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: 9.81). 07/2008; 105(24):8393-8. DOI: 10.1073/pnas.0801993105
Source: PubMed

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.

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Available from: Manuel Llinás, Aug 28, 2014
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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 · 4.04 Impact Factor
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    • "To identify transactivating regions within ApiAP2 proteins, we used the genetically tractable models yeast and T. gondii to more rapidly screen for active protein sequences. First, with a one-hybrid approach in Saccharomyces cerevisiae (Fields and Song, 1989), three ApiAP2 proteins from P. falciparum, PFF0200c/PfSIP2, PF11_0442, and PF14_0633 (De Silva et al., 2008; Flueck et al., 2010; Yuda et al., 2010; Yuda et al., 2009), were dissected into overlapping sections of 200 amino acids (Figure 1A), each fused to the C terminus of the GAL4 DNAbinding domain (GAL4-DBD), to test for transactivation. As a positive control, we used Aintegumenta (ANT), a well-characterized plant AP2 transcriptional activator involved in the floral development of Arabidopsis thaliana (Krizek and Sulli, 2006). "
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    ABSTRACT: A major obstacle in analyzing gene function in apicomplexan parasites is the absence of a practical regulatable expression system. Here, we identified functional transcriptional activation domains within Apicomplexan AP2 (ApiAP2) family transcription factors. These ApiAP2 transactivation domains were validated in blood-, liver-, and mosquito-stage parasites and used to create a robust conditional expression system for stage-specific, tetracycline-dependent gene regulation in Toxoplasma gondii, Plasmodium berghei, and Plasmodium falciparum. To demonstrate the utility of this system, we created conditional knockdowns of two essential P. berghei genes: profilin (PRF), a protein implicated in parasite invasion, and N-myristoyltransferase (NMT), which catalyzes protein acylation. Tetracycline-induced repression of PRF and NMT expression resulted in a dramatic reduction in parasite viability. This efficient regulatable system will allow for the functional characterization of essential proteins that are found in these important parasites.
    Cell host & microbe 12/2012; 12(6):824-34. DOI:10.1016/j.chom.2012.10.016 · 12.19 Impact Factor
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    • "The AP2 domain in RAV1 also binds a non-GC rich sequence CAACA (Kagaya et al., 1999). Two AP2 domain proteins from Plasmodium were found to bind the consensus sequences TGCATGCA and GTGCAC, which are different from the target sequences of all plant AP2 domain proteins characterized to date (De Silva et al., 2008). Collectively, these studies show that AP2 domains have wide ranging target specificities. "
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    ABSTRACT: Cell fate specification in development requires transcription factors for proper regulation of gene expression. In Arabidopsis, transcription factors encoded by four classes of homeotic genes, A, B, C and E, act in a combinatorial manner to control proper floral organ identity. The A-class gene APETALA2 (AP2) promotes sepal and petal identities in whorls 1 and 2 and restricts the expression of the C-class gene AGAMOUS (AG) from whorls 1 and 2. However, it is unknown how AP2 performs these functions. Unlike the other highly characterized floral homeotic proteins containing MADS domains, AP2 has two DNA-binding domains referred to as the AP2 domains and its DNA recognition sequence is still unknown. Here, we show that the second AP2 domain in AP2 binds a non-canonical AT-rich target sequence, and, using a GUS reporter system, we demonstrate that the presence of this sequence in the AG second intron is important for the restriction of AG expression in vivo. Furthermore, we show that AP2 binds the AG second intron and directly regulates AG expression through this sequence element. Computational analysis reveals that the binding site is highly conserved in the second intron of AG orthologs throughout Brassicaceae. By uncovering a biologically relevant AT-rich target sequence, this work shows that AP2 domains have wide-ranging target specificities and provides a missing link in the mechanisms that underlie flower development. It also sets the foundation for understanding the basis of the broad biological functions of AP2 in Arabidopsis, as well as the divergent biological functions of AP2 orthologs in dicotyledonous plants.
    Development 04/2012; 139(11):1978-86. DOI:10.1242/dev.077073 · 6.27 Impact Factor
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