Cis‐regulatory control of Hedgehog target genes (541.1)
The Hedgehog (Hh) signaling pathway is an important regulator of embryogenesis and tissue homeostasis during adulthood. This pathway is conserved from flies to humans, where it controls the transcription of key target genes. Cubitus interruptus (Ci), a member of the Gli family of transcription factors (TFs), regulates the transcription of Hh target genes by binding to regulatory sequences known as enhancers. In the Drosophila wing, preliminary data show that enhancers that contain consensus Ci binding sites, GACCACCCA, are repressed in cells that lack Ci. We hypothesized that an unidentified transcriptional repressor binds preferentially to consensus Ci binding sites to refine the expression patterns of some Hh target genes. In this project, we identified in silico nine related TFs that have similar Ci binding motifs, and tested whether they selectively recognize Ci binding sites in vitro. We transcribed and translated the DNA binding domains of Button head, Drosophila Specificity Protein 1, Lameduck (Lmd), Sugarbabe, Odd paired (Opa), Klumpfuss (Klu), Stripe, Scratch, and Longitudinals Lacking. Using Electrophoretic Mobility Shift Assays, we tested if these proteins differentially recognized consensus Ci binding sites versus non‐consensus binding sites, which are sequences that deviate from the consensus by one or more base pairs. We found that Opa, Klu, and Lmd recognized consensus Ci sites, but not non‐consensus sites. In addition, we performed in vivo genetic experiments to knockdown these proteins in the wing and the embryo. Knocking down Klu in the wing, resulted in a posterior cross vein phenotype, and knocking down Opa resulted in lethality. Knocking down Klu and Opa in embryos caused derepression of the Hedgehog target gene patched. These results posit a potential role for these TFs in refining the expression of Hh target genes..
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2014 |
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| Erschienen: |
2014 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:28 |
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| Enthalten in: |
The FASEB Journal - 28(2014) |
| Beteiligte Personen: |
Rivera‐Lugo, Rafael [VerfasserIn] |
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| BKL: |
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| Anmerkungen: |
© Federation of American Societies for Experimental Biology |
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| Umfang: |
1 |
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| doi: |
10.1096/fasebj.28.1_supplement.541.1 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a The Hedgehog (Hh) signaling pathway is an important regulator of embryogenesis and tissue homeostasis during adulthood. This pathway is conserved from flies to humans, where it controls the transcription of key target genes. Cubitus interruptus (Ci), a member of the Gli family of transcription factors (TFs), regulates the transcription of Hh target genes by binding to regulatory sequences known as enhancers. In the Drosophila wing, preliminary data show that enhancers that contain consensus Ci binding sites, GACCACCCA, are repressed in cells that lack Ci. We hypothesized that an unidentified transcriptional repressor binds preferentially to consensus Ci binding sites to refine the expression patterns of some Hh target genes. In this project, we identified in silico nine related TFs that have similar Ci binding motifs, and tested whether they selectively recognize Ci binding sites in vitro. We transcribed and translated the DNA binding domains of Button head, Drosophila Specificity Protein 1, Lameduck (Lmd), Sugarbabe, Odd paired (Opa), Klumpfuss (Klu), Stripe, Scratch, and Longitudinals Lacking. Using Electrophoretic Mobility Shift Assays, we tested if these proteins differentially recognized consensus Ci binding sites versus non‐consensus binding sites, which are sequences that deviate from the consensus by one or more base pairs. We found that Opa, Klu, and Lmd recognized consensus Ci sites, but not non‐consensus sites. In addition, we performed in vivo genetic experiments to knockdown these proteins in the wing and the embryo. Knocking down Klu in the wing, resulted in a posterior cross vein phenotype, and knocking down Opa resulted in lethality. Knocking down Klu and Opa in embryos caused derepression of the Hedgehog target gene patched. These results posit a potential role for these TFs in refining the expression of Hh target genes. | ||
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