Specific DNA-binding by apicomplexan AP2 transcription factors

Abstract

Malaria remains one of the most prevalent infectious diseases worldwide, affecting more than half a billion people annually. Despite many years of research, the mechanisms underlying transcriptional regulation in the malaria-causing Plasmodium spp., and in Apicomplexan parasites generally, remain poorly understood. In Plasmodium, few regulatory elements sufficient to drive gene expression have been characterized, and their cognate DNA-binding proteins remain unknown. This study characterizes the DNA-binding specificities of two members of the recently identified Apicomplexan AP2 (ApiAP2) family of putative transcriptional regulators from Plasmodium falciparum. The ApiAP2 proteins contain AP2 domains homologous to the well characterized plant AP2 family of transcriptional regulators, which play key roles in development and environmental stress response pathways. We assayed ApiAP2 protein-DNA interactions using protein-binding microarrays and combined these results with computational predictions of coexpressed target genes to couple these putative trans factors to corresponding cis-regulatory motifs in Plasmodium. Furthermore, we show that protein-DNA sequence specificity is conserved in orthologous proteins between phylogenetically distant Apicomplexan species. The identification of the DNA-binding specificities for ApiAP2 proteins lays the foundation for the exploration of their role as transcriptional regulators during all stages of parasite development. Because of their origin in the plant lineage, ApiAP2 proteins have no homologues in the human host and may prove to be ideal antimalarial targets.

Fig. 1.

Alignment of the AP2 domain from PF14_0633 (amino acids 63–123) to orthologues in five additional Plasmodium spp. and six Apicomplexan species. The AP2 domain (boxed) is highly conserved across all species. Conservation of residues is most significant in the three β-strands (shaded yellow) of the AP2 domain and is less significant in the α-helix (shaded blue). The AT-hook domain (shaded green) is found upstream of the AP2 domain in Plasmodium spp. (vertical black line). Absolutely conserved residues likely to be involved in DNA binding are highlighted in red. Secondary structure predictions were made by using Jnet (39). PF, P. falciparum; PVX, Plasmodium vivax; PKH, Plasmodium knowlesi; PB, Plasmodium berghei; PY, Plasmodium yoelii; PC, Plasmodium chabaudi; 583, T. gondii; TP, Theileria parvum; TA, Theileria annulata; BBOV, Babesia bovis; Chro, Cryptosporidium hominis; cgd2, Cryptosporidium parvum.

Fig. 2.

DNA motifs specifically bound by AP2 domains predicted using PBMs and computational analysis (FIRE algorithm). (A) The core nucleotides (boxed) in the motif specifically bound by the P. falciparum AP2 domain of PF14_0633 are highly similar to those bound by its C. parvum orthologue cgd2_3490 (Top and Middle). The motifs determined from the PBM are very similar to motifs predicted using the FIRE algorithm (Bottom, Predicted) (24). (B) The PBM-derived motif bound by the tandem AP2 domains of PFF0200c (Top) is highly similar to the motif bound by the first domain alone (Middle). Domain 2 of PFF0200c did not bind a specific DNA motif (data not shown). Both PBM-derived motifs for PFF0200c match the computationally predicted motif (Bottom).

Fig. 3.

Blood-stage gene expression profile of the PFF0200c ApiAP2 protein compared with the averaged expression profiles of putative FIRE-predicted target genes. The 48-hour gene expression profiles are positively correlated with a Pearson correlation coefficient of 0.97.

Fig. 4.

EMSA of AP2 domains. The AP2 domain from PF14_0633 (Upper) and the tandem AP2 domains from PFF0200c (Lower) were used to shift 40 bp of a radioactively labeled DNA probe derived from the upstream region of a computationally predicted target genes (PFI0540w and MAL7P1.119, respectively) (lane 2). Competition experiments show that an unlabeled specific competitor (SC) can deplete the labeled shifted band (lanes 3–6). An unlabeled mutant competitor (MC, lanes 7 and 8) or a random competitor (RC, lane 9) cannot deplete the shift.