1. Identification and Validation of Novel Hedgehog-Responsive Enhancers Predicted by Computational Analysis of Ci/Gli Binding Site Density.
- Author
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Gurdziel K, Lorberbaum DS, Udager AM, Song JY, Richards N, Parker DS, Johnson LA, Allen BL, Barolo S, and Gumucio DL
- Subjects
- Animals, Base Sequence, Binding Sites genetics, Chick Embryo, Drosophila melanogaster genetics, Embryo, Nonmammalian metabolism, Gene Expression Regulation, Developmental, Green Fluorescent Proteins genetics, Hedgehog Proteins metabolism, Neural Tube metabolism, Regulatory Sequences, Nucleic Acid, Sequence Analysis, DNA, Signal Transduction genetics, Wings, Animal embryology, Zinc Finger Protein GLI1, Computational Biology methods, DNA-Binding Proteins metabolism, Drosophila Proteins genetics, Drosophila Proteins metabolism, Drosophila melanogaster embryology, Hedgehog Proteins genetics, Oncogene Proteins metabolism, Trans-Activators metabolism, Transcription Factors metabolism
- 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.
- Published
- 2015
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