Rapid evolutionary rewiring of a structurally constrained eye enhancer

Abstract

Background: Enhancers are genomic cis-regulatory sequences that integrate spatiotemporal 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 that 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 noncone 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.

Figure 1. Despite Rapid Sequence Divergence, the Function and Cell-Type Specificity of the sparkling Enhancer Is Conserved

(A) Summary of the regulatory logic of the sparkling (spa) cone cell enhancer of dPax2. Colored bars indicate known binding sites for Su(H), PntP2/Yan, and Lozenge (Lz); essential novel regulatory sequences 1, 4, 5, and 6a are numbered. RCE indicates the Remote Control Element in region 1 [8]. (B) Comparison of pairwise ortholog sequence similarity between D. melanogaster (mel) and 11 other sequenced Drosophila species, for 16 developmental enhancers. sim, D. simulans; sec, D. sechellia; yak, D. yakuba; ere, D. erecta; ana, D. ananassae; pse, D. pseudoobscura; per, D. persimilis; wil, D. willistoni; moj, D. mojavensis; vir, D. virilis; gri, D. grimshawi. Similarity is measured as the percentage of mel enhancer sequence that is aligned to the orthologous region of the comparison genome by BLAT. See Supplemental Experimental Procedures for detailed information on these reference enhancers. (C) mel-pse pairwise sequence similarity for 16 developmental cis-regulatory sequences, measured as the percentage of mel enhancer sequence that is BLAST-alignable (x-axis) or BLAT-alignable (y-axis) to the pse ortholog. (D–E) The D. melanogaster (mel) and D. pseudoobscura (pse) orthologs of spa. Left, enhancer diagrams showing known or predicted Su(H), Ets, and Lz/Runx binding sites (red, yellow, and blue, respectively). Right, eye imaginal discs of transgenic mel larvae carrying mel spa-GFP (D) or pse spa-GFP (E). Insets show 24-hour transgenic pupal eyes stained with antibodies against GFP (green) and the cone cell nuclear marker Cut (magenta).

Figure 2. Divergent cis-Regulatory Organization of sparkling Between D. melanogaster and D. pseudoobscura

(A) Diagrams of mel-pse chimeric enhancer constructs, with GFP expression in cone cells of transgenic larvae summarized as follows: +++, wild-type levels of expression in cone cells; ++, moderately reduced; +, severely reduced; +/−, barely detectable or detectable in very few cells; –, no detectable expression; ++++, augmented levels of expression (greater than wild-type). (B–K) GFP expression in eye discs of transgenic third-instar larvae carrying selected chimeric spa reporters depicted in panel A. Insets show 24-hour transgenic pupal eyes stained with antibodies against GFP (green) and Cut (magenta).

Figure 3. Cross-Species Sequence Comparisons Identify Novel cis-Regulatory Motifs at Rapidly Changing Positions

(A) Distribution of Su(H), Ets, and Lz/Runx binding sites, along with putative novel regulatory motifs α, β, γ, δ, and ε, in D. mel and D. pse orthologs of spa. Sequence motifs are listed to the right. (B,C) In vivo mutational analysis of novel regulatory motifs in D. pse spa (B) and D. mel spa (C). (D) Experiments in which the Lz/Ets/Su(H) sites in mel are replaced with their orthologous pse sequences. Black arrowheads show D. mel Lz, Ets, and Su(H) sites (blue, yellow, and red bands) that have been replaced by orthologous D. pse sequences; green indicates that the orthologous D. pse sequence is not a predicted site. Red arrowheads show D. pse-specific Ets and ε sites added to D. mel spa, including the 5’ site Ets0. (E–K) GFP expression in eye discs of transgenic third-instar larvae carrying mutated or chimeric spa reporters depicted in panels B–D.

Figure 4. Low Binding Affinity for Su(H) Is Essential for Proper Cell Type Specificity

(A) The five previously identified Su(H) binding sites in D. mel spa [37], and the single identifiable putative Su(H) site in D. pse spa, deviate from the high-affinity consensus binding motif (non-matching bases are in red lowercase). (B,C) Levels of eye disc GFP expression driven by wild-type spa (B) and spa[Su(H)-HiAff] (C), in which the Su(H) sites were altered to the high-affinity sequence CGTGGGAA. (D) spa[Su(H)-HiAff]-GFP is ectopically expressed in a subset of photoreceptors. Left, GFP expression (green) precedes cone cell specification, as marked by Cut expression (magenta). Middle, GFP expression overlaps temporally and spatially with Elav, a photoreceptor marker (magenta). Right, GFP labels a varying subset of early-specified photoreceptors. (E) Unlike spa[wt], spa[Su(H)-HiAff]-GFP (green) is strongly active in Cut-negative, apically located, Notch-responsive primary pigment cells in the 24-hour pupal eye.

Figure 5. Evolutionary Dynamics of the Binding Site Grammar of sparkling

Diagram of selected cis-regulatory motifs within spa orthologs of several Drosophila species. (A) Grammar elements involving Lz, Ets, and/or RCE motifs. (B) Grammar elements involving Lz, Su(H), and/or ε motifs. Brackets, lettered a through n, show spatial relationships among binding sites (“grammar elements”) that are identifiable in multiple species; numbers indicate the base-pair spacing between two motifs. Gray ovals show the sequences included in the minimal mel and pse enhancer reporter constructs. Here, ε denotes a 5/6 or 6/6 match to the AGCCAG motif; selected sequences with a weaker match are designated ε*. The Lz site with a white asterisk in sim/sec indicates that that site is has a mismatch in sim only.

Figure 6. Inferred Evolutionary History of the Vocabulary and Grammar of the sparkling Enhancer

(A) Summary of conserved, divergent, and relocated cis-regulatory features of sparkling in D. melanogaster (top) and D. pseudoobscura (bottom). Symbols represent binding sites for known regulators (L, Lz/Runx; E, Ets; S, Su(H)), the Remote Control Element (R), and novel regulatory motifs α, β, γ, δ, and ε. Double-headed arrows show changes in position of the regulatory motifs comprising essential enhancer regions 1, 4, 5, and 6a (arrows). Selected lineage-specific innovations are indicated with black arrowheads. Letters in italics refer to grammar elements described in Figure 5. (B) A maximum parsimony tree describing the cis-regulatory features of the sparkling enhancer in the inferred last common ancestors (LCAs) of four Drosophila subtaxa, based on sequence analysis and functional assays. Acquired novel features—binding sites or grammar elements—are shown as white boxes crossing a particular lineage, while lost ancestral features are shown as black boxes. Arrows pointing up or down, next to the name of a transcription factor, indicate increased or decreased input of that regulator. Grammar elements are listed in 5’-3’ order.