Identification and Validation of Novel Hedgehog-Responsive Enhancers Predicted by Computational Analysis of Ci/Gli Binding Site Density

<div><p>The Hedgehog (Hh) signaling pathway directs a multitude of cellular responses during embryogenesis and adult tissue homeostasis. Stimulation of the pathway results in activation of Hh target genes by the transcription factor Ci/Gli, which binds to specific motifs in genomic enhancers. In <i>Drosophila</i>, only a few enhancers (<i>patched</i>, <i>decapentaplegic</i>, <i>wingless</i>, <i>stripe</i>, <i>knot</i>, <i>hairy</i>, <i>orthodenticle</i>) have been shown by <i>in vivo</i> functional assays to depend on direct Ci/Gli regulation. All but one (<i>orthodenticle</i>) contain more than one Ci/Gli site, prompting us to directly test whether homotypic clustering of Ci/Gli binding sites is sufficient to define a Hh-regulated enhancer. We therefore developed a computational algorithm to identify Ci/Gli clusters that are enriched over random expectation, within a given region of the genome. Candidate genomic regions containing Ci/Gli clusters were functionally tested in chicken neural tube electroporation assays and in transgenic flies. Of the 22 Ci/Gli clusters tested, seven novel enhancers (and the previously known <i>patched</i> enhancer) were identified as Hh-responsive and Ci/Gli-dependent in one or both of these assays, including: <i>Cuticular protein 100A</i> (<i>Cpr100A</i>); <i>invected</i> (<i>inv</i>), which encodes an <i>engrailed</i>-related transcription factor expressed at the anterior/posterior wing disc boundary; <i>roadkill (rdx)</i>, the fly homolog of vertebrate <i>Spop</i>; the segment polarity gene <i>gooseberry (gsb)</i>; and two previously untested regions of the Hh receptor-encoding <i>patched (ptc)</i> gene. We conclude that homotypic Ci/Gli clustering is not sufficient information to ensure Hh-responsiveness; however, it can provide a clue for enhancer recognition within putative Hedgehog target gene loci.</p>