Rapid Evolutionary Rewiring of a Structurally Constrained Eye Enhancer

Abstract

Summary Background Enhancers are genomic cis-regulatory sequences that integrate spatio-temporal signals to control gene expression. Enhancer activity depends on the combination of bound transcription factors, as well as—in some cases—the arrangement and spacing of binding sites for these factors. Here, we examine evolutionary changes to the sequence and structure of sparkling, a Notch/EGFR/Runx-regulated enhancer which activates the dPax2 gene in cone cells of the developing Drosophila eye. Results Despite functional and structural constraints on its sequence, sparkling has undergone major reorganization in its recent evolutionary history. Our data suggest that the relative strengths of the various regulatory inputs into sparkling change rapidly over evolutionary time, such that reduced input from some factors is compensated by increased input from different regulators. These gains and losses are at least partly responsible for the changes in enhancer structure that we observe. Furthermore, stereotypical spatial relationships between certain binding sites (“grammar elements”) can be identified in all sparkling orthologs—although the sites themselves are often recently derived. We also find that low binding affinity for the Notch-regulated transcription factor Su(H), a conserved property of sparkling, is required to prevent ectopic responses to Notch in non-cone cells. Conclusions Rapid DNA sequence turnover does not imply either the absence of critical cis-regulatory information or the absence of structural rules. Our findings demonstrate that even a severely constrained cis-regulatory sequence can be significantly rewired over a short evolutionary timescale.

Low-affinity transcription factor binding sites shape morphogen responses and enhancer evolution

“…To investigate the other inputs controlling dppD, we examined the sequence conservation of this element across 12 Drosophila species (see electronic supplementary material, figure S1b). Conserved TF binding motifs are considered likely to be functionally significant [94,95], although there are significant exceptions [22,23,61]. The dppD enhancer contains seven core HD binding motifs (TAAT), of which six are perfectly conserved throughout the genus (see electronic supplementary material, figure S1b).…”

“…Other studies use DNA sequence signatures, mainly evolutionary conservation and/or clustering of predicted TF binding motifs, to screen genomes for enhancers [16][17][18][19]. These methods have also been successful, although again, they are by no means foolproof: for example, not all functional enhancers show evidence of evolutionary sequence conservation-even if their function is conserved-and conversely, not all highly conserved sequences display regulatory activity [20][21][22][23].…”

Syntax compensates for poor binding sites to encode tissue specificity of developmental enhancers

“…enhancer | gene regulation | transcription | enhancer grammar | regulatory principles P revious studies have highlighted the importance of sequence constraints within developmental enhancers for tissue-specific patterns of gene expression in both Drosophila and Ciona embryos (1)(2)(3)(4)(5)(6). For example, the 69-bp orthodenticle homeobox (Otx)-a enhancer mediates restricted expression in the Ciona neural plate in response to pleiotropic fibroblast growth factor (FGF) signaling (7)(8)(9).…”

Pax7 is regulated by cMyb during early neural crest development through a novel enhancer

“…1A). Enhancers are generally identified in conserved sequences; however, growing evidence advocates for conservation of regulatory function of genomic regions across species without sequence conservation or even common TF-binding sites Romano and Wray, 2003;Ludwig et al, 2005; Fisher et al, 2006; Hare et al, 2008;Weirauch and Hughes, 2010;Swanson et al, 2011). Additionally, different species display slight variations in Pax7 expression.…”