Abstract
BackgroundDivergence within cis-regulatory sequences may contribute to the adaptive evolution of gene expression, but functional alleles in these regions are difficult to identify without abundant genomic resources. Among African cichlid fishes, the differential expression of seven opsin genes has produced adaptive differences in visual sensitivity. Quantitative genetic analysis suggests that cis-regulatory alleles near the SWS2-LWS opsins may contribute to this variation. Here, we sequence BACs containing the opsin genes of two cichlids, Oreochromis niloticus and Metriaclima zebra. We use phylogenetic footprinting and shadowing to examine divergence in conserved non-coding elements, promoter sequences, and 3'-UTRs surrounding each opsin in search of candidate cis-regulatory sequences that influence cichlid opsin expression.ResultsWe identified 20 conserved non-coding elements surrounding the opsins of cichlids and other teleosts, including one known enhancer and a retinal microRNA. Most conserved elements contained computationally-predicted binding sites that correspond to transcription factors that function in vertebrate opsin expression; O. niloticus and M. zebra were significantly divergent in two of these. Similarly, we found a large number of relevant transcription factor binding sites within each opsin's proximal promoter, and identified five opsins that were considerably divergent in both expression and the number of transcription factor binding sites shared between O. niloticus and M. zebra. We also found several microRNA target sites within the 3'-UTR of each opsin, including two 3'-UTRs that differ significantly between O. niloticus and M. zebra. Finally, we examined interspecific divergence among 18 phenotypically diverse cichlids from Lake Malawi for one conserved non-coding element, two 3'-UTRs, and five opsin proximal promoters. We found that all regions were highly conserved with some evidence of CRX transcription factor binding site turnover. We also found three SNPs within two opsin promoters and one non-coding element that had weak association with cichlid opsin expression.ConclusionsThis study is the first to systematically search the opsins of cichlids for putative cis-regulatory sequences. Although many putative regulatory regions are highly conserved across a large number of phenotypically diverse cichlids, we found at least nine divergent sequences that could contribute to opsin expression differences in cis and stand out as candidates for future functional analyses.
Highlights
Divergence within cis-regulatory sequences may contribute to the adaptive evolution of gene expression, but functional alleles in these regions are difficult to identify without abundant genomic resources
SWS1 is found alone on cichlid linkage group (LG) 17; SWS2A, SWS2B, and LWS are found together in a tandem array on LG 5 [44]; and RH2B, RH2Aa, and RH2Ab are found in a second tandem array on LG 5 approximately 30 cM from the SWS2-LWS array (KL Carleton, unpublished data) [44]
transcription factor binding sites (TFBS) search and phylogenetic shadowing of conserved non-coding element (CNE) We compared the 20 CNEs identified between O. niloticus and M. zebra and found many to be highly conserved; we found no identifiable orthologs between O. niloticus and M. zebra for CNEs 6 or 19
Summary
Divergence within cis-regulatory sequences may contribute to the adaptive evolution of gene expression, but functional alleles in these regions are difficult to identify without abundant genomic resources. Cisregulatory sequences can even reside within the untranslated regions (UTRs) of genes, where they alter the binding of microRNAs (miRNAs) that regulate gene expression following transcription [16,17] Where ever their location, two methods commonly used to identify cis-regulatory sequences and alleles are phylogenetic footprinting and phylogenetic shadowing [18]. One compares putative regulatory sequences among closely related taxa in hopes of identifying sequence polymorphisms correlated with the divergent expression of some target gene(s) Following their application, functional genomic analyses are necessary to validate the function of any candidate sequences or alleles identified by the phylogenetic footprinting and shadowing methods; but even by themselves, both methods can provide valuable insights into the location of potential cis-regulatory sequences and the transcription factors that bind them
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