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

Milla LA, Cortés CR, Hodar C, Oñate MG, Cambiazo V, Burgess SM, Palma V - BMC Genomics (2012)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Sciences, Universidad de Chile, Santiago, Chile.

ABSTRACT

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.

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

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Pharmacological gain of function of the Shh/Gli pathway activates transcription of novel target genes. A) C3H10T1/2 cells were cultured for 48 h in 10 μM pur or its control (vehicle DMSO) and processed for qPCR. c-myc shows significant differences in mRNA levels, whereas sfrp2 does not. ptc 1 is shown as a positive control. B) neo1 mRNA levels are increased after the same 48 h treatment of C3H10T1/2 cells, graph showed separately due to use of TaqMan probes.
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Figure 3: Pharmacological gain of function of the Shh/Gli pathway activates transcription of novel target genes. A) C3H10T1/2 cells were cultured for 48 h in 10 μM pur or its control (vehicle DMSO) and processed for qPCR. c-myc shows significant differences in mRNA levels, whereas sfrp2 does not. ptc 1 is shown as a positive control. B) neo1 mRNA levels are increased after the same 48 h treatment of C3H10T1/2 cells, graph showed separately due to use of TaqMan probes.

Mentions: All the selected candidate genes showed reductions in their mRNA levels after 24-hour cyc treatments except glra (Figure 2). To corroborate Hh regulation we performed gain of function experiments using purmorphamine (pur), a proven Smoothened agonist [15] for a subset of candidates, namely neo1, c-myc and sfrp2 based on their implication in developmental processes. Cells were treated for 24 hours and 48 hours before RNA extraction and qPCR processing. ptc1, a well known Hh transcriptional readout was used as positive control. For both c-myc and neo1 significant changes could be observed after 24-hours of treatment, nevertheless the increases was less pronounced in comparison to ptc1. In order to proof that the effects of Hh signaling were direct we included experiments with cycloheximide (CHX), obtaining similar results (Additional File 3). Increase in transcript levels of the selected target genes became clearly evident after 48 hours of treatment. Sfrp2 did not show any changes in expression for either time point (Figure 3). sfrp2 invariant mRNA levels possibly points to a context dependent case of regulation. Expression changes were calculated related to vehicle treatments (DMSO).


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

Milla LA, Cortés CR, Hodar C, Oñate MG, Cambiazo V, Burgess SM, Palma V - BMC Genomics (2012)

Pharmacological gain of function of the Shh/Gli pathway activates transcription of novel target genes. A) C3H10T1/2 cells were cultured for 48 h in 10 μM pur or its control (vehicle DMSO) and processed for qPCR. c-myc shows significant differences in mRNA levels, whereas sfrp2 does not. ptc 1 is shown as a positive control. B) neo1 mRNA levels are increased after the same 48 h treatment of C3H10T1/2 cells, graph showed separately due to use of TaqMan probes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285088&req=5

Figure 3: Pharmacological gain of function of the Shh/Gli pathway activates transcription of novel target genes. A) C3H10T1/2 cells were cultured for 48 h in 10 μM pur or its control (vehicle DMSO) and processed for qPCR. c-myc shows significant differences in mRNA levels, whereas sfrp2 does not. ptc 1 is shown as a positive control. B) neo1 mRNA levels are increased after the same 48 h treatment of C3H10T1/2 cells, graph showed separately due to use of TaqMan probes.
Mentions: All the selected candidate genes showed reductions in their mRNA levels after 24-hour cyc treatments except glra (Figure 2). To corroborate Hh regulation we performed gain of function experiments using purmorphamine (pur), a proven Smoothened agonist [15] for a subset of candidates, namely neo1, c-myc and sfrp2 based on their implication in developmental processes. Cells were treated for 24 hours and 48 hours before RNA extraction and qPCR processing. ptc1, a well known Hh transcriptional readout was used as positive control. For both c-myc and neo1 significant changes could be observed after 24-hours of treatment, nevertheless the increases was less pronounced in comparison to ptc1. In order to proof that the effects of Hh signaling were direct we included experiments with cycloheximide (CHX), obtaining similar results (Additional File 3). Increase in transcript levels of the selected target genes became clearly evident after 48 hours of treatment. Sfrp2 did not show any changes in expression for either time point (Figure 3). sfrp2 invariant mRNA levels possibly points to a context dependent case of regulation. Expression changes were calculated related to vehicle treatments (DMSO).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Sciences, Universidad de Chile, Santiago, Chile.

ABSTRACT

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.

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

Show MeSH