Specific DNA-binding by Apicomplexan AP2 transcription factors

Department of Molecular Biology and Lewis-Sigler Institute for Integrative Genomics, Princeton University, 246 Carl Icahn Laboratory, Princeton, NJ 08544, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2008; 105(24):8393-8. DOI: 10.1073/pnas.0801993105
Source: PubMed


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.

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Available from: Manuel Llinás, Aug 28, 2014
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    • "Moreover, GCATGC was identified as a PF14_0633-binding target in P. falciparum [47], an AP2-Sp-binding target in Plasmodium berghei [48], and a Toxoplasma Ribosomal Protein (TRP)-2-binding target in T. gondii [49]. GTGCAC is known as a subtelomeric variant gene promoter element (SPE)-2 [49] and a binding target of PFF0200c_DLD and PfSIP2 [47, 50, 51]. TAGCTA is also reportedly a binding target of Pb.AP2-O [52]. "
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    ABSTRACT: Background Babesia bovis is an apicomplexan parasite that causes babesiosis in infected cattle. Genomes of pathogens contain promising information that can facilitate the development of methods for controlling infections. Although the genome of B. bovis is publically available, annotated gene models are not highly reliable prior to experimental validation. Therefore, we validated a preproposed gene model of B. bovis and extended the associated annotations on the basis of experimentally obtained full-length expressed sequence tags (ESTs). Results From in vitro cultured merozoites, 12,286 clones harboring full-length cDNAs were sequenced from both ends using the Sanger method, and 6,787 full-length cDNAs were assembled. These were then clustered, and a nonredundant referential data set of 2,115 full-length cDNA sequences was constructed. The comparison of the preproposed gene model with our data set identified 310 identical genes, 342 almost identical genes, 1,054 genes with potential structural inconsistencies, and 409 novel genes. The median length of 5' untranslated regions (UTRs) was 152 nt. Subsequently, we identified 4,086 transcription start sites (TSSs) and 2,023 transcriptionally active regions (TARs) by examining 5' ESTs. We identified ATGGGG and CCCCAT sites as consensus motifs in TARs that were distributed around -50 bp from TSSs. In addition, we found ACACA, TGTGT, and TATAT sites, which were distributed periodically around TSSs in cycles of approximately 150 bp. Moreover, related periodical distributions were not observed in mammalian promoter regions. Conclusions The observations in this study indicate the utility of integrated bioinformatics and experimental data for improving genome annotations. In particular, full-length cDNAs with one-base resolution for TSSs enabled the identification of consensus motifs in promoter sequences and demonstrated clear distributions of identified motifs. These observations allowed the illustration of a model promoter composition, which supports the differences in transcriptional regulation frameworks between apicomplexan parasites and mammals. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-678) contains supplementary material, which is available to authorized users.
    BMC Genomics 08/2014; 15(1):678. DOI:10.1186/1471-2164-15-678 · 3.99 Impact Factor
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    • "(41) investigated the binding specificity of a single C. parvum domain that was highly conserved with a Plasmodium AP2 domain. They found that the binding specificities were absolutely conserved; however, of the 127 putative P. falciparum targets of regulation, only 26 are conserved in C. parvum, suggesting the transcriptional network itself has evolved considerably since Plasmodium and Cryptosporidium diverged (41). "
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    ABSTRACT: We provide the first comprehensive analysis of any transcription factor family in Cryptosporidium, a basal-branching apicomplexan that is the second leading cause of infant diarrhea globally. AP2 domain-containing proteins have evolved to be the major regulatory family in the phylum to the exclusion of canonical regulators. We show that apicomplexan and perkinsid AP2 domains cluster distinctly from other chromalveolate AP2s. Protein-binding specificity assays of C. parvum AP2 domains combined with motif conservation upstream of co-regulated gene clusters allowed the construction of putative AP2 regulons across the in vitro life cycle. Orthologous Apicomplexan AP2 (ApiAP2) expression has been rearranged relative to the malaria parasite P. falciparum, suggesting ApiAP2 network rewiring during evolution. C. hominis orthologs of putative C. parvum ApiAP2 proteins and target genes show greater than average variation. C. parvum AP2 domains display reduced binding diversity relative to P. falciparum, with multiple domains binding the 5′-TGCAT-3′, 5′-CACACA-3′ and G-box motifs (5′-G[T/C]GGGG-3′). Many overrepresented motifs in C. parvum upstream regions are not AP2 binding motifs. We propose that C. parvum is less reliant on ApiAP2 regulators in part because it utilizes E2F/DP1 transcription factors. C. parvum may provide clues to the ancestral state of apicomplexan transcriptional regulation, pre-AP2 domination.
    Nucleic Acids Research 06/2014; 42(13). DOI:10.1093/nar/gku500 · 9.11 Impact Factor
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    • "It was also previously identified as a potential cisregulatory element in C. parvum [22] in the upstream sequences of a subset of glycolysis pathway genes. De Silva et al. (2008) showed that orthologous ApiAP2 proteins from P. falciparum [PlasmoDB: PF14_0633; New ID "
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    ABSTRACT: There are very few molecular genetic tools available to study the apicomplexan parasite Cryptosporidium parvum. The organism is not amenable to continuous in vitro cultivation or transfection, and purification of intracellular developmental stages in sufficient numbers for most downstream molecular applications is difficult and expensive since animal hosts are required. As such, very little is known about gene regulation in C. parvum. We have clustered whole-genome gene expression profiles generated from a previous study of seven post-infection time points of 3,281 genes to identify genes that show similar expression patterns throughout the first 72 hours of in vitro epithelial cell culture. We used the algorithms MEME, Align ACE and FIRE to identify conserved, overrepresented DNA motifs in the upstream promoter region of genes with similar expression profiles. The most overrepresented motifs were E2F (5[prime]-TGGCGCCA-3[prime]); G-box (5[prime]-G.GGGG-3[prime]); a well-documented ApiAP2 binding motif (5[prime]-TGCAT-3[prime]), and an unknown motif (5[prime]-[A/C] AACTA-3[prime]). We generated a recombinant C. parvum DNA-binding protein domain from a putative ApiAP2 transcription factor [Crypto DB: cgd8_810] and determined its binding specificity using protein-binding microarrays. We demonstrate that cgd8_810 can putatively bind the overrepresented G-box motif, implicating this ApiAP2 in the regulation of many gene clusters. Several DNA motifs were identified in the upstream sequences of gene clusters that might serve as potential cis-regulatory elements. These motifs, in concert with protein DNA binding site data, establish for the first time the beginnings of a global C. parvum gene regulatory map that will contribute to our understanding of the development of this zoonotic parasite.
    BMC Genomics 07/2013; 14(1):516. DOI:10.1186/1471-2164-14-516 · 3.99 Impact Factor
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