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.
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ABSTRACT: Background Over 2700 genes are subject to stage-specific regulation during the intraerythrocytic development of the human malaria parasite Plasmodium falciparum. Bioinformatic analyses have identified a large number of over-represented motifs in the 5¿ flanking regions of these genes that may act as cis-acting factors in the promoter-based control of temporal expression. Triaging these lists to provide candidates most likely to play a role in regulating temporal expression is challenging, but important if we are to effectively design in vitro studies to validate this role.Methods We report here the application of a repeated search of variations of 5¿ flanking sequences from P. falciparum using the Finding Informative Regulatory Elements (FIRE) algorithm.ResultsOur approach repeatedly found a short-list of high scoring DNA motifs, for which cognate specific transcription factors were available, that appear to be typically associated with upregulation of mRNA accumulation during the first half of intraerythrocytic development.Conclusions We propose these cis-trans interactions may provide a combinatorial promoter-based control of gene expression to complement more global mechanisms of gene regulation that can account for temporal control during the second half of intraerythrocytic development.Parasites & Vectors 02/2015; 8(1):81. DOI:10.1186/s13071-015-0701-0 · 3.25 Impact Factor
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ABSTRACT: Understanding how sequence-specific protein-DNA interactions direct cellular function is of great interest to the research community. High-throughput methods have been developed to determine DNA-binding specificities; one such technique, the bacterial one-hybrid (B1H) system, confers advantages including ease of use, sensitivity and throughput. In this review, we describe the evolution of the B1H system as a tool capable of screening large DNA libraries to investigate protein-DNA interactions of interest. We discuss how DNA-binding specificities produced by the B1H system have been used to predict regulatory targets. Additionally, we examine how this approach has been applied to characterize two common DNA-binding domain families-homeodomains and Cys2His2 zinc fingers-both in organism-wide studies and with synthetic approaches. In the case of the former, the B1H system has produced large catalogs of protein specificity and nuanced information about previously recovered DNA targets, thereby improving our understanding of these proteins' functions in vivo and increasing our capacity to predict similar interactions in other species. In the latter, synthetic screens of the same DNA-binding domains have further refined our models of specificity, through analyzing comprehensive libraries to uncover all proteins able to bind a complete set of targets, and, for instance, exploring how context-in the form of domain position within the parent protein-may affect specificity. Finally, we recognize the limitations of the B1H system and discuss its potential for use in the production of designer proteins and in studies of protein-protein interactions. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: email@example.com.Briefings in functional genomics 12/2014; 14(1). DOI:10.1093/bfgp/elu048 · 3.43 Impact Factor
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ABSTRACT: Protein-DNA binding is central to specificity in gene regulation, and methods for characterizing transcription factor (TF)-DNA binding remain crucial to studies of regulatory specificity. High-throughput (HT) technologies have revolutionized our ability to characterize protein-DNA binding by significantly increasing the number of binding measurements that can be performed. Protein-binding microarrays (PBMs) are a robust and powerful HT platform for studying DNA-binding specificity of TFs. Analysis of PBM-determined DNA-binding profiles has provided new insight into the scope and mechanisms of TF binding diversity. In this review, we focus specifically on the PBM technique and discuss its application to the study of TF specificity, in particular, the binding diversity of TF homologs and multi-protein complexes. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: firstname.lastname@example.org.Briefings in functional genomics 11/2014; 14(1). DOI:10.1093/bfgp/elu046 · 3.43 Impact Factor