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. 2015 Oct 30:16:882.
doi: 10.1186/s12864-015-2070-7.

Functionally conserved enhancers with divergent sequences in distant vertebrates

Song Yang  1 , Nir Oksenberg  2   3 , Sachiko Takayama  4 , Seok-Jin Heo  5 , Alexander Poliakov  6 , Nadav Ahituv  7   8 , Inna Dubchak  9   10 , Dario Boffelli  11
Affiliations

Functionally conserved enhancers with divergent sequences in distant vertebrates

Song Yang et al. BMC Genomics. .

Abstract

Background: To examine the contributions of sequence and function conservation in the evolution of enhancers, we systematically identified enhancers whose sequences are not conserved among distant groups of vertebrate species, but have homologous function and are likely to be derived from a common ancestral sequence. Our approach combined comparative genomics and epigenomics to identify potential enhancer sequences in the genomes of three groups of distantly related vertebrate species.

Results: We searched for sequences that were conserved within groups of closely related species but not between groups of more distant species, and were associated with an epigenetic mark of enhancer activity. To facilitate inferring orthology between non-conserved sequences, we limited our search to introns whose orthology could be unambiguously established by mapping the bracketing exons. We show that a subset of these non-conserved but syntenic sequences from the mouse and zebrafish genomes have homologous functions in a zebrafish transgenic enhancer assay. The conserved expression patterns driven by these enhancers are probably associated with short transcription factor-binding motifs present in the divergent sequences.

Conclusions: We have identified numerous potential enhancers with divergent sequences but a conserved function. These results indicate that selection on function, rather than sequence, may be a common mode of enhancer evolution; evidence for selection at the sequence level is not a necessary criterion to define a gene regulatory element.

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Figures

Fig. 1
Fig. 1
Scheme of the analysis of orthologous introns bracketed by the orthologous exons in the rodent/rabbit and fish evolutionary groups of genomes. Conserved sequences (A1 and B, and A2 and C in multiple alignments of the groups 1 and 2 respectively) are compared, and highly similar A1 and A2 removed from the analysis. Sequences B and C are selected for experimental validation
Fig. 2
Fig. 2
Expression data in for positive zebrafish (blue) and mouse (brown) ECR constructs in transgenic zebrafish. The x-axis shows tissues with positive expression in at least one construct; the y-axis shows the number of fish expressing a construct in a given tissue (minus the number of fish with ectopic expression – see Additional file 2: Table S1 for details), reported as the proportion of fish alive at 24 hpf (light color) and 48 hpf (dark color). The identity of each construct is shown in the top right corner of each chart. The expression of the mouse ECR syntenic to zebrafish ECRs 10 and 11 is reported in both charts
Fig. 3
Fig. 3
Expression patterns driven by zebrafish and mouse syntenic ECRs with homologous activity in a zebrafish transgenic enhancer assay. ECRs are identified by the number at the bottom left of each panel, their genomic coordinates are in Table 4. Expression patterns were recorded at 24 hpf (ECR 7) or 48 hpf (ECRs 6, 10, 11, and 12) and detected by GFP expression (green). Labels indicate tissues in which homologous mouse and zebrafish sequences show consistent patterns of GFP expression (see Experimental Procedures for the definition of consistent pattern of expression). ECR 6: constructs from both species drive strong expression in several epidermal cells; the zebrafish construct also drives expression in the otic vesicle , weak staining at the periphery of the vesicle is visible with the mouse construct (see also Additional file 1: Figure S1). ECR 7: the mouse construct drives expression in the olfactory bulb, but expression by the zebrafish construct is restricted to the olfactory epithelium (the sensory component of the olfactory bulb – see also Additional file 1: Figure S1); constructs from both species drive expression in large neurons in the hindbrain of. ECR 10 and 11: one ECR is present in the mouse, and two ECRs are present in the orthologous zebrafish intron; constructs from both species drive clear expression in somitic muscle cells. ECR12: constructs from both species drive clear expression in several epidermal cells. Abbreviations – e: epidermis; ov: otic vesicle; oe: olfactory epithelium; ob: olfactory bulb; hb: hindbrain; sm: somitic muscle
Fig. 4
Fig. 4
Transcription factor binding sites detected in the five syntenic ECRs. a-e: each panel shows the five motifs with most significant E-value in the given ECR, and the sequence of the n-mer, identical in the zebrafish, mouse, and human sequence of the ECR, from which the motif was identified. The name of the transcription factor binding to a motif is shown above the motif, and the sequence of the motif is shown for the strand on which the n-mer was found. The arrangement of the motifs along the zebrafish (zf), mouse (mm), and human (hs) sequences is shown at the bottom of each panel (the scale, in base pairs, is the same for all panels and shown only for panel E). Motifs are color-coded according the legend at the bottom of the figure; motifs shown above and below the reference line are identified on the forward and reverse strands, respectively
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