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In silico discovery of transcription regulatory elements in Plasmodium falciparum.

Young JA, Johnson JR, Benner C, Yan SF, Chen K, Le Roch KG, Zhou Y, Winzeler EA - BMC Genomics (2008)

Bottom Line: When applied to promoter regions of genes contained within 21 co-expression gene clusters generated from P. falciparum life cycle microarray data using the semi-supervised clustering algorithm Ontology-based Pattern Identification, GEMS identified 34 putative cis-regulatory elements associated with a variety of parasite processes including sexual development, cell invasion, antigenic variation and protein biosynthesis.This GEMS analysis demonstrates that in silico regulatory element discovery can be successfully applied to challenging repeat-sequence-rich, base-biased genomes such as that of P. falciparum.The putative regulatory elements described represent promising candidates for future biological investigation into the underlying transcriptional control mechanisms of gene regulation in malaria parasites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, ICND 202, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. jayoung@scripps.edu

ABSTRACT

Background: With the sequence of the Plasmodium falciparum genome and several global mRNA and protein life cycle expression profiling projects now completed, elucidating the underlying networks of transcriptional control important for the progression of the parasite life cycle is highly pertinent to the development of new anti-malarials. To date, relatively little is known regarding the specific mechanisms the parasite employs to regulate gene expression at the mRNA level, with studies of the P. falciparum genome sequence having revealed few cis-regulatory elements and associated transcription factors. Although it is possible the parasite may evoke mechanisms of transcriptional control drastically different from those used by other eukaryotic organisms, the extreme AT-rich nature of P. falciparum intergenic regions (approximately 90% AT) presents significant challenges to in silico cis-regulatory element discovery.

Results: We have developed an algorithm called Gene Enrichment Motif Searching (GEMS) that uses a hypergeometric-based scoring function and a position-weight matrix optimization routine to identify with high-confidence regulatory elements in the nucleotide-biased and repeat sequence-rich P. falciparum genome. When applied to promoter regions of genes contained within 21 co-expression gene clusters generated from P. falciparum life cycle microarray data using the semi-supervised clustering algorithm Ontology-based Pattern Identification, GEMS identified 34 putative cis-regulatory elements associated with a variety of parasite processes including sexual development, cell invasion, antigenic variation and protein biosynthesis. Among these candidates were novel motifs, as well as many of the elements for which biological experimental evidence already exists in the Plasmodium literature. To provide evidence for the biological relevance of a cell invasion-related element predicted by GEMS, reporter gene and electrophoretic mobility shift assays were conducted.

Conclusion: This GEMS analysis demonstrates that in silico regulatory element discovery can be successfully applied to challenging repeat-sequence-rich, base-biased genomes such as that of P. falciparum. The fact that regulatory elements were predicted from a diverse range of functional gene clusters supports the hypothesis that cis-regulatory elements play a role in the transcriptional control of many P. falciparum biological processes. The putative regulatory elements described represent promising candidates for future biological investigation into the underlying transcriptional control mechanisms of gene regulation in malaria parasites.

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Gene-by-gene comparison of expression levels for mixed asexual versus sporozoite stages and mixed asexual parasites versus heat shock treated mixed asexual parasites. While expression levels for many genes vary widely between mixed asexual and sporozoite stages (dark gray points, Pearson's r = 0.284), little difference is observed in expression levels in mixed asexual parasites before and after heat shock treatment (white points, Pearson's r = 0.989) demonstrating a lack of robust transcriptional response to environmental perturbations at the level of transcription.
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Figure 8: Gene-by-gene comparison of expression levels for mixed asexual versus sporozoite stages and mixed asexual parasites versus heat shock treated mixed asexual parasites. While expression levels for many genes vary widely between mixed asexual and sporozoite stages (dark gray points, Pearson's r = 0.284), little difference is observed in expression levels in mixed asexual parasites before and after heat shock treatment (white points, Pearson's r = 0.989) demonstrating a lack of robust transcriptional response to environmental perturbations at the level of transcription.

Mentions: A long-standing question in malaria research is whether environmental or drug stimuli trigger transcriptional responses in genes active in the affected biological pathways. In S. cerevisiae, specific components of signaling pathways have been identified using a DNA microarray-profiling approach to analyze the yeast transcriptional responses to various drug pressures [43-45]. If P. falciparum behaves in a similar fashion to yeast, insights could be gained into the biological targets of certain anti-malarial drugs for little is known regarding specific modes of action. Since PfM15.1 was originally identified as the G-box motif using heat shock annotated genes, it provided us with an opportunity to test the biological specificity of transcriptional responses in P. falciparum to heat shock. If the application of heat shock resulted in a specific transcriptional response mediated by a cis-regulatory element, we hypothesized that we would be able to re-identify PfM15.1 from a list of differentially expressed genes obtained from experimental heat shock transcriptional response data. To test this hypothesis, we exposed a mixed asexual culture P. falciparum to a 42°C heat shock for one hour and identified differentially expressed genes relative to a negative control by hybridization to our whole genome high-density microarray. The greatest fold change for any one gene between heat shock and negative control was 4.03 demonstrating that transcriptional responses to environmental stimuli are not as robust in P. falciparum as has been reported in other organisms such as yeast and humans (Figure 8). However, by defining the top 75 differentially expressed genes identified by a non-parametric Mack-Skillings as a positive set [see Additional file 5] and searching the 2000 bases upstream of these genes, GEMS analysis was able to rediscover a motif similar to PfM15.1/G-box motif (ATGGGGCC, log10PPf = -5.06) [46]. Although this motif was the 12th best scoring motif in this analysis, the fact that GEMS was able to rediscover the G-box equivalent motif even from this modest heat shock transcriptional response was encouraging. Since versions of PfM15.1 were also discovered from clusters of genes that show similar expression patterns to ribosomal genes, namely mitotic cell cycle (PfM15.2, AAAGGGA, log10PPf = -8.72) and tRNA metabolism (PfM15.3, TAGGGGAA, log10PPf = -7.41) [see Additional file 3], as well as from intron regions of genes involved in tRNA metabolism (PfMIntron13.2, CCTCCCCC, log10PPf = -3.29) and the ribosome (PfMIntron16.2, ACGGGGG, log10PPf = -3.91) [see Additional file 4], it is possible that in general the G-box motif is primarily associated with highly expressed trophozoite-specific metabolic genes and that identification of the G-box through heat shock gene promoter analysis by Militello et al. was merely fortuitous as heat shock genes are highly-expressed during these same stages.


In silico discovery of transcription regulatory elements in Plasmodium falciparum.

Young JA, Johnson JR, Benner C, Yan SF, Chen K, Le Roch KG, Zhou Y, Winzeler EA - BMC Genomics (2008)

Gene-by-gene comparison of expression levels for mixed asexual versus sporozoite stages and mixed asexual parasites versus heat shock treated mixed asexual parasites. While expression levels for many genes vary widely between mixed asexual and sporozoite stages (dark gray points, Pearson's r = 0.284), little difference is observed in expression levels in mixed asexual parasites before and after heat shock treatment (white points, Pearson's r = 0.989) demonstrating a lack of robust transcriptional response to environmental perturbations at the level of transcription.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268928&req=5

Figure 8: Gene-by-gene comparison of expression levels for mixed asexual versus sporozoite stages and mixed asexual parasites versus heat shock treated mixed asexual parasites. While expression levels for many genes vary widely between mixed asexual and sporozoite stages (dark gray points, Pearson's r = 0.284), little difference is observed in expression levels in mixed asexual parasites before and after heat shock treatment (white points, Pearson's r = 0.989) demonstrating a lack of robust transcriptional response to environmental perturbations at the level of transcription.
Mentions: A long-standing question in malaria research is whether environmental or drug stimuli trigger transcriptional responses in genes active in the affected biological pathways. In S. cerevisiae, specific components of signaling pathways have been identified using a DNA microarray-profiling approach to analyze the yeast transcriptional responses to various drug pressures [43-45]. If P. falciparum behaves in a similar fashion to yeast, insights could be gained into the biological targets of certain anti-malarial drugs for little is known regarding specific modes of action. Since PfM15.1 was originally identified as the G-box motif using heat shock annotated genes, it provided us with an opportunity to test the biological specificity of transcriptional responses in P. falciparum to heat shock. If the application of heat shock resulted in a specific transcriptional response mediated by a cis-regulatory element, we hypothesized that we would be able to re-identify PfM15.1 from a list of differentially expressed genes obtained from experimental heat shock transcriptional response data. To test this hypothesis, we exposed a mixed asexual culture P. falciparum to a 42°C heat shock for one hour and identified differentially expressed genes relative to a negative control by hybridization to our whole genome high-density microarray. The greatest fold change for any one gene between heat shock and negative control was 4.03 demonstrating that transcriptional responses to environmental stimuli are not as robust in P. falciparum as has been reported in other organisms such as yeast and humans (Figure 8). However, by defining the top 75 differentially expressed genes identified by a non-parametric Mack-Skillings as a positive set [see Additional file 5] and searching the 2000 bases upstream of these genes, GEMS analysis was able to rediscover a motif similar to PfM15.1/G-box motif (ATGGGGCC, log10PPf = -5.06) [46]. Although this motif was the 12th best scoring motif in this analysis, the fact that GEMS was able to rediscover the G-box equivalent motif even from this modest heat shock transcriptional response was encouraging. Since versions of PfM15.1 were also discovered from clusters of genes that show similar expression patterns to ribosomal genes, namely mitotic cell cycle (PfM15.2, AAAGGGA, log10PPf = -8.72) and tRNA metabolism (PfM15.3, TAGGGGAA, log10PPf = -7.41) [see Additional file 3], as well as from intron regions of genes involved in tRNA metabolism (PfMIntron13.2, CCTCCCCC, log10PPf = -3.29) and the ribosome (PfMIntron16.2, ACGGGGG, log10PPf = -3.91) [see Additional file 4], it is possible that in general the G-box motif is primarily associated with highly expressed trophozoite-specific metabolic genes and that identification of the G-box through heat shock gene promoter analysis by Militello et al. was merely fortuitous as heat shock genes are highly-expressed during these same stages.

Bottom Line: When applied to promoter regions of genes contained within 21 co-expression gene clusters generated from P. falciparum life cycle microarray data using the semi-supervised clustering algorithm Ontology-based Pattern Identification, GEMS identified 34 putative cis-regulatory elements associated with a variety of parasite processes including sexual development, cell invasion, antigenic variation and protein biosynthesis.This GEMS analysis demonstrates that in silico regulatory element discovery can be successfully applied to challenging repeat-sequence-rich, base-biased genomes such as that of P. falciparum.The putative regulatory elements described represent promising candidates for future biological investigation into the underlying transcriptional control mechanisms of gene regulation in malaria parasites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Biology, ICND 202, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. jayoung@scripps.edu

ABSTRACT

Background: With the sequence of the Plasmodium falciparum genome and several global mRNA and protein life cycle expression profiling projects now completed, elucidating the underlying networks of transcriptional control important for the progression of the parasite life cycle is highly pertinent to the development of new anti-malarials. To date, relatively little is known regarding the specific mechanisms the parasite employs to regulate gene expression at the mRNA level, with studies of the P. falciparum genome sequence having revealed few cis-regulatory elements and associated transcription factors. Although it is possible the parasite may evoke mechanisms of transcriptional control drastically different from those used by other eukaryotic organisms, the extreme AT-rich nature of P. falciparum intergenic regions (approximately 90% AT) presents significant challenges to in silico cis-regulatory element discovery.

Results: We have developed an algorithm called Gene Enrichment Motif Searching (GEMS) that uses a hypergeometric-based scoring function and a position-weight matrix optimization routine to identify with high-confidence regulatory elements in the nucleotide-biased and repeat sequence-rich P. falciparum genome. When applied to promoter regions of genes contained within 21 co-expression gene clusters generated from P. falciparum life cycle microarray data using the semi-supervised clustering algorithm Ontology-based Pattern Identification, GEMS identified 34 putative cis-regulatory elements associated with a variety of parasite processes including sexual development, cell invasion, antigenic variation and protein biosynthesis. Among these candidates were novel motifs, as well as many of the elements for which biological experimental evidence already exists in the Plasmodium literature. To provide evidence for the biological relevance of a cell invasion-related element predicted by GEMS, reporter gene and electrophoretic mobility shift assays were conducted.

Conclusion: This GEMS analysis demonstrates that in silico regulatory element discovery can be successfully applied to challenging repeat-sequence-rich, base-biased genomes such as that of P. falciparum. The fact that regulatory elements were predicted from a diverse range of functional gene clusters supports the hypothesis that cis-regulatory elements play a role in the transcriptional control of many P. falciparum biological processes. The putative regulatory elements described represent promising candidates for future biological investigation into the underlying transcriptional control mechanisms of gene regulation in malaria parasites.

Show MeSH
Related in: MedlinePlus