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. Author manuscript; available in PMC: 2014 Jun 23.
Published in final edited form as: Cell Host Microbe. 2012 Dec 13;12(6):739–750. doi: 10.1016/j.chom.2012.11.004

Nuclear Repositioning Precedes Promoter Accessibility and Is Linked to the Switching Frequency of a Plasmodium falciparum Invasion Gene

Bradley I Coleman 1,3, Ulf Ribacke 1,3, Micah Manary 2, Amy K Bei 1, Elizabeth A Winzeler 2, Dyann F Wirth 1, Manoj T Duraisingh 1,*
PMCID: PMC4066821  NIHMSID: NIHMS579336  PMID: 23245319

SUMMARY

Variation of surface adhesins, such as the Plasmodium falciparum erythrocyte invasion ligand PfRh4, is critical for virulence and immune evasion in many microbes. While phenotypic switching is linked to transcriptional changes and chromatin function, the determinants of switching frequency remain poorly defined. By expressing a prokaryotic DNA methylase in P. falciparum, we directly assayed accessibility of transcriptionally active and silent chromatin at the PfRh4 locus. Parasites selected for in vivo PfRh4 activation show a reversible increase in promoter accessibility and exhibit perinuclear repositioning of the locus from a silent to a conserved activation domain. Forced activation of a proximal gene results in a similar repositioning of the PfRh4 locus, with a concomitant increase in PfRh4 activation in a subpopulation of parasites and promoter accessibility correlating with actively transcribed loci. Thus, nuclear repositioning is associated with increased P. falciparum switching frequency, while promoter accessibility is tightly linked to clonally active PfRh4 promoters.

INTRODUCTION

The virulence of diverse bacterial, fungal, and apicomplexan pathogens lies in their ability to colonize a specific niche while evading mechanical and immunological clearance by the host. Frequently, this is facilitated by clonally variant expression of genes within expanded families, which generates diverse adhesive and antigenic phenotypes. Trypanosoma brucei, Giardia lamblia, and Plasmodium falciparum all undergo rapid phenotypic switches of surface-exposed proteins (Deitsch et al., 2009), implying that this represents a particularly successful adaptation for a parasitic lifestyle. In P. falciparum, the most clinically significant cause of human malaria, clonally variant expression of parasite adhesins drives major processes critical for infection. The var family encodes polymorphic PFEMP1 proteins that mediate the adhesion of parasite-infected erythrocytes to host blood vessels (Scherf et al., 1998), and the members of the PfRh and PfEBA families encode phenotypically diverse erythrocyte invasion ligands (Baum et al., 2005).

Variant expression of PfRh and PfEba protein ligands provides P. falciparum with the ability to bind alternative erythrocyte receptors, defining pathways for the invasion of host cells (Bei et al., 2007; Duraisingh et al., 2003; Nery et al., 2006). The efficiency of invasion into host erythrocytes has been correlated with the severity of clinical disease (Chotivanich et al., 2000). The antigenic diversity generated by clonal variation of PfRh and PfEBA expression also provides a mechanism for the escape of the parasite from invasion-blocking antibodies (Gaur et al., 2007; Persson et al., 2008). PfRh4 has recently been shown to bind complement receptor 1 (CR1) on the erythrocyte surface (Tham et al., 2010), the primary alternative to sialic acid-containing receptors such as glycophorin A. Clonally variant expression of PfRh4 is epigenetically controlled (Stubbs et al., 2005). As with var gene expression (Duffy et al., 2005), the epigenetic switch between active and silent PfRh4 expression bestows parasites with distinct adhesive phenotypes (Dolan et al., 1990).

Chromatin states and their associated transcriptional profiles and modifying enzymes have been primarily characterized as they relate to euchromatin or heterochromatin. Clonally variant genes in P. falciparum, most notably var, have been best described in the heterochromatin-like silent state, which involves H3K9me3 enrichment (Chookajorn et al., 2007; Lopez-Rubio et al., 2009) in association with the heterochromatin proteins Sir2a and HP1 (Duraisingh et al., 2005; Flueck et al., 2009; Freitas-Junior et al., 2005; Pérez-Toledo et al., 2009), with the absence of histone H2A.Z (Petter et al., 2011) and localization to specific perinuclear repression sites (Dzikowski et al., 2007; Ralph et al., 2005; Voss et al., 2006). However, an understanding of the transition from the silent to the active transcriptional state, particularly the determinants of the rate of phenotypic switching, remains elusive.

Here we present a study of the determinants of high-frequency switching of the PfRh4 invasion gene. By expressing a prokaryotic DNA methylase in Plasmodium falciparum, we have directly assayed the accessibility of transcriptionally active and silent chromatin at this locus. We additionally correlated these states with conserved peripheral nuclear regulatory domains using FISH. By directly perturbing activation and silencing, we generated a population of parasites that, while largely maintaining silent PfRh4 at the population level, transition to the active state with increased frequency. In this population we find that nuclear repositioning is associated with increased P. falciparum switching frequency, while promoter accessibility is tightly linked to clonally active PfRh4 promoters.

RESULTS

Epigenetic Transcriptional Regulation Involves Facultative Changes in Promoter Accessibility

To investigate the regulatory scheme that generates phenotypic plasticity in P. falciparum, we focused on the high-frequency facultative switching of the PfRh4 gene between transcriptionally active and silent states. Though PfRh4 is predominantly silent in the W2mef strain, a small subpopulation of PfRh4-expressing parasites is maintained in a clonal fashion (Soubes et al., 1997). We isolated a uniform population of PfRh4-expressing parasites (W2mef/N) by growing W2mef in sialic acid-depleted erythrocytes (Figure 1A). We performed ChIP on these parasites to confirm that activation was accompanied by loss of the heterochromatin-associated histone modification H3K9me3 within the PfRh4 promoter and ORF (Figure 1B) (Jiang et al., 2010) and extended these findings to the neighboring genes. Eba165, a psuedogene which shares a bidirectional promoter with PfRh4, was similarly H3K9me3 enriched, while PfRh5, which is indispensible for P. falciparum invasion (Crosnier et al., 2011), was not. The facultative heterochromatin formed in association with this phenotypic switch is therefore restricted to the Eba165/PfRh4 locus.

Figure 1. The Accessibility of Epigenetically Regulated Chromatin Can Be Probed with an Ectopically Expressed E. coli DNA Methylase.

Figure 1

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(A) The sialic acid dependence of P. falciparum invasion varies in a reversible, PfRh4-dependent fashion in W2mef, as demonstrated by neuraminidase treatment of target cells to remove surface sialic acid.

(B) H3K9me3 enrichment of the PfRh4 locus in sialic acid-dependent (W2mef) and -independent (W2mef/N) parasites was assayed by ChIP. Mean plus range are shown from two independent experiments. An H3K9me3-associated gene (var2csa), a silent gene not associated with H3K9me3 (PF14_0491), and a constitutively expressed gene (gbp130) are controls. See also Table S1.

(C)EcDammethylates adenine within the sequence GATC. DpnI cleaves methylated sequences while DpnII is completely blocked by m6A.

(D) Schematic of the methylase overexpression construct pEcDamHi.

(E) Schematic of the methylase overexpression construct pEcDamLo.

(F) Dam methylase from E. coli was expressed in P. falciparum, and genomic DNA was digested with DpnII. Parasites were cultured in the presence or absence of 0.5 µM Shld1 and 100 nM TSA, as indicated. EtBr signal is graphed to the right of each panel. Disrupting heterochromatin with TSA causes an increase in methylated DNA that is not cut by DpnII (arrow), which correlates with increasingly accessible chromatin. See also Figure S1 and Table S1.

Heterochromatin differs fundamentally from euchromatin in that it is largely inaccessible to DNA binding proteins. To directly probe the structure of active and silent chromatin at the PfRh4 locus, we expressed an E. coli DNA adenine methyltrasferase (EcDam) in P. falciparum. In contrast to nuclease-based methods that rely upon the purification of intact chromatin, EcDam marks accessible chromatin in vivo by stably methylating the N-6 position of adenine (m6A) within the target sequence GATC in proliferating cells (Gottschling, 1992; Singh and Klar, 1992; Wines et al., 1996). Methylation is dependent on the binding of EcDam to the double helix, making m6A a direct corollary of chromatin accessibility. The P. falciparum genome does not contain detectible m6A (Hattman, 2005), which facilitates the detection of EcDam signals with the restriction enzymes DpnI and DpnII that require or are blocked by m6A, respectively (Figure 1C).

The W2mef clone Dd2attB was stably transfected with a highly expressed copy of EcDam (pEcDamHi) (Figure 1D), and methylase activity was assayed by digesting genomic DNA with DpnII, which is blocked by m6A. Unlike wild-type genomic DNA, Dd2attB-pEcDamHi gDNA was refractory to DpnII digestion (Figure 1F, top and middle panels). This corresponds to saturation of the genome with m6A. As in Saccharomyces and Drosophila, high levels of m6A did not affect Plasmodium growth or morphology (data not shown) (Fehér et al., 1988; Wines et al., 1996). To assay relative levels of accessibility, nonsaturating levels of EcDam expression were required. While a destruction domain (DD) tag was fused to the N terminus of the pEcDamHi construct to regulate methylase activity (Banaszynski et al., 2006), DpnII digestion was blocked even in the absence of the stabilizing molecule, Shld1. This may result from high stability of the N-terminal DD-EcDam fusion or the strength of the calmodulin promoter. Appropriately low levels of EcDam expression were achieved with a C-terminal EcDam-DD fusion downstream of a P. berghei terminator sequence with no known promoter activity, termed pEcDamLo (Figure 1E). In Dd2attB-pEcDamLo parasites cultured without Shld1, m6A was undetectable by the DpnII assay (Figure 1F, bottom panel). The addition of Shld1 to the cultures generated a slight increase in the size of DpnII-digested fragments, but the levels of methylation remained far below that of Dd2attB-pEcDamHi.

To verify that changes in chromatin accessibility could be detected as changes in m6A, we chemically perturbed accessibility by treating Dd2attB-pEcDamLo with trichostatin A (TSA), a histone deacetylase inhibitor that causes hyperacetylation (Andrews et al., 2008) and increases chromatin accessibility (Görisch et al., 2005). Indeed, genomic DNA from TSA-treated parasites showed a shift toward larger molecular weight fragments, including the partial return of undigested species (Figure 1F, bottom panel). While TSA treatment is known to affect transcription in subsets of genes (Andrews et al., 2012), we find that this is likely not a strict consequence of increased chromatin accessibility. Instead, we universally detect an increase in chromatin accessibility following TSA treatment, independent of the magnitude or direction of transcriptional changes induced by the treatment at that locus (see Figure S1 online). These data confirm the ability of the EcDam system to detect changes in chromatin accessibility in P. falciparum.

The in vivo accessibility of active and silent PfRh4 chromatin was measured by Southern blots assaying two DpnI restriction sites 5′ of the PfRh4 ORF (Table S2). DpnI cuts only in the presence of methyladenine and can therefore be used to determine the methylated proportion of a genomic DNA sample (Figure 1C). One DpnI site on either side of the TSS was interrogated, at −215 and −1,153 base pairs from the start codon (Figure 2A). In the representative series of blots in Figure 2C, Dd2attB-pEcDamLo methylation was observed at 6.3% of the DNA at −215 and 11.5% at −1,153. Methylation increased to 21.7% and 30.5% in PfRh4-expressing D/N parasites (Figure 2B). Upon reversion to the sialic acid-dependent state in which PfRh4 is silent (D/R), the PfRh4 promoter again became less accessible (5.5% and 8.2% methylation). Across independent selections and Southern digests, activation of the PfRh4 promoter was consistently accompanied by an approximately 3-fold increase in EcDam accessibility (Figure 2D). These reversible changes in chromatin accessibility link alterations of P. falciparum invasion phenotype to facultative heterochromatin formation. A similar increase was observed at the very low methylase expression levels generated in the absence of Shld1 (Figures S2B and S2C). Accessibility was unchanged in the promoter of PfRh1 (Figures 2C and 2D), a PfRh family member whose expression is not effected by PfRh4 activation (Figure S2A). Accessibility was also unchanged in the promoter of the highly expressed control gene PfActin-1 and the heterochromatically silenced control var2csa (Figures 2C and 2D, Figure S2D). PfRh4 activation had no effect on chromatin accessibility beyond the promoter (Figure S2E), demonstrating that while clonally silenced genes in P. falciparum are widely associated with H3K9me3 marks (Lopez-Rubio et al., 2009), chromatin accessibility plays a promoter-specific regulatory role in vivo.

Figure 2. Phenotypic Switching of Sialic Acid Dependence Is Accompanied by a Reversible Increase in PfRh4 Promoter Accessibility.

Figure 2

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(A) Schematic of a Southern blot using m6A-sensitive* restriction enzymes to measure chromatin accessibility at the PfRh4 promoter (K, KpnI; D, DpnI*; B, BglII; R, RsaI). The transcriptional start site and +1 nucleosome (black oval) are indicated. Gray boxes represent probe sequences.

(B) qRT-PCR analysis of PfRh4 expression in pEcDamLo schizonts, normalized to Dd2-pEcDamLo. Values represent mean and standard deviation of the quotient from an experiment performed in triplicate.

(C) Representative methylation-sensitive Southern blots assaying accessibility of DpnI sites. Position relative to ATG in parentheses. Percent methylation, shown below each graph, was calculated from band densities as (density + DpnI)/(density+DpnI + density−DpnI)*100. Arrowhead points to an additional band believed to represent a reported duplication of PfRh1 in the W2mef parasite lineage (Triglia et al., 2005).

(D) Levels of methylation in PfRh4-expressing and silenced parasites normalized to unselected Dd2-pEcDamLo. Values represent mean and range from two biological replicates for PfRh4 −215 and var2csa, mean and SD of four replicates for PfRh4 −1153 and a single experiment for PfRh1. See also Figure S2 and Table S2.

Repositioning to a Conserved Subnuclear Domain for P. falciparum Virulence Gene Expression

The P. falciparum nucleus is a heterogeneous organelle that contains multiple active and silent chromatin domains within a partially compacted periphery (Freitas-Junior et al., 2005; Ralph et al., 2005; Weiner et al., 2011). To examine the relationship between subnuclear position and facultative activation of invasion gene expression, we performed DNA FISH on W2mef and W2mef/N parasites. PfRh4 and PfRh1 loci were scored for colocalization with Rep20, a marker of heterochromatin-associated telomere clusters (Figure 3A) (Freitas-Junior et al., 2000). In contrast to PfRh4, PfRh1 is expressed in both W2mef and W2mef/N parasites (Figure S2A). Both genes were localized to the nuclear periphery in ~90% of cells, independent of transcriptional status (Figure 3B). PfRh1 and PfRh4 lie ~100 kb from different arms of chromosome 4. Their peripheral localization may be explained by the general compaction observed in subtelomeres or by active tethering to the nuclear lamina (Freitas-Junior et al., 2005; Ralph et al., 2005).

Figure 3. Subnuclear Localization of PfRh4 Varies with Invasion Phenotype Switching.

Figure 3

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(A) Representative images of DNA FISH measuring the colocalization of active and silent PfRh loci (green) with Rep20 (red).

(B) Nuclear position of PfRh loci was scored as peripheral or within the center of the nucleus.

(C) Loci were scored for colocalization with the Rep20 marker (p < 0.0001, Fisher’s exact test).

(D) Schematic and representative images of DNA FISH localizing PfRh4 (green) and a plasmid containing transcriptionally active hDHFR (red) and localized to the nuclear periphery with Rep20 (gray).

(E) Nuclei were scored for colocalization of PfRh4 and the active, hDHFR-containing plasmid (p < 0.0001, Fisher’s exact test).

PfRh4 frequently colocalized with Rep20 loci (78%; n = 100) in W2mef nuclei (Figure 3C), suggesting preferential recruitment of silent PfRh4 to a transcriptionally repressive heterochromatic zone. Colocalization was significantly lower in W2mef/N, where PfRh4 is actively transcribed (34%; n = 102). Those levels were similar to those of PfRh1 in both strains, in which it is equivalently expressed.

Active genes within the clonally variant var and rifin families are preferentially localized to a transcriptionally permissive subdomain that contains histone-modifying enzymes and nucleic acid binding proteins (Duraisingh et al., 2005; Dzikowski et al., 2007; Howitt et al., 2009; Ralph et al., 2005; Volz et al., 2012, Voss et al., 2006). Adapting a strategy previously used to identify this active site (Duraisingh et al., 2005), we transfected W2mef with a Rep20/hDHFR (human dihydrofolate reductase) plasmid, targeted the plasmid to the active site with the antifolate drug WR99210, and scored colocalization of hDHFR with PfRh4 (Figure 3D). The PfRh4 locus was significantly more likely to colocalize with the active hDHFR promoter in W2mef/N (64.9%; n = 202) than in unselected W2mef (38.5%; n = 192) (Figure 3E). Epigenetic activation of PfRh4 is therefore accompanied by its movement from a heterochromatic silencing domain into a conserved nuclear site linked to virulence gene activation.

Disrupting Silencing within the PfRh4 Locus Perturbs Its Transcriptional Regulation

To determine the sequence dependence of activation and silencing at the PfRh4 locus, we inserted the hDHFR marker immediately downstream of the endogenous PfRh4 gene (Figure 4A, Figure S3). Positive integrants, termed W2mef-F11, were selected for WR99210 resistance. Without drug pressure, resistant parasites (F11R) reverted to the WR99210-sensitive state (F11S). The kinetics of this sequence-independent silencing were similar to reversion of PfRh4 to the silent state after W2mef/N are returned to untreated erythrocytes (Figure 4B).

Figure 4. Trans-Activation within the Silent PfRh4 Locus Drives Phenotypic Conversion.

Figure 4

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(A) W2mef-F11 parasites contain the hDHFR drug resistance cassette downstream of the endogenous PfRh4 locus. This allows independent selection for sialic acid independence (PfRh4 activation, F11S/N) and WR99210 resistance (hDHFR activation, F11R). PfRh5 is insulated from transcription or silencing within the PfRh4 locus (white circle). See also Figure S3.

(B) W2mef-F11 parasites are resistant to WR99210 (F11R) due to hDHFR expression. Resistance is lost in the absence of drug pressure (F11S), but a resistant population can be regenerated from a silent clone.

(C) qRT-PCR on schizont stage cDNA obtained from WR99210- or neuraminidase-selected F11 parasites normalized to F11S. Values represent mean and standard deviation of the quotient from an experiment performed in triplicate.

(D) The growth of F11S and F11R populations seeded at 0.5% parasitemia in the presence or absence of host cell sialic acid as counted daily by flow cytometry.

(E) WR99210 IC50 as determined by the incorporation of 3H hypoxanthine. The IC50 of F11S/N was significantly higher than F11S (p = 0.0003). A parametric curve is fitted to the data represented as mean ± standard deviations from triplicate experiments. See also Figure S3 and Table S3.

Given the similarities we observed between the dynamics of WR99210 sensitivity and those of W2mef sialic acid dependence, we hypothesized that the F11S population was actually a mosaic of F11S and F11R clones. Indeed, F11R parasites could be selected from within the F11S population with WR99210. These parasites showed a 142-fold increase in hDHFR expression relative to F11S. However, these drug-selected F11R parasites demonstrated only a 6.5-fold increase of PfRh4 expression, which represents just 15.3% of the PfRh4 expression peak observed in F11S/N (Figure 4C). Selecting for maximal hDHFR expression did not, therefore, trigger maximal PfRh4 expression. Despite this intermediate effect, growth of F11R in neuraminidase-treated host erythrocytes, which is strictly dependent on PfRh4 expression (Stubbs et al., 2005), was increased relative to F11S (Figure 4D). The increased PfRh4 expression we observed following hDHFR activation thus altered the ability of F11 parasites to invade this population of host erythrocytes.

Though the relationship between hDHFR transcription and WR99210 resistance can be confounded by the ability of both human and Plasmodium DHFR proteins to inhibit translation of their transcripts (Zhang and Rathod, 2002), selection for maximal expression of PfRh4 affected both transcription of hDHFR and the drug resistance phenotype of the associated parasites. Sialic acid depletion led to a 314-fold increase in PfRh4 transcripts but a partial, 8-fold increase in hDHFR message (Figure 4C). This modest increase in transcriptional activity was associated with a 4-fold increase in WR99210 IC50 (Figure 4E). While transcription of Eba165 paralleled PfRh4, PfRh5 was unaffected by either PfRh4 or hDHFR activation (Figure 4C).

Clonal Activation Frequency Is Associated with Nuclear Repositioning and Closely Tracks with Promoter Accessibility

This incomplete transcriptional activation and the associated phenotypes we observed could be the result of modest population-wide activation or of full activation in a clonal subset of parasites. To distinguish between these possibilities, we measured the sialic acid-independent growth of individual parasites within F11S and F11R populations (Soubes et al., 1997). In six independent experiments the proportion of PfRh4-expressing parasites within an F11R population was consistently higher (10.5% ± 4.9%) than that within F11S (2.5% ± 1.1%) (Figure 5A, Table S4). The increased sialic acid independence of F11R is thus attributable to an increase in the proportion of the population in which PfRh4 has been maximally activated. To unambiguously correlate this phenotype with clonal alterations in PfRh4 transcriptional activation, we directly queried the presence and absence of PfRh4 transcripts with both single-cell qPCR and RNA FISH. When normalized to the presence of PfCoronin, a stage-matched control gene, our qPCR assay identified PfRh4 transcripts in 11.1% ± 0.3% of F11R parasites, compared to 3% ± 1.2% of F11S (Figure 5B, Figures S4A–S4C). Similar results were obtained in multiplex reactions with PfRh1 and PfRh4. RNA FISH likewise revealed that the number of PfCoronin-positive nuclei that were also positive for PfRh4 increased from less than 1% in F11S to 16.2% in F11R (Figure 5C). As with DNA FISH, PfRh4 transcripts were found overwhelmingly in the periphery of the nucleus. Surprisingly, PfRh4 transcripts colocalized with those of PfCoronin at a high rate, implying that these two genes are frequently targeted to the same activation domain. Through these three approaches, it is clear that activation of hDHFR within the silent PfRh4 locus increases the rate of clonal PfRh4 activation and that this drives a phenotypic switch to sialic acid-independent host cell invasion in these clones.

Figure 5. Subnuclear Positioning Is Associated with Altered Frequency of Clonal Activation while Chromatin Accessibility Tracks with Active Loci.

Figure 5

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(A) F11S and F11R populations were cloned into neuraminidase-treated and untreated erythrocytes as indicated. Positive wells were scored by microscopy 14 days later. Bars represent mean percent positivity ±95%CI for six independent experiments. Calculated proportions of sialic acid-independent parasites within each population (mean ±95%CI) are shown.

(B) Proportion of single cells transcribing PfRh4 was determined by single-cell exon-exon junction qPCR using PfCoronin (dark gray bars) and PfRh1 (light gray bars) as endogenous positive controls. Total number of cells assayed was 1,170, 964, and 507 for F11S, F11R, and F11S/N, respectively. Data are presented as mean percentage PfRh4-positive cells, and error bars represent ranges of two biological replicates.

(C) Percent PfRh4-transcript-positive nuclei were determined by RNA FISH from 126, 142, and 132 PfCoronin-positive F11S, F11R, and F11S/N parasites, respectively. Peripheral localization and colocalization to PfCoronin were determined from the PfRh4-positive cells (F11S; n = 1, F11R; n = 23, F11S/N; n = 111). Representative images of a PfCoronin+/PfRh4− nucleus (top panel) and a PfCoronin+/PfRh4+ nucleus with PfRh4 in the nuclear periphery and colocalized with PfCoronin (lower panel).

(D) PfRh4 promoter (−215) accessibility was measured by methylation-specific TaqMan real-time qPCR in F11-pEcDamLo parasites. Amplification of DpnII-digested PfRh4 promoter DNA was measured by a restriction site spanning TaqMan probe and normalized to the amplification of an exonic part of PfRh4 lacking a DpnII site. Values represent mean ± range from two independent experiments, each assayed in six replicates.

(E) DNA FISH measuring colocalization of PfRh4 and Rep20 in W2mef-F11. Activation of hDHFR within the PfRh4 locus excludes PfRh4 from silent regions of the nuclear periphery (p < 0.0001, Fisher’s exact test). See also Figure S4 and Table S4.

To dissect the mechanistic contributions of chromatin accessibility and perinuclear repositioning to the increased clonal activation we observe in F11R parasites, we transfected the pEcDamLo plasmid into W2mef-F11 to examine the accessibility of the PfRh4 promoter within these populations (Figure S4D). While we were again able to detect an ~4-fold change between F11S and F11S/N, no such change was seen with F11R (Figures S4E and S4F). We hypothesized that this was due to the limited ability of a Southern blot to detect small changes and developed a TaqMan real-time qPCR assay as a more sensitive alternative. By comparing the accessibility of position −215 of the PfRh4 promoter, this assay reproducibly detected a 1.27 ± 0.07-fold increase in the accessibility of the PfRh4 promoter in F11R relative to F11S (Figure S4G). In F11S/N, where all parasites express PfRh4, accessibility was 3.90 ± 0.11-fold higher than in F11S (Figure 5D). These values mirror the increases in clonal PfRh4 activation we have observed, indicating that increased promoter accessibility tracks closely with the proportion of active promoters within a population.

Promoter accessibility is likely to be a critical component of transcriptional activation at a single promoter. In an F11R population, however, promoter accessibility is largely unchanged, and it is the rate at which activation occurs within the population that has been perturbed. To investigate the potential role of nuclear organization in this upstream regulatory event, we colocalized PfRh4 and silent Rep20 loci in F11S and F11R with DNA FISH. F11S parasites colocalized with Rep20 at levels similar to those observed for wild-type W2mef (73.0%; n = 152). However, colocalization of PfRh4 and Rep20 in F11R parasites (39.9%; n = 158) was similar to that in F11S/N (31.3%; n = 150) (Figure 5E). Even though the activation of PfRh4 within the F11R population was clonal, and therefore only occurred in a small subset of the nuclei we observed, repositioning occurred at levels approximating a F11S/N population in which all loci are being transcribed.

DISCUSSION

We have found that regulated promoter accessibility and the repositioning of loci within the parasite nucleus are critical elements of the epigenetic control of the PfRh4 invasion gene of P. falciparum. Silent PfRh4 loci exist as H3K9me3-enriched heterochromatin and localize to a repressive domain within the nuclear periphery. This silent state is reminiscent of the silencing of var loci and other clonally variant P. falciparum genes (Lopez-Rubio et al., 2009). The epigenetic regulation of PfRh4 has previously been characterized by facultative activation from the silent state (Stubbs et al., 2005). We additionally demonstrate that active PfRh4 loci reside within an activation domain. A portion of the nucleus marked by this same approach was previously shown to contain active var loci (Duraisingh et al., 2005), implying that clonal activation of P. falciparum virulence genes involves repositioning of loci among universal functional domains within a heterogeneous parasite nucleus. Analagous domains have been implicated in the monoallelic expression of vsg genes in Trypanosoma brucei (Landeira and Navarro, 2007). This may represent a widespread adaptation for the regulation of expression of genes with high switch rates involved in the antigenic and polymorphic requirements of a parasitic lifestyle. The strong colocalization of PfRh4 transcripts with those of the invariant gene PfCoronin, however, may point to a less restrictive requirement for activation domain residence in P. falciparum. The recent identification of chromatin components marking the P. falciparum active site has begun the work of understanding the molecules involved in transcriptional activation within these specialized compartments (Volz et al., 2012).

We generated an in vivo dam methylase system in P. falciparum and with it identified a reversible epigenetic switch in promoter accessibility accompanying the transition between silent and active PfRh4. This contrasts the distribution of histone modifications marking these transcriptional states, which extend well into the coding sequence (Lopez-Rubio et al., 2009). We expect that the EcDam approach will become a constructive tool for the investigation of transcriptional regulation and chromatin structure in P. falciparum. As has been observed in other systems, when comparing across loci we found a positive but imperfect correlation between promoter accessibility and transcription (Figures S1E and S1F), but outside of promoters no such relationship was found. Genome-wide application of the EcDam system will shed more light on these relationships and serve as a valuable complement to the growing number of ChIP-on-chip data sets cataloging the locations of histone modifications and chromatin proteins.

The EcDam method assays the accessibility of target regions to a DNA binding protein. The positive correlation between accessibility and transcription we observe may result from the increased binding of regulatory proteins within these accessible regions. The principle transcription factors in P. falciparum are the ~27 members of the ApiAP2 family of DNA binding proteins (De Silva et al., 2008). The PfRh4/Eba165 promoter contains 23 putative ApiAP2 binding motifs (Campbell et al., 2010) (Table S5), including two sites associated with the var- and heterochromatin-associated PFF0200c (Flueck et al., 2010). Roughly half of the present motifs are bound by ApiAP2 proteins with schizont-specific expression profiles, though the roles of these or other transcription and chromatin proteins in clonal activation and the regulation of high frequency switching will need to be experimentally determined.

Through a functional perturbation of the PfRh4 locus we have additionally separated nuclear repositioning from promoter accessibility, with the former being linked to the frequency of clonal activation within a population, and the latter specifically tracking to activation of individual promoters. Using a drug-selectable promoter, we created a population (F11R) in which the PfRh4 locus was forced from the silent region independent of PfRh4 activation. This functionally uncoupled transcriptional activation and heterochromatin-mediated silencing in these parasites, demonstrating that clonally variant loci do not exist in a default active state. They instead appear to require a second activation step following the loss of silencing. The rate at which this activation occurs is dependent on residence within specific subnuclear domains but not upon the accessibility of the promoter, which we find to be strictly associated with active transcription.

The ability to facultatively switch between virulence phenotypes via epigenetic transcriptional activation is a critical feature of P. falciparum erythrocyte invasion and cytoadherence. The data presented here support a tiered model of the high-frequency gene activation underlying these phenomena (Figure 6). We suggest that nuclear repositioning is an early regulatory event that is not directly linked to transcription but instead, by shifting PfRh4 loci into a permissive environment, increases the frequency with which clonal transcriptional activation at individual promoters may occur. A promoter within a repressive nuclear subdomain can become accessible and transcriptionally active, but does so at a low rate. Relocalization of the locus away from this region does not universally drive transcriptional activation but instead increases the frequency of activation. The forces that localize and maintain specific loci in these subdomains are not well characterized, though a recent publication has demonstrated a critical role for actin in these processes (Zhang et al., 2011; Volz et al., 2012). Another recent publication noted that the rate of var gene switching could be affected by repressing the transcription of a clonally active member (Fastman et al., 2012). We hypothesize that, as we observed for PfRh4, this may be due to movement of transcriptionally repressed loci between heterogeneous compartments of the parasite nucleus. Following relocalization, a second, independent regulatory event is then required to enter a transcriptionally active state, which we characterize as containing a highly accessible promoter in vivo. Nuclear repositioning, not the inaccessibility of a heterochromatic locus, is the main driver of the frequency of clonally variant transcriptional activation. Thus, we find a multistep regulatory scheme for the high-frequency switching of P. falciparum multigene families that maintains both the adaptability and virulence of these parasites.

Figure 6. A Model Suggesting Nuclear Repositioning Alters the Frequency of PfRh4 Transcriptional Activation.

Figure 6

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In the absence of phenotypic selection (W2mef, F11S), PfRh4 activation is infrequent (~2% of parasites, green) and is primarily silenced (red). Selection for a sialic acid-independent invasion phenotype (W2mef/N, F11S/N) yields a population in which 100% of parasites express PfRh4. An intermediate state can be captured by disrupting F11S silencing using a drug-selectable marker in the absence of phenotypic selection (F11R, yellow). Here, clonal activation of PfRh4 is significantly increased but remains incomplete (~11%). Activated PfRh4 loci are found in a conserved activation domain (cyan) and have promoters accessible to DNA binding proteins, just as in F11S and F11S/N (right center, host erythrocyte omitted for clarity). In contrast to F11S, where silent PfRh4 loci are associated with a heterochromatic silencing domain (left center), inactive loci in F11R are found outside of the silencing domain even as the promoter remains in the inaccessible state and no activation occurs (middle center). The derepression that accompanies relocalization of these loci leads to an increase in the frequency of clonal activation that is observed as an increase in the proportion of F11R parasites expressing PfRh4 and as greater independence of the population from the requirement for host cell surface sialic acid. See also Table S5.

EXPERIMENTAL PROCEDURES

Parasite Culture

W2mef was obtained from the Walter and Eliza Hall Institute (Melbourne, Australia). Dd2attB parasites were obtained from David Fidock. P. falciparum were cultured as previously described (Trager and Jensen, 1976).

Generation of Transgenic P. falciparum

To generate pEcDamHi, E. coli Dam was amplified via PCR (Table S1) and cloned into the bxb1 integrase-based vector pLN-DD. To generate pEcDamLo, the EcDam ORF was subcloned into pLN-GFP-HA-DD. The PfCaM promoter was then replaced with the PbDT terminator. Transfection into attB parasites was performed as previously described (Nkrumah et al., 2006). The terminal 1.2kb of PfRh4 was amplified (Table S1) and subcloned into pHHT-TK to generate pHHT-TK-Rh4-3′. Transgenic parasites were selected via homologous recombination by cycling on and off WR99210.

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were performed as previously described (Comeaux et al., 2011). 108 schizonts were used to generate chromatin fractions for each strain tested. Immunoprecipitations were performed using 5 µl of αH3 (06–755, Millipore), αH3K9me3 (07–442, Millipore), or normal rabbit IgG (12–370, Millipore) for 4 hr at 4°C. Quantification of immunoprecipitated DNA was accomplished by qRT-PCR using the primers indicated (Table S1). Values were normalized to percent input, and a ratio of H3K9me3 percent input to that of H3 was calculated.

Microarray Analysis

Early trophozoite stage Dd2-pEcDamLo parasites were incubated with 0.5 µM Shld1 and either 100 nM TSA or DMSO for 6 hr. Parasites were harvested in Trizol and frozen at −80°C. Identical samples were harvested for qRT-PCR. RNA was amplified, biotinylated, and hybridized to a custom expression microarray containing probes for 5,132 genes in the P. falciparum genome. Data were analyzed as previously described (Le Roch et al., 2003) and deposited in NCBI’s Gene Expression Omnibus (accession number GSE41567). For a detailed methodology, see the Supplemental Information.

Quantitative Reverse-Transcriptase PCR

Schizont stage cDNA was prepared and qRT-PCR analysis performed as previously described (Comeaux et al., 2011). PCR primers used for amplification were tested for specificity and efficiency (Table S2). Analysis was performed using the comparative Ct method according to the Applied Bio-systems user bulletin 2.

For a detailed methodology of single-cell qPCR, see the Supplemental Information. In brief, exon-exon junction-spanning probes with accompanying primers were designed for PfRh4, PfRh1, and PfCoronin (Table S1). Appropriate qPCR performances were optimized and confirmed, with a sensitivity of detection below 0.2 parasites (Figure S5). RNA from single schizont stage parasites was reverse transcribed, preamplified, and subsequently assayed for PfRh4/PfCoronin-positive wells in singleplex and PfRh4/PfRh1 multiplex qPCR reactions. Frequency of parasites with activated PfRh4 was computed from the number of cells transcribing PfCoronin and PfRh1.

Sialic Acid-Dependence Assays

Sialic acid was removed from erythrocytes by treatment with Neuraminidase from V. cholerae (Roche). Parasite growth into treated cells was monitored by flow cytometry as previously described (Bei et al., 2010) and analyzed using FloJo (Tree Star). Erythrocytes were gated by forward and side scatter and infected cells counted by SYBR Green I signal in the FITC channel.

In Vivo Chromatin Accessibility Assays

Ring stage Dd2-pEcDamLo parasites were treated with 0.5 µM Shld1 for 48 hr to stabilize the methylase. gDNA was prepared with the DNA Blood Mini Kit (QIAGEN). For Southern blot analysis of individual loci, 500 ng of genomic DNA were digested with DpnI and the appropriate methyl-insensitive restriction enzymes (Table S2). Hybridized membranes were exposed to phosphorimager screens, and band intensities were determined using Quantity One (Bio-Rad). For global assays, 6 hr treatment with 100 nM TSA was performed on early schizonts. gDNA (250 ng) was digested with DpnII overnight and run on a 0.8% TAE agarose gel. For TaqMan real-time qPCR determination of PfRh4 promoter accessibility, genomic DNA was digested with DpnII and assayed using primers and probes listed in Table S1. The signal from the restriction digest-spanning probe (−215) was normalized (ΔCt) to signal from an endogenous control probe detecting a PfRh4 exonic fragment lacking relevant restriction sites. Relative levels of accessibility were determined by the ΔΔCt method using F11S as the calibrator sample. Sensitivity and specificity were confirmed on dilution series and artificial mixes of DpnII-digested gDNA from F11S and F11S/N (Figure S6D).

Fluorescence In Situ Hybridization

FISH was adapted from Brolin et al. (2009) with modifications. For detailed protocols, see the Supplemental Information. In brief, DNA FISH was done on parasites (28–32 hr postinvasion) hybridized with DNA probes toward PfRh4, PfRh1, hDHFR, and Rep20 labeled with either Biotin Hi-Prime or Fluorescein Hi-Prime (Roche Applied Science). RNA FISH was performed using parasites (28–36 hr postinvasion) hybridized with DIG and biotin-labeled RNA probes for the stage-matched PfCoronin and PfRh4. HRP-conjugated anti-DIG and anti-biotin antibodies and tyramide signal amplification were used to stain the transcripts. Hybridizations were visualized with fluorescent microscopy and subnuclear localization (peripheral versus nonperipheral and colocalizations) scored blind. Colocalization was defined as any overlapping signal between the red and the green channels, and peripheral nuclear localization was defined as any part of signal overlapping with or abutting the edge of the nucleus as defined by DAPI staining (Ralph et al., 2005). Any other localization in the DAPI-stained nucleus was judged nonperipheral. Statistical significance was calculated with Fisher’s exact test for these binomial, unpaired data with n > 100.

Clonal Sialic Acid-Independence Frequency

F11S and F11R parasites were serially diluted to one and ten parasites per well in neuraminidase-treated and untreated erythrocytes. Positivity was scored by microscopy at day 14 (Table S3). Mean parasites per well and percent sialic acid independence were calculated as previously described (Soubes et al., 1997).

Supplementary Material

Supplementary Information
Table S1
Table S2
Table S4

ACKNOWLEDGMENTS

This work was supported by a National Science Foundation Graduate Research Fellowship (B.I.C.) and a Burroughs Wellcome Fund New Investigator in the Pathogenesis of Infectious Diseases Fellowship (M.T.D.). We thank Danesh Moazed for critical reading of the manuscript.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes four figures, five tables, array full list, Supplemental Experimental Procedures, and Supplemental References and can be found with this article at http://dx.doi.org/10.1016/j.chom.2012.11.004.

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Associated Data

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Supplementary Materials

Supplementary Information
NIHMS579336-supplement-Supplementary_Information.pdf (13.2MB, pdf)
Table S1
NIHMS579336-supplement-Table_S1.xlsx (10.6KB, xlsx)
Table S2
NIHMS579336-supplement-Table_S2.xls (381KB, xls)
Table S4
NIHMS579336-supplement-Table_S4.xlsx (11.2KB, xlsx)

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