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 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Vic. 3050, Australia b Department of Biochemistry and Molecular Biology, Bio21 Molecular Science & Biotechnology Institute, University of Melbourne, Melbourne, Vic. 3050, Australia c MacFarlane Burnet Institute, 85 Commercial Road, Melbourne, Vic., Australia article info 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 abstract The life cycle of the malaria parasite Plasmodium falciparum involves dramatic morphological and molec- ular changes required for infection of insect and mammalian hosts. Stage-specific gene expression is cru- cial, 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 fam- ilies, 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 sin- gle 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 identifica- tion 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 reg- ulators 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 stage- specific gene expression regulating cell cycle progression and cel- lular differentiation. Additionally, antigenic variation takes place, during which individual members of large, multi-copy gene fami- lies are expressed in a mutually exclusive manner. The switched expression of different forms of a protein, which are usually ex- posed to the host immune system, leads to persistence and viru- lence of a P. falciparum infection. The molecular mechanism by which gene expression is regu- lated in P. falciparum is not yet understood. It is known that the ba- sal 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 gen- ome 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 dis- covered (Voss et al., 2003; De Silva et al., 2008). This led to the gen- erally 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 medi- ated by mutually exclusive expression of one member of the large var gene family which encodes P. falciparum erythrocytic mem- brane 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 * 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: crabb@burnet.edu.au (B.S. Crabb), cowman@wehi.edu.au (A.F. Cowman). 1 These authors contributed equally to this work. International Journal for Parasitology 40 (2010) 109–121 Contents lists available at ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara