Yeast-based assay identifies novel Shh/Gli target genes in vertebrate development

Abstract

BackgroundThe 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.ResultsThe 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.ConclusionA recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in different contexts of vertebrate embryonic development.

Beyond the Binding Site: In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development

“…In this work, we employed a yeast-based one-hybrid genome-wide screen to identify mouse and zebrafish DNA sequences that are bound by CNBP in a cellular context. The same yeast-based method had been successfully applied to identify transcription factor target genes [13,22]. Identified sequences were analyzed by bioinformatics allowing the definition of a consensus DNAbinding site, as well as a set of putative target genes, whose expression profiles were assessed in vivo in a vertebrate model.…”

Sonic Hedgehog in cancer stem cells: a novel link with autophagy

“…This suggests that autophagy regulation by SHH could be driven by a transcripcional control of specifi c key autophagic genes. Importantly, using a yeast-reverse one-hybrid system (Milla et al, 2012) and bioinformatics we were able to detect non -consensus GLI binding sites (GBS) in the fi rst and seventh intron of the mouse atg5. We also found multiple GBS in the human ATG5 promoter (unpublished results).…”

Neogenin1 is a sonic hedgehog target in medulloblastoma and is necessary for cell cycle progression

“…To address the molecular repertoire of Shh target genes, we and other groups have identified a number of transcriptional targets active during embryonic development and in cancer . In the cerebellum, these Shh targets include Ptc1 , Ptc2 , Gli1 , Nmyc , CyclinD1 , Bmi , Gpr153 , Foxo6 , Yap1 and Ncor2 .…”