Potential epigenetic regulatory proteins localise to distinct nuclear sub-compartments in Plasmodium falciparum

Abstract

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.

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 stage-specific 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 exposed 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., 1996, Baruch et al., 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 sub-nuclear 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 tri- methylation 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, Lopez-Rubio et al., 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 chromosome-internal 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.