Abstract
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 Drosophila, only a few enhancers (patched, decapentaplegic, wingless, stripe, knot, hairy, orthodenticle) have been shown by in vivo functional assays to depend on direct Ci/Gli regulation. All but one (orthodenticle) 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 patched enhancer) were identified as Hh-responsive and Ci/Gli-dependent in one or both of these assays, including: Cuticular protein 100A (Cpr100A); invected (inv), which encodes an engrailed-related transcription factor expressed at the anterior/posterior wing disc boundary; roadkill (rdx), the fly homolog of vertebrate Spop; the segment polarity gene gooseberry (gsb); and two previously untested regions of the Hh receptor-encoding patched (ptc) 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.
Highlights
The Hedgehog (Hh) signaling pathway plays multiple roles in embryonic organ development and adult tissue homeostasis across animal phyla [1,2,3]
To test if clustering of Ci/Gli sites could be used to predicted Hh enhancers, we developed a computational strategy to identify all regions of the genome that contain clusters of 3–10 Ci/ Gli sites that are enriched above chance expectation
The randomized (Model 1) and shuffled 3-mer (Model 2) strategies significantly change the GC context around Ci/Gli sites, while the Flip GC/AT model (Model 3), by its nature, faithfully replicates the GC context of Ci/Gli sites in the real genome; this model was selected for use
Summary
The Hedgehog (Hh) signaling pathway plays multiple roles in embryonic organ development and adult tissue homeostasis across animal phyla [1,2,3]. Despite the functional importance and high conservation of the Hh pathway, surprisingly little is known about its target genes in any organism. These target genes and their associated enhancers, which are responsible for the genomic response to Hh in development and disease, have significant potential therapeutic and diagnostic value. To identify regions of the genome that exhibit a higher density of Ci/Gli sites than would be expected by chance, we compared the actual distribution of Ci/Gli sites to a randomized background model. On the basis of the data shown in Results (S1 Fig), only the Flip GC/AT model generates background genomes that most closely represent the GC content surrounding Ci/Gli sites in the native genome. Using the Flip GC/AT strategy, background models were generated separately for the Dm and Dp genomes for comparison to each native genome
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