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

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.

Competition between two phosphatases fine-tunes Hedgehog signaling

“…Most Hh targets rely on the presence of clustered Ci/Gli consensus sites for binding of Hh signaling transcription factors ( Gurdziel et al, 2015 ). Upon scanning the PpV locus using the Genomatix MatInspector algorithm ( http://www.genomatix.de ), we identified two clusters of putative Ci/Gli binding sites, as well as three scattered sites spanning a 4-kb region upstream of the PpV transcription start site ( PpV 5′; Fig.…”

“…The DNA-binding zinc-finger domain of the Ci protein (Ci ZnF), which binds efficiently to verified Ci binding sites ( ptc-5 ) in the ptc locus ( Fig. 8 D ; Gurdziel et al, 2015 ), was purified and used in the electrophoretic mobility shift assay. Our DNA binding and competition assays (shown in Fig.…”

A low affinity cis-regulatory BMP response element restricts target gene activation to subsets of Drosophila neurons

“…Moreover, our discovery here of a novel motif with predictive value only increases the promise that more BMP-REs with predictive value will be discovered. However, as demonstrated by numerous studies, accounting for low affinity sites has proven to be beneficial in identifying functional enhancers ( Zandvakili et al, 2018 ; Gurdziel et al, 2015 ). Therefore, it remains important to identify novel BMP-responsive motifs with demonstrated predictive value in BMP-RE discovery.…”

Intercellular calcium signaling is regulated by morphogens duringDrosophilawing development

“…The A/P patterning of ICW amplitudes suggests a potential biological function of ICWs in conveying or storing spatial information in wing discs. One possible mechanism to be tested in the future is that Hh signalling regulates ICWs through Plc21C activity, which may be a direct regulatory target of Ci, as the Plc21C enhancer has Ci binding sites, and is responsive to Hh signalling 41 . Further, canonical Hh signalling has many target genes, including the Decapentaplegic and Wingless pathways.…”