International Journal for Parasitology 40 (2010) 109–121
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Potential epigenetic regulatory proteins localise to distinct nuclear sub-compartments in Plasmodium falciparum Jennifer Volz a,1, Teresa G. Carvalho a,1, Stuart A. Ralph b, Paul Gilson a,c, Jenny Thompson a, Christopher J. Tonkin a, Christine Langer a, Brendan S. Crabb a,c,*, Alan F. Cowman a,* a b c
The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic. 3050, Australia Department of Biochemistry and Molecular Biology, Bio21 Molecular Science & Biotechnology Institute, University of Melbourne, Melbourne, Vic. 3050, Australia MacFarlane Burnet Institute, 85 Commercial Road, Melbourne, Vic., Australia
a r t i c l e
i n f o
Article history: Received 3 August 2009 Received in revised form 9 September 2009 Accepted 9 September 2009
Keywords: Plasmodium Malaria Nucleus Epigenetic regulators PHD-finger domain SET domain CHROMO domain
a b s t r a c t The life cycle of the malaria parasite Plasmodium falciparum involves dramatic morphological and molecular changes required for infection of insect and mammalian hosts. Stage-specific gene expression is crucial, yet few nuclear factors, including potential epigenetic regulators, have been identified. Epigenetic mechanisms play an important role in the switched expression of members of species-specific gene families, which encode proteins exported into the cytoplasm and onto the surface of infected erythrocytes. This includes the large virulence-associated var gene family, in which monoallelic transcription of a single member and switching to other var genes leads to a display of different surface ligands with distinct antigenic and adhesive properties. Using a bio-informatic approach we identified 24 putative nuclear proteins. Tagging with sequences encoding GFP or haemagglutinin (HA) epitopes allowed for identification and localisation analysis of 12 nuclear proteins that are potential regulators of P. falciparum gene expression. These proteins specifically localise to distinct areas of the nucleus, reaching from the centre towards the nuclear envelope, giving new insights into the apicomplexan nuclear architecture. Proteins presenting a punctate distribution in the perinuclear sub-compartments are potential virulence gene regulators as silenced and active var genes reside at the nuclear periphery either clustered or in small expression sites, respectively. These analyses demonstrated an ordered compartmentalisation, indicating a complex sub-nuclear organisation that contributes to the complexity of transcriptional regulation in P. falciparum. Ó 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Malaria is a major human health problem and as a consequence is an impediment to economic and social development in countries where it is endemic (Sachs and Malaney, 2002; Snow et al., 2005). Plasmodium falciparum is the parasite responsible for the most lethal form of malaria in humans. The parasite undergoes different developmental stages during its sexual and asexual life cycle in its hosts, which require stagespecific gene expression regulating cell cycle progression and cellular differentiation. Additionally, antigenic variation takes place, during which individual members of large, multi-copy gene families are expressed in a mutually exclusive manner. The switched expression of different forms of a protein, which are usually ex-
* Corresponding authors. Tel.: +61 3 92822123; fax: +61 3 92822126 (B.S. Crabb); tel.: +61 3 93452446; fax: +61 3 93470852 (A.F. Cowman). E-mail addresses:
[email protected] (B.S. Crabb),
[email protected] (A.F. Cowman). 1 These authors contributed equally to this work.
posed to the host immune system, leads to persistence and virulence of a P. falciparum infection. The molecular mechanism by which gene expression is regulated in P. falciparum is not yet understood. It is known that the basal transcription machinery such as proteins associated with RNA polymerase II are conserved in P. falciparum (Callebaut et al., 2005; Kyes et al., 2007). However, analysis of the completed genome sequence failed to detect many canonical transcription factors (Aravind et al., 2003; Coulson et al., 2004) and only recently has the apicomplexan AP2 (ApiAP2) transcription factor family been discovered (Voss et al., 2003; De Silva et al., 2008). This led to the generally accepted hypothesis that P. falciparum may be unusually reliant on epigenetic mechanisms to control gene expression. As a result, much emphasis has been placed on the dissection of epigenetic mechanisms controlling the antigenic variation mediated by mutually exclusive expression of one member of the large var gene family which encodes P. falciparum erythrocytic membrane protein 1 (PfEMP1) (Baruch et al., 1995; Su et al., 1995; Mok et al., 2007). This protein is exported to and displayed on the surface of the parasite-infected erythrocyte (Baruch et al.,
0020-7519/$36.00 Ó 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2009.09.002
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1996, 1997). Also, members of the rifin, stevor and Pfmc-2tm gene families, located in the subtelomeric regions where the majority of var genes reside, are expressed in a restrictive manner, but their functional role is not yet clear (Lavazec et al., 2007; Mok et al., 2007; Niang et al., 2009). The promoters of var genes appear to be the key elements in nucleation of transcriptional silencing, activation and maintenance of allelic var gene exclusion (Dzikowski et al., 2006; Voss et al., 2006); although the var gene intron may play a role in gene silencing (Deitsch et al., 2001; Voss et al., 2006). Recent evidence demonstrates that the var genes are located at the ‘transcriptionally silent’ nuclear periphery (Duraisingh et al., 2005; Ralph et al., 2005). The var genes located in subtelomeric regions are influenced by telomere position effect (TPE) as genetic disruption of the silencing factor PfSIR2A or PfSIR2B results in loss of the allelic exclusion mechanism by activating var gene subsets, such as some rifin genes (Duraisingh et al., 2005; Tonkin et al., 2009). These findings demonstrate an important role of the nuclear periphery as well as the need for multi-factorial regulators for var gene silencing and activation. Although largely associated with electron dense heterochromatin-like material, the P. falciparum nuclear periphery also contains electron-sparse gaps as observed in electron microscopy studies (Ralph et al., 2005). It has been speculated that these nuclear peripheral regions contain active euchromatin, as is the case with mammalian cells and budding yeast (for review, see Akhtar and Gasser (2007)), and that var activation involves repositioning of the active locus into this zone. This hypothesis is supported by fluorescent in situ hybridization (FISH) experiments showing perinuclear repositioning of transcriptionally active loci (Duraisingh et al., 2005; Ralph et al., 2005). Further, a study on transgenic parasite lines, in which two var genes were simultaneously activated, determined the strict localization of these genes to a specific subnuclear site (Dzikowski et al., 2007). In recent years, post-translational modification of histones has been linked to var gene regulation (Freitas-Junior et al., 2005; Chookajorn et al., 2007; Lopez-Rubio et al., 2007). Histone modifications commonly associated with active transcription, such as acetylation at lysine 9 of histone subunit H3 (H3K9ac) and trimethylation at histone H3 lysine 4 (H3K4m3) have been linked to transcriptionally active var genes, whereas a silent var gene was found to be enriched in the typically silencing modification H3K9m3 (Lopez-Rubio et al., 2007, 2009). It has also been hypothesized that histone 3 lysine 4 di-methylation (H3K4m2) serves as a mark for cellular memory as it associates with var gene promoters poised for transcription. In a recent mass-spectrometry analysis (Trelle et al., 2009) an additional number of histone modifications in P. falciparum have been described, however, their roles in gene control still await clarification. To date, the identity and molecular function of histone modifying and binding proteins in P. falciparum are not well understood. Although the P. falciparum genome contains a large repertoire of chromatin-modifying proteins, suggesting major involvement in gene regulation, to date only a handful of nuclear factors have been characterized, such as histone lysine methyltransferases and demethylases (Cui et al., 2008a), the histone acetyl transferase PfGCN5 (Miao et al., 2006; Cui et al., 2008b), the protein arginine methyltransferase I (Fan et al., 2009) and the class III histone deacetylase Sir2 proteins (Duraisingh et al., 2005; Freitas-Junior et al., 2005; Lopez-Rubio et al., 2009; Tonkin et al., 2009). A recent study has revealed P. falciparum Heterochromatin Protein 1 (PfHP1) as a major heterochromatin component, which occupies the full complement of subtelomeric and chromosomeinternal var genes and is extended to other protein families exported or predicted to be exported to the erythrocyte surface (Flueck et al., 2009). Interestingly, PfHP1 is also associated with genes involved in erythrocyte invasion, which support recent studies that
indicate involvement of epigenetic mechanisms in their regulation (Cortes et al., 2007). Variation in the expression of protein families responsible for erythrocyte invasion (i.e. eba, rhop1/clag, PfRh gene families) is linked to alternative invasion pathways involving different ligand–receptor interactions (Duraisingh et al., 2003; Stubbs et al., 2005). In this study, we made use of the availability of the P. falciparum genome and systems-biology datasets to identify potential nuclear factors that may be involved in control of gene expression. Since suites of enzymes with similar functions in disparate organisms share several domains that are present throughout most eukaryotes, we have used a bio-informatic approach to identify candidate genes containing known domains involved in gene regulation and epigenetic mechanisms. We have analysed the in vivo localisation of these proteins to confirm their nuclear localisation and examine sub-nuclear organisation. These analyses demonstrated an ordered compartmentalisation, indicating a complex sub-nuclear organisation that contributes to the complexity of transcriptional regulation in P. falciparum.
2. Materials and methods 2.1. Bio-informatic analysis of PlasmoDB Candidates for nuclear proteins involved in transcriptional regulation were chosen by a combination of bio-informatic queries. Plasmodium falciparum proteins containing domains associated with transcriptional regulation were sought using protein family and domain searches at PlasmoDB (www.plasmodb.org), Pfam (www.ebi.ac.uk/pfam) and interpro (www.ebi.ac.uk/interpro). Experimentally and computationally derived interactome sets (LaCount et al., 2005; Suthram et al., 2005) were also interrogated to select proteins predicted to belong to networks involved in transcriptional regulation. OrthoMCL (www.orthomcl.org) was used to examine predicted orthologs of candidates, as well as to identify P. falciparum-specific candidates. Whole genome transcriptome data sets (Bozdech et al., 2003; Le Roch et al., 2003) were examined to enrich for candidates transcribed during the asexual intraerythrocytic life cycle. Nuclear localisation signals in Plasmodium spp. appear not to conform to the motifs defined in model eukaryotes to date. Candidates were therefore searched to eliminate those with any non-nuclear targeting signals, as well as enriching for basic rich motifs that may serve as nuclear localisation signals. 2.2. Plasmids PCR amplifications were performed on P. falciparum strain 3D7 genomic DNA using oligonucleotides (listed in Supplementary Table S1) to obtain either full-length or 30 end gene fragments. Fragments were specifically digested (Supplementary Table S1) prior to cloning into a tetracycline-inducible GFP expression vector (Meissner et al., 2005) or into a GFP- or haemagglutinin(HA)-fusion vector, respectively. All of the vectors contain the human dihydrofolate reductase (hDHFR) gene, which confers resistance to the antifolate inhibitor WR99210 (Fidock and Wellems, 1997), under the control of 1 kbp of Calmodulin 50 untranslated region (UTR), and 0.6 kbp of histidine rich protein 2 (hrps) 30 UTR (Crabb and Cowman, 1996). 2.3. Transfection and analysis of P. falciparum recombinant parasites Transfection of parasites was carried out as previously described (Crabb and Cowman, 1996) and cultures were maintained with 5 nM of WR99210 (an antifolate drug) (Fidock and Wellems,
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1997). In the case of tetracycline-inducible expression vectors, a final concentration 5 lg/ml of anhydrotetracycline (ATc) was also added to the culture to prevent expression of the GFP-fusion protein (Meissner et al., 2005). After one to three cycles of drug in the case of the GFP and HA 3’ replacement lines, or after ATc removal in the case of ATc-inducible lines, GFP expression was detected by fluorescence live microscopy (Zeiss Axiovert microscope) or by immunofluorescence assay using a monoclonal mouse or rat anti-HA-tag antibody (Roche, clone 3F10). Integration events of the 30 gene replacement constructs were subjected to Southern blot analyses (Supplementary Fig. S1 and Supplementary Table S2). 2.4. Generation of polyclonal Nup100 and polyclonal CenPA antiserum Antibodies to Nup100 (a nuclear pore protein) were created by immunizing rabbits with the peptide N-512 TSHDITDVQNSENKC-C conjugated to keyhole limpet cyanin (KLH). Antisera was purified using protein-G Sepharose. The anti-centromere protein A (CenPA) (or anti-histone H3) antibody was generated by cloning the 50 193 nucleotides of the histone H3 gene (PF13_0185) into the pGEX 4T expression vector to produce a GST fusion protein in BL21 cells. Rabbits were immunised with the fusion protein and antibodies were purified by affinity chromatography. All work using rabbits described was formally approved by the Animal Ethics Committee of the Royal Melbourne Hospital Research Foundation and care and maintenance of animals was in accordance with the National Health and Medical Research Council of Australia guidelines. 2.5. Immunoblotting Nuclear and cytoplasmic protein fractions from non-synchronized 3D7 cultures were obtained as previously described (Voss et al., 2002). Proteins were resolved by 12% SDS–PAGE, transferred to a nitrocellulose membrane, blocked with 10% milk, and detected using Nup100 and CenPA rabbit antisera at 1:400 and 1:50 dilution, respectively. Anti-rabbit IgG conjugated to horseradish peroxidase (Chemicon) was used as a secondary antibody (1:2000), and blots were developed using a Western blot Chemiluminescence Reagent Plus kit (Amersham). Protein loadings were monitored in parallel using a rabbit anti-heat shock protein 70 (hsp70) (1:4000) and corresponding bands were detected as described above. 2.6. Immunofluorescence assay Red blood cells containing non-synchronized parasites were fixed in 0.0075% glutaraldehyde/4% paraformaldehyde (EM Grade ProSciTech) for 30 min at room temperature. After membrane permeabilization with 0.1% Triton-X in PBS for 5 min at room temperature, samples were blocked with 3% BSA for 1 h. Primary and secondary antibodies were diluted in 3% BSA and samples were incubated successively at room temperature for 1 h. Dilutions of antibodies were used as follows: HA-tag 1:500 (anti-mouse/antirat, Roche), Nup100 1:200 (anti-rabbit), CenPA 1:200 (anti-rabbit), H3K9m3 1: 1000 (anti-rabbit, Abcam), H3K9ac 1:1000 (antimouse, Abcam), BiP (endoplasmic reticulum chaperone) 1:500 (anti-mouse, MR4), ERD2 (endoplasmic reticulum KDEL receptor) 1:200 (anti-mouse, MR4), Aldolase 1:300 (anti-mouse) (Baum et al., 2006), secondary antibodies 1:1000 (Molecular Probes). Samples were mounted with Vectorshield (Vector 540 Laboratories Inc., USA) containing DAPI (Roche Applied Science 1:2000) for nuclear cell staining. Z-stack series of the cells were taken using a Carl
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Zeiss Axiovert 200 M (motorized) microscope with an AxioCam MRm camera. Images were deconvolved using Axiovision v 4.2 software (Carl Zeiss Pty. Ltd.).
3. Results 3.1. Identification and structural characterization of putative P. falciparum nuclear proteins In order to identify P. falciparum genes containing functional domains related to protein–DNA interaction or chromatin status modification we used hidden Markov model-based identifiers such as pfam and interpro. Additionally, we incorporated information both from a Plasmodium high-throughput yeast two-hybrid (Y2H) study that identified a network of putative chromatin-interacting proteins (LaCount et al., 2005) and from a study that analyzed the degree of conservation between the Plasmodium protein networks with those of model organisms (Suthram et al., 2005). The resulting list of genes was screened for characteristics consistent with a potential role in gene regulation such as (i) targeting information consistent with nuclear localisation and (ii) transcriptional array evidence for expression in asexual blood-stage forms. This analysis resulted in a list of 24 proteins (Fig. 1) that included putative Plasmodium chromatin-modifying enzymes homologous to those found in other eukaryotes, as well as some Plasmodium-specific proteins, likely to be involved in Plasmodium-specific processes such as antigenic variation. The majority of proteins in our study are putative histonemodifying enzymes, including eight putative histone methyltranferases characterized by the presence of a SET-domain, and one putative histone demethylase. Histone methyltransferases catalyse methylation of lysine residues on histones H3 and H4, which is a well-characterized epigenetic mark either for gene activation or repression depending on the methylated residue’s position on the histone tail. Recently, the existence of a histone code for transcriptional activation and silencing has been demonstrated in P. falciparum (Miao et al., 2006; Lopez-Rubio et al., 2007), and six of the SET-domain proteins we included in our analysis have been predicted for their histone site specificity during the course of our study (Cui et al., 2008a). PFF1440w, PF13_0293 and PFI0485c are proposed to be putative H3K4 methyltransferases, with the latter two similar to SET- and MYND-domain (SMYD)-containing proteins. PFF1440w has been predicted to be part of a network of chromatin related proteins (LaCount et al., 2005). MAL13P1.122 has been proposed to be a putative H3K36 methyltransferase and in an in vitro histone-methylation assay functionally shown to target histone 3 (Cui et al., 2008a). Applying the same functional assay, PFD0190w has been demonstrated to target non-methylated, mono- or di-methylated lysine 20 on histone 4 (H4K20) (Cui et al., 2008a). PF08_0012 was predicted to be the P. falciparum Su(var)3–9 histone methyltransferase with H3K9 specificity, a primary modification mark in heterochromatin (Cui et al., 2008a). Recent studies have demonstrated its localisation to the nuclear periphery (Lopez-Rubio et al., 2009). Methylators of other histone 3 residues were also included in this study (Fig. 1). The putative histone demethylase PFL0575w is similar to the lysine-specific histone demethylase 1 (LSD1) (Iyer et al., 2008). LSD1 appears to have a dual role by promoting both gene repression, via its H3K4 demethylase activity, and transcriptional activation (reviewed in Forneris et al. (2008)). Further, LSD1 has specificity for non-histone substrates such as p53 (Huang et al., 2007). Besides putative histone methylases and a demethylase, we have identified proteins that present chromatin remodelling activities, structural proteins and
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Fig. 1. Plasmodium falciparum putative nuclear proteins identified using a bio-informatic approach. We identified 24 P. falciparum putative nuclear proteins and grouped them into histone-modifying enzymes such as putative histone methyltransferases and demethylases, chromatin remodellers, structural proteins, putative transcription factors and P. falciparum-specific proteins. The presence of a SET-domain, PHD-finger domain(s), CHROMO-domain(s) and P. falciparum specificity is indicated by a tick in the corresponding column. The next column describes proteins previously predicted to be part of experimentally and computationally derived interactome sets (1, LaCount et al. (2005); 2, Suthram et al. (2005)). Additional information on homology, putative function, predicted histone targets and domains are included in the next column (see text for references). Finally, the last column summarizes the expression profiles of most genes during the asexual intraerythrocytic life cycle (Bozdech et al., 2003).
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putative transcription factors. The P. falciparum nucleosomeassembly protein (NAPS) PFI0930c has been initially characterized as a cytoplasmic member of the SET/TAF family (Dobson et al., 2003) and its nuclear localisation has since been demonstrated (Chandra et al., 2005). PFI0930c is able to deposit histones onto DNA and it is likely to be an integral part of the chromatin assembly motor in the parasite nucleus (Chandra et al., 2005). An important component of the nucleosome remodelling factor complex is ISWI, a SNF2 helicase, a homologue of which is present in P. falciparum (PFF1185w). Apicomplexan SWI/SNF factors are characterized by fusion to PHD fingers (Iyer et al., 2008) and are most likely core subunit of large functional complexes that include other chromatin-modifiers such as acetylases, methylases or ubiquinating enzymes, which facilitate the perturbation of chromatin structure in vitro in an ATP-dependant manner (Ji and Arnot, 1997). ISWI has been proposed to recognize H3K4 methylated tails and mediate transcriptional activation (SantosRosa et al., 2003). PFF1185w has been previously identified to be part of a transcription/chromatin remodelling network (Suthram et al., 2005) together with the NAP PFI0930c and PF10_0232 (LaCount et al., 2005). PF10_0232 is another putative P. falciparum SNF2 helicase, included in this study and which is the homologue of chromodomain helicase DNA-binding protein 1 (CHD1). CHD proteins have been described as either transcription activators or repressors (Stokes et al., 1996; Shimono et al., 2003; Tai et al., 2003; Li et al., 2005) with a common involvement in ATP-dependent chromatin remodelling (reviewed in Hall and Georgel (2007)). The plant homeodomain (PHD-finger) is thought to be involved in chromatin-mediated transcription regulation and it has been shown to bind to all nucleosomal histones (Eberharter et al., 2004). It is prevalent in chromatin proteins and different versions mediate distinct interactions with histones or other chromatin proteins. PF11_0429 has been predicted to be involved in chromatin remodelling and potentially interacts with PfISWI (PFF1185w) (LaCount et al., 2005). It also contains an ELM2 domain, a component of transcription and chromatin-regulatory complexes containing one or more histone deacetylases (Ding et al., 2003). The second PHD-finger domain protein of this study, PFC0425w, potentially interacts with the putative transcription regulator THO2 (PFL2390c) (LaCount et al., 2005). Our bio-informatic analysis led to the identification of three CHROMO-domain containing proteins (PF11_0418, PFD0920w, PFL1005c). These proteins can alter the structure of chromatin to the condensed morphology of heterochromatin. Sequence analysis of PFL1005c suggests that this protein is the P. falciparum Protein 1 (HP1) orthologue (Flueck et al., 2009; Perez-Toledo et al., 2009). In addition, three proteins have been included in our study, of which two are putative bZIP transcription factors (PFI0175w and PFL0290w) and one is predicted for its chromosome association (PFD0685c). Yeast two-hybrid studies suggest an interaction of PFI0175w and PFL0290w with a hypothetical transcription factor containing AP2 domains (PFF0670w) and the transcription regulator PfNOT1 (PF11_0049), respectively (LaCount et al., 2005). PFD0685c has been predicted to be involved in chromatin remodelling as part of a P. falciparum-specific complex that includes the ISWI homologue PFF1185c, the NAPS PFI0930c and PF11_0429 (Suthram et al., 2005) as described above. Finally, in the search for virulence gene regulators, we have included four potentially nuclear P. falciparum-specific proteins (PFB0540w, PF14_0745, PFI0330c, PFL2560c). To date, these proteins do not contain any known functional domains, however, PFB0540w has been proposed to be part of a Plasmodium-specific pathway that regulates gene expression (LaCount et al., 2005).
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3.2. The P. falciparum nucleus consists of compartments defined by Nup100, CenPA and DAPI staining In P. falciparum chromosome end clusters and centromeres have previously been shown to specifically localise to the nuclear periphery (Freitas-Junior et al., 2000; Marty et al., 2006; Li et al., 2008), however, the paucity of well-characterized nuclear structures means that most studies utilise DAPI labelling as a means to define the nucleus. To examine the localisation of the identified nuclear proteins in this study in relation to the nuclear envelope we used two nuclear periphery markers. One of the antibodies targets nucleoporin 100 (Nup100), a protein of the nuclear pore (Wente et al., 1992). Western blot analysis showed that Nup100 was present only in nuclear fractions and has a molecular weight of 235 kDa (Fig. 2A). The second antibody we used was directed against the P. falciparum CenPA protein (PF13_0185), a variant histone H3 centromere-specific protein. In eukaryotes, CenPA is required for nucleation and maintenance of centromeric chromatin (Okamoto et al., 2007). The P. falciparum CenPA has a molecular weight of 19.6 kDa and was also present only in the nuclear fraction (Fig. 2A). Nup100 localises in close proximity to BiP, a marker for the endoplasmic reticulum (ER) (Kumar et al., 1991) and does not overlap with DAPI, confirming that it defines the outer limits of the nucleus (Fig. 2B). In fact, the DAPI labelled area was restricted to a small central location of the Nup100/BiP delimited region. CenPA and Nup100 do not co-localise; however, the former protein extends beyond the DAPI staining area of the nucleus suggesting that it is located within the periphery but inside the Nup100 delimited area of the nucleus (Fig. 2C). Using Nup100 and CenPA as nuclear markers in addition to DAPI, it was apparent that this organelle occupies a significant portion of the P. falciparum cell and extends considerably beyond the DAPI staining region. Different histone modifications have been shown to associate with active transcription and with silenced genes. Indeed, silent var genes have been found to be enriched in the typically silencing modification H3K9m3 whereas H3K9ac is found associated with active transcription in general (Lopez-Rubio et al., 2007, 2009). In order to map the localisation of these histone marks within the nucleus we co-localised them with CenPA (Fig. 2D). This indicated that H3K9m3 localised mostly to the nuclear periphery outside the DAPI stained area, which was consistent with the location of chromosome clusters containing silenced var genes. H3K9ac localised more with the DAPI stained area of the nucleus suggesting the DAPI stained area is broadly the transcriptionally active zone. It is tempting to speculate that in the P. falciparum nucleus the DAPI staining delineates a central region that encompasses most transcriptionally active genes whilst the generally transcriptionally inactive nuclear periphery extends beyond this region and is bordered by the Nup100-defined envelope. 3.3. Live cell imaging and immunofluorescence analysis of P. falciparum reveals distinct localisation of 12 novel proteins within the centre and periphery of the nucleus To determine whether the proteins identified in the bio-informatic screen are located within the nucleus, we attached GFP or HA epitope tags to each corresponding gene, using transfection, so that we could visualise the protein within the parasite. This was done using either a tetracycline-inducible expression system (Meissner et al., 2005) in which the protein was expressed from a transfected episome or alternatively, in the case of large genes not easily amenable to this analysis, we modified the C-termini of endogenous proteins using a 30 gene replacement single crossover homologous recombination approach (Supplementary Fig. S1). Using these approaches we successfully tagged 13 of the 24 candidate nuclear proteins with GFP or HA; five by transgene
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Fig. 2. Characterization of two nuclear periphery markers: Nup100 and CenPA. (A) Western blot analysis using Nup100 and CenPA (or histone 3) antibody on cytoplasmic (C) and nuclear (N) fractions of wild-type 3D7 asynchronous parasite cultures reveals their exclusive presence in the nucleus. Detected protein bands corresponding to the predicted sizes are indicated with asterisks (). We always observed two protein bands for Nup100 which points to unspecific protein degradation. Hsp70 was used as a loading control. (B) Nup100 and BiP immunofluorescence analysis reveals the localisation of Nup100 outside the DAPI stained area. (C) Localisation of Nup100 and CenPA outside the DAPI stained area. (D) Localisation of the histone marks H3K9m3 with CenPA and H3K9ac in the nucleus. Ea Trophozoite, Early Trophozoite; La Trophozoite, Late Trophozoite.
expression and eight by C-terminal tagging of the endogenous gene (Figs. 3–6).
Live microscopic visualisation of the GFP-tagged proteins or immunofluorescence analysis of one candidate protein
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Fig. 3. The proteins encoded by the genes PFI0930c and MAL13.P1.122 localise to the far nuclear periphery. (A,B) PFI0930c is either tagged with GFP or Cherry, respectively. Live imaging (a) and co-localisation studies by immunofluorescence (b) reveal its localisation to the nuclear periphery, which is marked by CenPA and delimited by the nuclear envelope marker Nup100 (A), but not to the cytoplasmic portion of the cell marked by aldolase and ERD2 (B). (C) MAL13P1.122 is a SET-domain protein, which localises to the nuclear periphery surrounding the DAPI stained portion within the same region of CenPA and the histone mark H3K9m3. Live imaging (a) and co-localisation studies by immunofluorescence (b) are shown.
(PF08_0012) tagged with HA in the transgenic parasites showed that 12 localised to the nucleus (Figs. 3–6A) with one residing in the mitochondria (Fig. 6B). This high hit rate in predicting nu-
clear located proteins strongly validates our bio-informatic approach and suggests that the other proteins not yet tagged are likely to also be located to this organelle (Fig. 1). One of the nu-
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Fig. 4. SET-domain proteins PF08_0012 and PF13_0293 localise to the nuclear periphery marked by CenPA. (A) Localisation of PF08_0012 reveals a clustered localisation pattern present throughout the nuclear periphery of the cell. PF08_0012 is HA-tagged at the C-terminus and detected with anti-HA antibodies. (B) Live imaging of PF13_0293 shows localisation to a distinct area in the nuclear periphery. Live imaging (a) and co-localisation studies by immunofluorescence (b) are shown.
clear proteins (PFL1005c) initially identified through our bioinformatic approach has been shown to be the P. falciparum Heterochromatin Protein 1 (HP1) orthologue (Flueck et al., 2009; Perez-Toledo et al., 2009). Six of the nuclear proteins identified localise to the periphery of the DAPI labelled nuclear area suggesting they represent a subcompartment of the organelle (Figs. 3–5), whereas a second group of six proteins was shown to predominantly co-localise with the DAPI stained area of the nucleus (Fig. 6A). Combining the live imaging with immunofluorescence analysis using markers for the nuclear periphery as described above allowed us to gain insight into the P. falciparum nuclear architecture. Two of the proteins in our study, a nucleosome-assembly protein PFI0930c (Chandra et al., 2005) and one histone-methyltransferase (MAL13P1.122) (Cui et al., 2008a) localise to the periphery of the nucleus either partially (PFI0930c) or fully (MAL13P1.122) surrounding the DAPI stained area (Fig. 3). PFI0930c encompasses a large region of the nuclear periphery that is distinctly outside the DAPI stained area, close to the nuclear envelope marked by Nup100 and within the region of CenPA location (Fig. 3A). In order to confirm the nuclear localisation of this new area, as opposed to a cytoplasmic location in close proximity to the external nuclear membrane, we made use of a stable transgenic parasite line in which endogenous PFI0930c is fused to the fluorescent mCherry
protein (Shaner et al., 2004) (Fig. 3B). As already shown in the PFI0930c GFP-tagged parasite line (Fig. 3A), PFI0930c-cherry localises to the far nuclear periphery and surrounds the DAPI staining (Fig. 3B). Co-localisation of PFI0930c-cherry and aldolase, used as a cytoplasmic marker (Buscaglia et al., 2003), showed clear separation of nuclear and cytoplasmic regions in the P. falciparum cell (Fig. 3B). Co-localisation analysis with Nup100 and ERD2, a marker for the cis- and trans-Golgi (Elmendorf and Haldar, 1993), showed the same overlapping localisation of PFI0930c-cherry and Nup100, both in an internal location relative to ERD2 (Fig. 3B). Also close to the nuclear envelope is the SET-domain protein MAL13P1.122, which is spread over the nuclear periphery in a speckled pattern (Fig. 3C). It appears from co-localisation analyses that MAL13P1.122 resides within the same nuclear zones as CenPA and the histone mark H3K9m3 and is peripheral to the histone H3K9ac, which is more restricted to the DAPI stained portion of the nucleus (Fig. 3B). We have further identified two SET-domain proteins which localise to the nuclear periphery in distinct regions (Fig. 4). PF08_0012 is present in cluster-like formations, which spread from the central part of the nucleus to a region near the nuclear envelope (Fig. 4A). Co-localisation analyses demonstrate its distribution in the heterochromatic nuclear periphery, which is marked by CenPA and H3K9m3. PF13_0293 localises in a more defined region within the same area (Fig. 4B).
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Fig. 5. PFL1010c and PFL0575w localise to the nuclear periphery in close proximity to the DAPI stained portion of the nucleus. (A) Live imaging of the SET-domain protein PFL1010c shows localisation in one distinct spot, which is characterized by the presence of both histone marks H3K9m3 and H3K9ac as shown in immunofluorescence analyses. Live imaging (a) and co-localisation studies by immunofluorescence (b) are shown. (B) The putative LSD1 homolog PFL0575w localises in speckles mostly around and partially overlapping the DAPI stained portion of the nucleus within the area of the histone mark H3K9ac. Live imaging (a) and co-localisation studies by immunofluorescence (b) are shown.
The localisation analyses of the SET-domain protein PFL1010c and the putative LSD1 homolog (PFL0575w) (Iyer et al., 2008) have revealed a region close to and partially overlapping with the DAPI, respectively, which is characterized by the presence of both histone marks H3K9m3 and H3K9ac (Figs. 2 and 5). PFL1010c does not show any co-localisation with the Nup100 or CenPA (Fig. 5A) and PFL0575w is distributed within the same area in speckles (Fig. 5B). A group of six nuclear proteins were located with the DAPI stained area in a variety of patterns (Fig. 6A). Two proteins (PFI0485c, PF10_0232) specifically accumulate in a punctuate pattern, whereas PFF1185w and PFL2560c appear evenly distributed. Except for a P. falciparum-specific protein (PFL2560c), each of the
other proteins are predicted enzymes, such as a histone methyltransferase (PFI0485c), the homolog to CHD1 (PF10_0232) or the ISWI homolog (PFF1185c). In contrast to this broad distribution, the PHD-finger domain protein PF11_0429 occupies a third of the central nuclear area. Finally, a P. falciparum-specific protein, PFB0540w, shows a distinct pattern as it is present in the central nucleus and partially localises to the nuclear periphery. As expected, co-localisation analyses with the two peripheral markers Nup100 and CenPA did not reveal any overlap (data not shown). The protein (PFL0690c) that localised to the mitochondria (Fig. 6B) was not analysed further. In summary, these data show that nuclear proteins in P. falciparum are located within distinct regions of the nucleus.
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4. Discussion
Fig. 6. Live imaging of GFP-transgenic Plasmodium falciparum lines reveals the localisation of (A) six nuclear proteins, which localise to the central part of the nucleus stained by DAPI (blue panel) and one (B), which localises to the mitochondrium. For each nuclear protein, we have included a schematic protein representation. Insets show one enlarged schizont nucleus.
Our current knowledge of P. falciparum gene regulation, particularly in relation to virulence gene families, is scant. At this point we can only hypothesize the co-existence of various molecular mechanisms but lack the identification and functional characterization of the players involved. In this study, we used a bio-informatic screen of the P. falciparum genome to identify genes encoding nuclear proteins, some of which, likely the ones localizing to the nuclear periphery, are candidates for virulence gene regulation. We identified and tagged 24 potential nuclear candidates. Live cell imaging and immunofluorescence analyses validated this approach confirming that 12 of the 13 proteins successfully tagged localise to the nucleus. Among the 24 candidates we were unable to GFP-tag eight genes, most likely because this interfered with the function of the protein causing a lethal phenotype. The use of a smaller tag such as HA presents an alternative approach and has so far proven to be successful for at least one nuclear protein, which we had previously been unable to fuse to GFP (PF08_0012). Out of the 12 nuclear proteins, six are present in the central area of the nucleus, which is commonly stained with DAPI. Three proteins (ISWI homologue PFF1185w, CHD1 homologue PF10_0232 and PF11_0429) are related, suggesting an involvement in chromatin remodelling (LaCount et al., 2005; Suthram et al., 2005). Further, ISWI and the putative histone methyltransferase PFI0485c, which was localised in a scattered manner throughout the nuclear centre, may share the same histone target (H3K4) (Santos-Rosa et al., 2003; Cui et al., 2008a). PFB0540w has also been proposed to be involved in the chromatin or ubiquitin metabolism (LaCount et al., 2005) and is localised in a cluster-like formation within and partially outside of the DAPI stain. We have localised two proteins (PFL0575w and PFL1010c) that reside in an area around and partially overlapping with the DAPI stain, and which may define a specific nuclear compartment. PFL0575w has been predicted to be the LSD1 homologue, which demethylates H3K4 sites (reviewed in Forneris et al. (2008)). The localisation within the region of transcriptionally active chromatin (as marked by H3K9ac) may underline that prediction. The investigation of a functional link between PFL0575w and the more centrally located PFI0485c or PFF1185c should be addressed in future studies. The Plasmodium-specific SET-domain protein PFL1010c shows an intriguing localisation exclusively to one spot within the nuclear periphery close to the DAPI stained portion. Recent studies in yeast show that functional compartments within the nuclear periphery have been associated with active genes (reviewed in Akhtar and Gasser (2007)). As it is similarly believed that P. falciparum var gene activation requires re-location of a silent var gene into a transcriptionally competent sub-nuclear compartment (Duraisingh et al., 2005), PFL1010c would be an interesting candidate for this localisation and present analyses are ongoing to determine its putative involvement in var gene regulation. Also PF13_0293, a putative H3K4-specific histone methyltransferase (Cui et al., 2008a), localises exclusively to one area that is different to that stained by DAPI. It is possible that this protein localises to the nucleolus. Previous studies demonstrate nucleolar localisation for three telomere-associated proteins (Figueiredo and Scherf, 2005; Freitas-Junior et al., 2005; Mancio-Silva et al., 2008). Therefore, if PF13_0293 is confirmed to be nucleolar, which is presently being examined, this compartment could not only serve as a reservoir for telomere-associated proteins as previously speculated (Mancio-Silva et al., 2008), but also contain proteins with other nuclear functions.
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PF08_0012 has been predicted to be the Su(var)3–9 histone methyltransferase with H3K9 specificity (Cui et al., 2008a). Its clustered presence within the nuclear periphery and partial overlap with the histone mark H3K9m3, CenPA and the nuclear envelope underlines its predicted function and association with heterochromatin, which has been previously suggested (Lopez-Rubio et al., 2009). A more peripheral nuclear area was characterized by the presence of two proteins (PFI0930c, MAL13P1.122). Recent localisation of PFI0930c in P. falciparum (Chandra et al., 2005) and of its Plasmodium berghei orthologue (Pace et al., 2006) revealed it is a nuclear protein, which is consistent with our live cell imaging and immunofluorescence analyses of both GFP- and cherry-tagged PFI0930c. Although direct interaction between PFI0930c and ISWI (described above) has been suggested as part of a chromatinremodelling complex (Suthram et al., 2005), our in vivo imaging results do not suggest a spatial overlap of these two proteins. Indeed PFI0930c was restricted to the nuclear periphery whereas the ISWI homologue PFF1185w was mainly localised within the central nuclear region. The protein encoded by MAL13P1.122 appears to be closely associated with the nuclear envelope as it appears to have a similar localisation to Nup100 and this suggests speculations about its putative function. It is known that in budding yeast and mammalian cells a special relationship between the nuclear envelope and chromatin can be observed. The inner side of the nuclear membrane is characterized by the presence of transcriptionally inactive heterochromatin, which contains clustered telomeres, thereby creating a functional perinuclear environment. Similarly, in P. falciparum centromeres and telomeres, that remain condensed
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throughout the cell cycle, localise to the nuclear periphery, likely within a region of condensed heterochromatin. In that context, the presence of MAL13P1.122 adjacent to the nuclear envelope points to a potential role in maintaining a particular chromatin environment. It is clear that the nucleus of P. falciparum is divided into subcompartments and this is likely to have important functional implications (Duraisingh et al., 2005; Ralph et al., 2005; Issar et al., 2008; Lopez-Rubio et al., 2009). DAPI staining defines a small central core of the nucleus; however, the nucleus extends well beyond this domain. A histone mark (H3K9ac) was localised with the DAPI stained area of the nucleus suggesting this was generally more transcriptionally active (Lopez-Rubio et al., 2007, 2009; Issar et al., 2008). The regions outside this domain most likely correspond to the electron dense zone of the nucleus where chromosome clusters are located, which includes the var gene family (Ralph et al., 2005). This suggests that this region contains heterochromatin with silenced genes and is consistent with the localisation of the histone mark H3K9m3 at the nuclear periphery outside the DAPI stained area. However, it has been shown that in addition to silenced var genes, the single activated var is also present within this perinuclear domain, suggesting that there is a permissive zone for var gene transcription (Duraisingh et al., 2005; Ralph et al., 2005). Therefore, the nuclear proteins that we have localised to this perinuclear domain are potential candidates for regulators of silencing or activation of virulence gene families. Live cell imaging combined with immunofluorescence studies of putative nuclear proteins revealed novel insights into the
Fig. 7. Schematic representation of the Plasmodium falciparum nuclear architecture. A schematic representation of an infected red blood cell (RBC) and an enlargement of the P. falciparum nucleus are shown. From the centre towards the periphery we have defined three distinct areas (1–3) in which nuclear proteins specifically distribute. A summary is shown in which trophozoite stages have been analysed. Proteins, previously predicted to functionally interact, are shown in red. Proteins previously predicted for their histone target sites are indicated by grey boxes. A, apicoplast. M, mitochondria, No, nucleolus.
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nuclear architecture of P. falciparum. We are now able to compartmentalize the nucleus into at least three distinct areas (Fig. 7). From the centre towards the periphery we have defined (i) a central area commonly stained with DAPI and where we localised six proteins; (ii) an area surrounding the DAPI stain characterized by the presence of three proteins with distinct localisation patterns (PFL1010c, PFL0575w and PF13_0293); and (iii) a nuclear periphery region containing the centromeres, the nucleosome-assembly protein PFI0930c, the putative histone methyltransferase PF08_0012, and MAL13P1.122. Finally, this work provides the tools to dissect the function of potential gene regulators and epigenetic factors. By doing so we intend to determine functional nuclear subcompartments and identify the molecular components of active and silent transcription pockets at the nuclear periphery. Acknowledgements We thank the Red Cross Blood Service (Melbourne, Australia) for supply of red cells and serum. J.V. was supported by the Human Frontier Science Program and TC was supported by the Fondation pour la Recherche Medicale. This work was supported by the National Health and Medical Research Council of Australia (NHMRC). AFC is a Howard Hughes International Scholar and an Australia Fellow of the NHMRC.
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