A genome-scale analysis of the cis-regulatory circuitry underlying sonic hedgehog-mediated patterning of the mammalian limb.
Sonic hedgehog (Shh) signals via Gli transcription factors to direct digit number and identity in the vertebrate limb. We characterized the Gli-dependent cis-regulatory network through a combination of whole-genome chromatin immunoprecipitation (ChIP)-on-chip and transcriptional profiling of the developing mouse limb. These analyses identified approximately 5000 high-quality Gli3-binding sites, including all known Gli-dependent enhancers. Discrete binding regions exhibit a higher-order clustering, highlighting the complexity of cis-regulatory interactions. Further, Gli3 binds inertly to previously identified neural-specific Gli enhancers, demonstrating the accessibility of their cis-regulatory elements. Intersection of DNA binding data with gene expression profiles predicted 205 putative limb target genes. A subset of putative cis-regulatory regions were analyzed in transgenic embryos, establishing Blimp1 as a direct Gli target and identifying Gli activator signaling in a direct, long-range regulation of the BMP antagonist Gremlin. In contrast, a long-range silencer cassette downstream from Hand2 likely mediates Gli3 repression in the anterior limb. These studies provide the first comprehensive characterization of the transcriptional output of a Shh-patterning process in the mammalian embryo and a framework for elaborating regulatory networks in the developing limb.
Vokes
Steven A
SA
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Ji
Hongkai
H
Wong
Wing H
WH
McMahon
Andrew P
AP
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000502:physiology; D001665:Binding Sites / Q000235:genetics; D019521:Body Patterning / Q000235:genetics* / Q000502:physiology; D047369:Chromatin Immunoprecipitation; D016207:Cytokines; D004742:Enhancer Elements, Genetic; D005121:Extremities / Q000196:embryology*; D005260:Female; D053263:Gene Regulatory Networks; D005816:Genetic Complementation Test; D023281:Genomics; D053823:Hedgehog Proteins / Q000235:genetics* / Q000502:physiology; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology; D000074462:Positive Regulatory Domain I-Binding Factor 1; D011247:Pregnancy; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology; D000074290:Zinc Finger Protein Gli3
D051792:Basic Helix-Loop-Helix Proteins; D016207:Cytokines; D053823:Hedgehog Proteins; D036341:Intercellular Signaling Peptides and Proteins; D051741:Kruppel-Like Transcription Factors; D009419:Nerve Tissue Proteins; D000074462:Positive Regulatory Domain I-Binding Factor 1; D014157:Transcription Factors; D000074290:Zinc Finger Protein Gli3; C113178:Grem1 protein, mouse; C465571:Gli3 protein, mouse; C498766:Hand2 protein, mouse; C513258:Prdm1 protein, mouse; C465570:Shh protein, mouse