SUMMARY Enhancers integrate spatiotemporal information to generate precise patterns of gene expression. How complex is the regulatory logic of a typical developmental enhancer, and how important is its internal organization? Here, we examine in detail the structure and function of sparkling, a Notch- and EGFR/MAPK-regulated, cone cell-specific enhancer of the Drosophila Pax2 gene, in vivo. In addition to twelve previously identified protein binding sites, sparkling is densely populated with previously unmapped regulatory sequences, which interact in complex ways to control gene expression. One segment is essential for activation at a distance, yet dispensable for other activation functions and for cell-type patterning. Unexpectedly, rearranging sparkling’s regulatory sites converts it into a robust photoreceptor-specific enhancer. Our results show that a single combination of regulatory inputs can encode multiple outputs, and suggest that the enhancer’s organization determines the correct expression pattern by facilitating certain short-range regulatory interactions at the expense of others.

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.

In developing organisms, divergence from the canonical cell division cycle is often necessary to ensure the proper growth, differentiation, and physiological function of a variety of tissues. An important example is endoreplication, in which endocycling cells alternate between G and S phase without intervening mitosis or cytokinesis, resulting in polyploidy. Although significantly different from the canonical cell cycle, endocycles use regulatory pathways that also function in diploid cells, particularly those involved in S phase entry and progression. A key S phase regulator is the Cyclin E-Cdk2 kinase, which must alternate between periods of high (S phase) and low (G phase) activity in order for endocycling cells to achieve repeated rounds of S phase and polyploidy. The mechanisms that drive these oscillations of Cyclin E-Cdk2 activity are not fully understood. Here, we show that the Drosophila Cyclin E-Cdk2 inhibitor Dacapo (Dap) is targeted for destruction during S phase via a PIP degron, contributing to oscillations of Dap protein accumulation during both mitotic cycles and endocycles. Expression of a PIP degron mutant Dap attenuates endocycle progression but does not obviously affect proliferating diploid cells. A mathematical model of the endocycle predicts that the rate of destruction of Dap during S phase modulates the endocycle by regulating the length of G phase. We propose from this model and our in vivo data that endo S phase-coupled destruction of Dap reduces the threshold of Cyclin E-Cdk2 activity necessary to trigger the subsequent G-S transition, thereby influencing endocycle oscillation frequency and the extent of polyploidy.

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