Structural Rules and Complex Regulatory Circuitry Constrain Expression of a Notch- and EGFR-Regulated Eye Enhancer

Abstract

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.

Multiple layers of complexity in cis‐regulatory regions of developmental genes

“…Over many years, these analyses have shed light on the logic of cis-regulation (Istrail and Davidson, 2005). Furthermore, they have provided invaluable insights into the structural rules (Erives and Levine, 2004;Panne et al, 2007;Swanson et al, 2010) and evolutionary histories (Ludwig and Kreitman, 1995;Crocker et al, 2008) of enhancers. Nevertheless, it is widely recognized in the cis-regulation community that, often, small enhancer elements do not faithfully recapitulate native expression patterns (Barolo, 2012).…”

Enhancer turnover and conserved regulatory function in vertebrate evolution

“…each transcription factor can recognize a variety of binding site sequences. In addition, although some examples of rigid enhancer organization exist [2,3], the order of TFBSs on a given enhancer can be quite flexible [4]. Finally, enhancers act at a distance and can even be embedded within neighbouring loci [5][6][7][8][9], indicating a large degree of positional freedom of enhancers in relation to the genes they control.…”

Mutational analysis of a Drosophila neuroblast enhancer governing nubbin expression during CNS development

“…Although previous studies have shown that enhancers contain clusters of transcription factor (TF) binding sites within blocks of conserved sequences (Berman et al, 2004; Brody et al, 2012; Davidson & Erwin, 2006; Swanson, Evans, & Barolo, 2010), it is not yet well understood just how the distribution of these sites provides a basis for combinatorial logic of enhancer function. One entry point into deciphering the rules that govern an enhancer's ability to direct gene expression is to manipulate the internal organization of enhancers, that is, position, frequency, and/or order of functionally relevant sequences and examine the effects on cis ‐regulatory behavior.…”

“…One entry point into deciphering the rules that govern an enhancer's ability to direct gene expression is to manipulate the internal organization of enhancers, that is, position, frequency, and/or order of functionally relevant sequences and examine the effects on cis ‐regulatory behavior. For example, altering the TF binding sites and other sequences within the sparkling enhancer of shaven (dPax), a gene that encodes a key regulator of photoreceptor fate specification in the developing Drosophila retina, switched the specificity of the enhancer from cone to rod photoreceptors (Swanson et al, 2010). A second study has shown that an enhancer of the Drosophila Suppressor of Hairless gene is composed of overlapping elements termed submodules that can function independently to activate enhancer activity (Liu & Posakony, 2014).…”