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PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in PROP1 are the most common cause of hypopituitarism in humans; therefore, unraveling its mechanism of action is highly relevant from a therapeutic perspective. Our current understanding of the role of PROP1 in the pituitary gland is limited to the repression and activation of the pituitary transcription factor genes Hesx1 and Pou1f1, respectively. To elucidate the comprehensive PROP1-dependent gene regulatory network, we conducted genome-wide analysis of PROP1 DNA binding and effects on gene expression in mutant mice, mouse isolated stem cells and engineered mouse cell lines. We determined that PROP1 is essential for stimulating stem cells to undergo an epithelial to mesenchymal transition-like process necessary for cell migration and differentiation. Genomic profiling reveals that PROP1 binds to genes expressed in epithelial cells like Claudin 23, and to EMT inducer genes like Zeb2, Notch2 and Gli2. Zeb2 activation appears to be a key step in the EMT process. Our findings identify PROP1 as a central transcriptional component of pituitary stem cell differentiation.

Doi:: http://dx.doi.org/10.7554/eLife.14470.001

No MeSH data available.


PROP1 is a regulator of genes involved in EMT.(A) HOMER analysis of new motif enrichment within PROP1 peaks. (B) Canonical Pathway Analysis of the putative PROP1 target genes found on the ChIP-Seq. (C) Streptavidin ChIP-Seq enrichment profiles. Enrichment peaks corresponding to Gli2, Notch2, Zeb2 and Cldn23 are indicated with asterisks. (D) Quantitative ChIP assay on candidate promoters. For each gene, primers were designed to amplify region where the PROP1 peak was detected. Note that enrichment of DNA fragments was specific to BirA, Prop1Tag cells, validating the specificity of ChIP-Seq. Experiments were done using three samples for each genotype (N = 3). Data are mean + SEM. OWA and * indicates p<0.05 relative to controls. Each ChIP experiment was repeated at least twice with similar results. (E) Table containing coordinates of the peaks found on each gene and the enriched motif found by HOMER. See also Figure 9—source data 1.DOI:http://dx.doi.org/10.7554/eLife.14470.01510.7554/eLife.14470.016Figure 9—source data 1.Top list of putative PROP1 target genes that were associated with strong peaks in ChIP-Seq.Related to Figure 9.DOI:http://dx.doi.org/10.7554/eLife.14470.016
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fig9: PROP1 is a regulator of genes involved in EMT.(A) HOMER analysis of new motif enrichment within PROP1 peaks. (B) Canonical Pathway Analysis of the putative PROP1 target genes found on the ChIP-Seq. (C) Streptavidin ChIP-Seq enrichment profiles. Enrichment peaks corresponding to Gli2, Notch2, Zeb2 and Cldn23 are indicated with asterisks. (D) Quantitative ChIP assay on candidate promoters. For each gene, primers were designed to amplify region where the PROP1 peak was detected. Note that enrichment of DNA fragments was specific to BirA, Prop1Tag cells, validating the specificity of ChIP-Seq. Experiments were done using three samples for each genotype (N = 3). Data are mean + SEM. OWA and * indicates p<0.05 relative to controls. Each ChIP experiment was repeated at least twice with similar results. (E) Table containing coordinates of the peaks found on each gene and the enriched motif found by HOMER. See also Figure 9—source data 1.DOI:http://dx.doi.org/10.7554/eLife.14470.01510.7554/eLife.14470.016Figure 9—source data 1.Top list of putative PROP1 target genes that were associated with strong peaks in ChIP-Seq.Related to Figure 9.DOI:http://dx.doi.org/10.7554/eLife.14470.016

Mentions: Streptavidin (SA)-based ChIP was performed on GHFT1 cells containing BirA alone or in combination with Prop1Tag. This takes advantage of the extremely high affinity of avidin for biotinylated proteins (Viens et al., 2004). A quantitative DNA-PCR assay was used to assess enrichment for occupancy of Prop1Tag at a known PROP1 binding site in the Pou1f1 gene. To assess nonspecific background PROP1-DNA binding, a quantitative DNA-PCR assay was carried out for a site in the Hoxd10 promoter, which is not expressed in the pituitary or other structures anterior to the hindbrain (Figure 8C). The known PROP1 binding site in Pou1f1 was highly enriched relative to input and to the controls. The DNA recovered by SA-ChIP was sequenced with a HiSeq 2000 sequencing analyzer and the results analyzed with a standard bioinformatics pipeline. The enrichment profile near the Pou1f1 gene identified the known PROP1-binding sites at the early enhancer and the promoter, as well as another, more prominent site at -6 kb, demonstrating the efficacy of our approach for identification of novel PROP1-binding sites (Figure 8D and E). We carried out sequence motif searches on the ChIP-Seq peaks (center ± 200 bp) that were recovered throughout the genome using HOMER algorithms. This analysis identified a consensus sequence: TAATNNNATTA in 60% of peaks (p = 1e-1599) (Figure 9A). This consensus sequence is the first to be experimentally determined based on genome-wide data and is consistent with the paired homeodomain core consensus expected for PROP1 (Sornson et al., 1996). Interestingly, additional enriched transcription factor binding motifs were also identified in approximately 30–38% of peaks (Figure 9A). The best match for the motifs TGAGTCAT and CGATTAGC are Jun-AP1 and CRX, respectively. Because CRX (cone rod homeobox) is not expressed in the pituitary gland, the CGATTAGC motif is likely bound by a different OTX-like paired homeodomain protein that cooperates with PROP1 to direct transcriptional programs associated with target genes (Reed et al., 2008).10.7554/eLife.14470.015Figure 9.PROP1 is a regulator of genes involved in EMT.


PROP1 triggers epithelial-mesenchymal transition-like process in pituitary stem cells
PROP1 is a regulator of genes involved in EMT.(A) HOMER analysis of new motif enrichment within PROP1 peaks. (B) Canonical Pathway Analysis of the putative PROP1 target genes found on the ChIP-Seq. (C) Streptavidin ChIP-Seq enrichment profiles. Enrichment peaks corresponding to Gli2, Notch2, Zeb2 and Cldn23 are indicated with asterisks. (D) Quantitative ChIP assay on candidate promoters. For each gene, primers were designed to amplify region where the PROP1 peak was detected. Note that enrichment of DNA fragments was specific to BirA, Prop1Tag cells, validating the specificity of ChIP-Seq. Experiments were done using three samples for each genotype (N = 3). Data are mean + SEM. OWA and * indicates p<0.05 relative to controls. Each ChIP experiment was repeated at least twice with similar results. (E) Table containing coordinates of the peaks found on each gene and the enriched motif found by HOMER. See also Figure 9—source data 1.DOI:http://dx.doi.org/10.7554/eLife.14470.01510.7554/eLife.14470.016Figure 9—source data 1.Top list of putative PROP1 target genes that were associated with strong peaks in ChIP-Seq.Related to Figure 9.DOI:http://dx.doi.org/10.7554/eLife.14470.016
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fig9: PROP1 is a regulator of genes involved in EMT.(A) HOMER analysis of new motif enrichment within PROP1 peaks. (B) Canonical Pathway Analysis of the putative PROP1 target genes found on the ChIP-Seq. (C) Streptavidin ChIP-Seq enrichment profiles. Enrichment peaks corresponding to Gli2, Notch2, Zeb2 and Cldn23 are indicated with asterisks. (D) Quantitative ChIP assay on candidate promoters. For each gene, primers were designed to amplify region where the PROP1 peak was detected. Note that enrichment of DNA fragments was specific to BirA, Prop1Tag cells, validating the specificity of ChIP-Seq. Experiments were done using three samples for each genotype (N = 3). Data are mean + SEM. OWA and * indicates p<0.05 relative to controls. Each ChIP experiment was repeated at least twice with similar results. (E) Table containing coordinates of the peaks found on each gene and the enriched motif found by HOMER. See also Figure 9—source data 1.DOI:http://dx.doi.org/10.7554/eLife.14470.01510.7554/eLife.14470.016Figure 9—source data 1.Top list of putative PROP1 target genes that were associated with strong peaks in ChIP-Seq.Related to Figure 9.DOI:http://dx.doi.org/10.7554/eLife.14470.016
Mentions: Streptavidin (SA)-based ChIP was performed on GHFT1 cells containing BirA alone or in combination with Prop1Tag. This takes advantage of the extremely high affinity of avidin for biotinylated proteins (Viens et al., 2004). A quantitative DNA-PCR assay was used to assess enrichment for occupancy of Prop1Tag at a known PROP1 binding site in the Pou1f1 gene. To assess nonspecific background PROP1-DNA binding, a quantitative DNA-PCR assay was carried out for a site in the Hoxd10 promoter, which is not expressed in the pituitary or other structures anterior to the hindbrain (Figure 8C). The known PROP1 binding site in Pou1f1 was highly enriched relative to input and to the controls. The DNA recovered by SA-ChIP was sequenced with a HiSeq 2000 sequencing analyzer and the results analyzed with a standard bioinformatics pipeline. The enrichment profile near the Pou1f1 gene identified the known PROP1-binding sites at the early enhancer and the promoter, as well as another, more prominent site at -6 kb, demonstrating the efficacy of our approach for identification of novel PROP1-binding sites (Figure 8D and E). We carried out sequence motif searches on the ChIP-Seq peaks (center ± 200 bp) that were recovered throughout the genome using HOMER algorithms. This analysis identified a consensus sequence: TAATNNNATTA in 60% of peaks (p = 1e-1599) (Figure 9A). This consensus sequence is the first to be experimentally determined based on genome-wide data and is consistent with the paired homeodomain core consensus expected for PROP1 (Sornson et al., 1996). Interestingly, additional enriched transcription factor binding motifs were also identified in approximately 30–38% of peaks (Figure 9A). The best match for the motifs TGAGTCAT and CGATTAGC are Jun-AP1 and CRX, respectively. Because CRX (cone rod homeobox) is not expressed in the pituitary gland, the CGATTAGC motif is likely bound by a different OTX-like paired homeodomain protein that cooperates with PROP1 to direct transcriptional programs associated with target genes (Reed et al., 2008).10.7554/eLife.14470.015Figure 9.PROP1 is a regulator of genes involved in EMT.

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in PROP1 are the most common cause of hypopituitarism in humans; therefore, unraveling its mechanism of action is highly relevant from a therapeutic perspective. Our current understanding of the role of PROP1 in the pituitary gland is limited to the repression and activation of the pituitary transcription factor genes Hesx1 and Pou1f1, respectively. To elucidate the comprehensive PROP1-dependent gene regulatory network, we conducted genome-wide analysis of PROP1 DNA binding and effects on gene expression in mutant mice, mouse isolated stem cells and engineered mouse cell lines. We determined that PROP1 is essential for stimulating stem cells to undergo an epithelial to mesenchymal transition-like process necessary for cell migration and differentiation. Genomic profiling reveals that PROP1 binds to genes expressed in epithelial cells like Claudin 23, and to EMT inducer genes like Zeb2, Notch2 and Gli2. Zeb2 activation appears to be a key step in the EMT process. Our findings identify PROP1 as a central transcriptional component of pituitary stem cell differentiation.

Doi:: http://dx.doi.org/10.7554/eLife.14470.001

No MeSH data available.