Unravelling cis-Regulatory Elements in the Genome of the Smallest Photosynthetic Eukaryote: Phylogenetic Footprinting in Ostreococcus
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We used a phylogenetic footprinting approach, adapted to high levels of divergence, to estimate the level of constraint in intergenic regions of the extremely gene dense Ostreococcus algae genomes (Chlorophyta, Prasinophyceae). We first benchmarked our method against the Saccharomyces sensu stricto genome data and found that the proportion of conserved non-coding sites was consistent with those obtained with methods using calibration by the neutral substitution rate. We then applied our method to the complete genomes of Ostreococcus tauri and O.lucimarinus, which are the most divergent species from the same genus sequenced so far. We found that 77% of intergenic regions in Ostreococcus still contain some phylogenetic footprints, as compared to 88% for Saccharomyces, corresponding to an average rate of constraint on intergenic region of 17% and 30%, respectively. A comparison with some known functional cis-regulatory elements enabled us to investigate whether some transcriptional regulatory pathways were conserved throughout the green lineage. Strikingly, the size of the phylogenetic footprints depends on gene orientation of neighboring genes, and appears to be genus-specific. In Ostreococcus, 5′ intergenic regions contain four times more conserved sites than 3′ intergenic regions, whereas in yeast a higher frequency of constrained sites in intergenic regions between genes on the same DNA strand suggests a higher frequency of bidirectional regulatory elements. The phylogenetic footprinting approach can be used despite high levels of divergence in the ultrasmall Ostreococcus algae, to decipher structure of constrained regulatory motifs, and identify putative regulatory pathways conserved within the green lineage.
KeywordsPhylogenetic footprinting Non-coding DNA cis-regulatory elements Saccharomyces Ostreococcus
We would like to thank an anonymous referee for constructive comments on a previous version and Eric Bonnet for help with alignment software and sequence shuffling. We would also like to thank Severine Jancek, Nigel Grimsley, Stéphane Rombauts, Pierre Rouzé, David Waxman, and Jan Wuyts for critical comments and stimulating discussions. This collaboration was founded by Tournesol. G.P. was granted an EMBO short-term fellowship and a “Marine Genomics Europe” GAP fellowship (European Network of Excellence 2004–2008 GOCE-CT-2004-505403). K.V. is a postdoctoral fellow of the Fund for Scientific Research, Flanders. This work was supported by the Belgian Federal Science Policy Office: IUAP P6/25 (BioMaGNet).
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