Mechanisms of gene regulation in Plasmodium

Recent investigations into gene expression in malarial systems suggest that locus-specific, promoter-based transcriptional control is not the dominant mode of regulation in Plasmodium. Although global transcript and protein profiles across the life cycle of Plasmodium imply significant control of developmental progression,^1,2 the exact mechanisms underlying this pattern are largely unknown, and appear to be unconventional compared with those of most model organisms. As such, our understanding of transcriptional, post-transcriptional, and epigenetic mechanisms in Plasmodium, as well as their relative contributions to gene regulation, lags far behind that of other eukaryotic systems. We seek a comprehensive characterization of the mechanisms that Plasmodium species have evolved to control gene expression during their complex life cycles. To address these concerns, a panel of experts recently convened for an initial consultation at the Broad Institute (Cambridge MA) in September 2006. Both the integration of existing data in a manner that will be most useful to the malarial community and the types of information currently lacking in the field were discussed. Preliminary recommendations include further delineation of the parasite's genomic structure, as well as elucidation of chromatin structure and epigenetic features; a systematic comparison of global expression data from nuclear run-on, RNA expression microarrays, and proteomic based assays; and the re-evaluation and functional testing of putative regulatory nucleic acid motifs, including characterization of their binding proteins. Detailed examination of regulation on a whole-genome scale, as well as among specific loci, will ultimately make way for control measures aimed at interfering with processes crucial for parasite survival, such as differentiation, antigenic variation, and development of drug resistance. Overview. Plasmodium, the causal agent of malaria, afflicts more than half a billion people worldwide. Despite global efforts to curb malaria, the spread of drug and insecticide resistance and the continuing lack of an effective vaccine contribute to its persistence as a major health burden. A fundamental understanding of how parasite genes governing transmission success, immune evasion, and drug resistance are regulated is critical to developing novel therapeutic strategies against these processes. Although focused studies on candidate genes have provided some insights, we remain largely ignorant of the mechanisms underlying gene control, and the relative role of transcriptional and post-transcriptional/translational regulation in the parasite. For example, although genes appear to be monocistronically transcribed, no clear canonical promoter has been defined to date and the handful of functional cis-acting elements uncovered in Plasmodium^3-9 is unique to this system. The dearth of annotated transcription factors in the genome of Plasmodium falciparum^10,11 in conjunction with phased expression of stage-specific transcripts¹ suggests that post-transcriptional control may be a major means of regulating gene expression, as supported by a recent study documenting the significant role of translation repression in sexual differentiation.¹² The successful application of high-throughput approaches in malarial systems now provides the opportunity to fundamentally transform the pace of post-genomic research on gene regulation in Plasmodium. For example, both microarray and proteomics-based assays document significant regulation of transcript and protein expression profiles across asexual and sexual stages of development.^1,2,13,14 Such technologies have opened the door to bioinformatically investigating the co-regulation of genes, with the potential to define sequence elements that may be behind this control. Variation in global rates of RNA decay during the intraerythrocytic developmental cycle contributes to mounting evidence supporting a more prominent role for post-transcriptional control in the specific regulation of transcript subsets, as do more recent experiments uncovering global control of transcriptional activity. This provocative combination follows a growing emphasis on chromatin remodeling^15,16 and gene-silencing, ^4,15,17 and suggests a complex, multi-layered regulatory network in the parasite. Collectively, these studies underscore the need for a comprehensive, concerted effort to gain the same level of understanding in Plasmodium as has been achieved in the model eukaryotic systems for which these gene regulatory mechanisms were first elucidated. Objectives/Conclusions. We first describe the state of the field in Plasmodium, with specific emphasis on what is known about the regulation of steady-state RNA levels during the life cycle and exposure to stress. Although microarray studies clearly demonstrate a "hard-wired" pattern of expression tied to developmental staging, perturbation with small molecules has rendered conflicting results, leading to difficulties in interpretation of this data. We next describe efforts to bioinformatically mine these steady-state RNA data sets to uncover civ-acting sequence motifs that may control expression of gene clusters. Preliminary evidence that more global mechanisms of regulating transcriptional activity may dominate this paradigm is also presented. The single exception to this model characterized to date, the antigenic var gene family, and its regulatory features are subsequently detailed. The growing body of evidence for post-transcriptional regulation in Plasmodium species is then discussed, including function-dependent changes in mRNA decay rate across the life cycle, and translational repression and proteomic regulation in P. berghei gametocytes. Finally, we propose the following recommendations for the next steps to be taken in our multilateral investigation: 1) determination of chromatin organization and structure; 2) comparison and further characterization of global expression profiles from nuclear run-on, microarray, and proteomic-based assays; and 3) identification, functional testing, and re-evaluation of potentially regulatory nucleic acid binding proteins and their binding sites. In conclusion, we articulate a way forward in the field of malarial gene expression, and propose experiments that are needed to pursue these goals systematically. This complex topic demands that experts from the malaria field as well as authorities on transcriptional, post-transcriptional, and epigenetic gene regulation in other systems be brought together. With this initial consultation, we have taken the first step for forming and funding such a consortium of scientists.