Blots were probed with a radiolabelled kahrp fragment.
C. Schematic map of the endogenous kahrp locus.
D–F. Schematic maps of the integration events in 3D7/pBKmin (D), 3D7/pBC1Kmin and 3D7/pBC2Kmin (E) and 3D7/pBC3Kmin (F). BglII and HindIII restriction sites and length of the corresponding fragments are indicated.
Fig. S2. Transcriptional initiation form an alternative upsC upstream TSS. The promoters in pBC and pBC4 are schematically depicted on top. Semi-quantitative analysis of protein and transcript abundance by Western and Northern blot in a time-course experiment. Total protein and RNA were harvested simultaneously from synchronized 3D7/pBC and 3D7/pBC4 parasites at three consecutive time points during intra-erythrocytic development (ring stages, 8–18 hpi; late ring stages/early trophozoites, 16–26 hpi; late trophozoites/early schizonts, 24–34 hpi). Expression of hDHFR-GFP and GAPDH (loading control) was detected with anti-GFP and anti-GAPDH antibodies respectively (upper panels). Steady-state hdhfr-gfp and hsp86 (loading control) transcripts were detected using radiolabelled hdhfr and hsp86 probes respectively.
Fig. S3. Competition EMSAs. All EMSAs were carried out using radiolabelled MEE2 and parasite nuclear extract.
A. Mutational analysis of MEE2. Competition was carried out in presence of a 25- and 100-fold molar excess of unlabelled DNA. The nucleotide sequences of wild-type and mutated MEE2 elements are indicated on the right. The ATAGATTA core motif is underlined. Mutated 8mers are highlighted in red.
B. Competition of the MEE2 complex by a MEE2-related upsB sequence element. Competition was carried out in presence of a 25-, 100-, 250- and 500-fold molar excess of unlabelled DNA. The nucleotide sequences of wild-type and scrambled MEE2 and the MEE2-related upsB element are indicated on the right. The ATAGATTA core motif is underlined. The differences in the upsB-derived motif compared with MEE2 are highlighted in red.
C. The ATAGATTA core motif is not sufficient for complex formation. Competition was carried out in presence of a 25-, 100- and 500-fold molar excess of unlabelled DNA. The ATAGATTA core motif is underlined. The nucleotide sequences of wild-type and scrambled MEE2 and two unrelated sequence elements that contain the ATAGATTA core motif are indicated on the right.
Fig. S4. The MEE2 core motif occurs in a conserved position upstream of 44 var genes.
A. The schematic shows the presence and relative position of the (A/T)(A/T)(A/T)GA(A/T)TA consensus sequence found upstream of 44 var genes. This motif forms the core of the 47 bp MEE2 element that is bound by a nuclear factor in a sequence-specific manner (see Figs 4 and S3). Red boxes indicate the position of the motif in each upstream region. Numbers on the right represent the position of the first nucleotide of the motif relative to the translation initiation ATG. Gene accession numbers were retrieved from PlasmoDB version 7.2 (http://www.plasmoDB.org) and are indicated on the left. The colour code clusters var genes into the different var gene subgroups upsA, upsB, upsC, upsE, upsB/C and upsB/A (Lavstsen et al., 2003).
B. Alignment of MEE2-related sequences that are centred around the (A/T)(A/T)(A/T)GA(A/T)TA core consensus element in 44 var upstream regions. The original MEE2 motif identified upstream of the upsC var gene PFL1960w is shown as the first sequence in the alignment. The local context of the MEE2-related core motifs shows a high level of sequence similarity that includes a prominent upstream poly-dT stretch. Gene accession numbers are indicated on the left and are colour-coded as in Fig. S4A. Orientation of the motif is indicated on the right (+, upper strand; −, lower strand). The red bar on top highlights the position of the core motif.
Table S1. All primers used in this study are listed. Restriction sites are indicated in bold.
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