Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Basic Helix-Loop-Helix Transcription Factors / genetics
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Basic Helix-Loop-Helix Transcription Factors / physiology
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Binding Sites / genetics
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Body Patterning / genetics*
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Body Patterning / physiology
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Chromatin Immunoprecipitation
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Cytokines
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Enhancer Elements, Genetic
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Extremities / embryology*
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Female
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Gene Regulatory Networks
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Genetic Complementation Test
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Genomics
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Hedgehog Proteins / genetics*
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Hedgehog Proteins / physiology
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Intercellular Signaling Peptides and Proteins / genetics
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Intercellular Signaling Peptides and Proteins / physiology
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Kruppel-Like Transcription Factors / genetics
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Kruppel-Like Transcription Factors / physiology
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Mice
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Mice, Transgenic
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Models, Biological
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / physiology
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Positive Regulatory Domain I-Binding Factor 1
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Pregnancy
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Signal Transduction
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Transcription Factors / genetics
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Transcription Factors / physiology
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Zinc Finger Protein Gli3
Substances
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Basic Helix-Loop-Helix Transcription Factors
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Cktsf1b1 protein, mouse
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Cytokines
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Gli3 protein, mouse
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Hand2 protein, mouse
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Hedgehog Proteins
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Intercellular Signaling Peptides and Proteins
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Kruppel-Like Transcription Factors
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Nerve Tissue Proteins
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Prdm1 protein, mouse
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Shh protein, mouse
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Transcription Factors
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Zinc Finger Protein Gli3
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Positive Regulatory Domain I-Binding Factor 1