Yeast-based assay identifies novel Shh/Gli target genes in vertebrate development.
BACKGROUND
The increasing number of developmental events and molecular mechanisms associated with the Hedgehog (Hh) pathway from Drosophila to vertebrates, suggest that gene regulation is crucial for diverse cellular responses, including target genes not yet described. Although several high-throughput, genome-wide approaches have yielded information at the genomic, transcriptional and proteomic levels, the specificity of Gli binding sites related to direct target gene activation still remain elusive. This study aims to identify novel putative targets of Gli transcription factors through a protein-DNA binding assay using yeast, and validating a subset of targets both in-vitro and in-vivo. Testing in different Hh/Gli gain- and loss-of-function scenarios we here identified known (e.g., ptc1) and novel Hh-regulated genes in zebrafish embryos.


RESULTS
The combined yeast-based screening and MEME/MAST analysis were able to predict Gli transcription factor binding sites, and position mapping of these sequences upstream or in the first intron of promoters served to identify new putative target genes of Gli regulation. These candidates were validated by qPCR in combination with either the pharmacological Hh/Gli antagonist cyc or the agonist pur in Hh-responsive C3H10T1/2 cells. We also used small-hairpin RNAs against Gli proteins to evaluate targets and confirm specific Gli regulation their expression. Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates. Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches. We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo.


CONCLUSIONS
A recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in different contexts of vertebrate embryonic development.
Faculty of Sciences, Universidad de Chile, Santiago, Chile.
Luis A
LA
Milla
Claudio R
CR
Cortés
Christian
C
Hodar
Maritza G
MG
Oñate
Veronica
V
Cambiazo
Shawn M
SM
Burgess
Verónica
V
Palma
D000818:Animals; D002460:Cell Line; D004625:Embryo, Nonmammalian / Q000187:drug effects / Q000378:metabolism; D047108:Embryonic Development; D018507:Gene Expression Regulation, Developmental; D005821:Genetic Techniques / Q000592:standards; D053823:Hedgehog Proteins / Q000819:agonists / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D015513:Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014704:Veratrum Alkaloids / Q000494:pharmacology; D015027:Zebrafish / Q000254:growth & development; D029961:Zebrafish Proteins / Q000378:metabolism; D000071676:Zinc Finger Protein GLI1; D000094742:Secreted Frizzled-Related Proteins
D053823:Hedgehog Proteins; D008565:Membrane Proteins; D015513:Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014704:Veratrum Alkaloids; D029961:Zebrafish Proteins; D000071676:Zinc Finger Protein GLI1; C515511:Sfrp2 protein, zebrafish; D000094742:Secreted Frizzled-Related Proteins; C554167:URA3 protein, S cerevisiae; C091279:neogenin; C000541:cyclopamine