title: 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.
authors.affiliation: Department of Cell and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109, United States of America.|Department of Computational Medicine and Bioinformatics, The University of Michigan, Ann Arbor, MI 48109, United States of America.
authors.forename: Katherine
authors.initials: K
authors.lastname: Gurdziel
authors.affiliation: Department of Cell and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109, United States of America.|Cellular and Molecular Biology Program, The University of Michigan, Ann Arbor, MI 48109, United States of America.
authors.forename: David S
authors.initials: DS
authors.lastname: Lorberbaum
authors.affiliation: Department of Cell and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109, United States of America.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Udager
authors.affiliation: Department of Cell and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109, United States of America.|Cellular and Molecular Biology Program, The University of Michigan, Ann Arbor, MI 48109, United States of America.
authors.forename: Jane Y
authors.initials: JY
authors.lastname: Song
authors.affiliation: Department of Cell and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109, United States of America.
authors.forename: Neil
authors.initials: N
authors.lastname: Richards
authors.affiliation: Department of Cell and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109, United States of America.
authors.forename: David S
authors.initials: DS
authors.lastname: Parker
authors.affiliation: Department of Cell and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109, United States of America.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Johnson
authors.affiliation: Department of Cell and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109, United States of America.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Allen
authors.affiliation: Department of Cell and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109, United States of America.
authors.forename: Scott
authors.initials: S
authors.lastname: Barolo
authors.affiliation: Department of Cell and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109, United States of America.
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Gumucio
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002642:Chick Embryo; D019295:Computational Biology / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000196:embryology* / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics; D053823:Hedgehog Proteins / Q000235:genetics* / Q000378:metabolism; D054259:Neural Tube / Q000378:metabolism; D015513:Oncogene Proteins / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014921:Wings, Animal / Q000196:embryology; D000071676:Zinc Finger Protein GLI1
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D053823:Hedgehog Proteins; D015513:Oncogene Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; C092937:ci protein, Drosophila; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C077245:hh protein, Drosophila