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Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules.

Nakamura Y, de Paiva Alves E, Veenstra GJ, Hoppler S - Development (2016)

Bottom Line: Here, a direct comparison of genome-wide occupancy of β-catenin with a stage-matched Wnt-regulated transcriptome reveals that only a subset of β-catenin-bound genomic loci are transcriptionally regulated by Wnt signalling.The transcriptional response to Wnt signalling depends on additional mechanisms, such as BMP or FGF signalling for the particular genes we investigated, which do not influence β-catenin recruitment.Chromatin association of β-catenin, even to functional Wnt-response elements, can no longer be considered a proxy for identifying transcriptionally Wnt-regulated genes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, Foresterhill Health Campus, University of Aberdeen, Aberdeen AB25 2ZD, UK.

No MeSH data available.


Related in: MedlinePlus

β-catenin ChIP-seq analysis of Xenopus early gastrulae. (A) Experimental design of β-catenin ChIP-seq analysis. Early gastrulae were collected and fixed. Following chromatin shearing, β-catenin antibodies were used to selectively precipitate DNA fragments bound by β-catenin-containing protein complexes. Subsequently, the precipitated DNA fragments were sequenced. (B) Genome view of example β-catenin target gene hoxd1. Note the clear β-catenin ChIP-seq peaks (β-peaks) downstream (to the left) of the hoxd1 locus. (C) Scatter plot combining peak calling analysis by SPP [considering signal strength, applying false discovery rate (FDR)≤0.1] and MACS2 (considering fold change, applying P≤0.01) software, with black dots indicating 10,638 β-peaks reproducibly called [applying an irreproducible discovery rate (IDR)≤0.01]. (D,E) β-peaks are associated with sequences throughout the genome (D) but enriched close to and just upstream (putative promoter) of the transcriptional start site (TSS) of nearby genes (E; analysed in 500 bp bins). Pie chart (D) shows the percentage of β-peaks according to their location relative to TSS (within 1 kb, 1-5 kb, 5-10 kb, 10-50 kb, over 50 kb upstream or downstream of TSS). (F) Heat map illustrating genome-wide association of β-peaks with histone modifications and transcription co-factor binding sites indicative of cis-regulatory modules (CRMs; such as promoters and enhancers) in patterns that can be clustered into ten groups. Each horizontal line represents the 5 kb downstream and upstream regions of ChIP-seq data around a β-peak. (G) Enriched motifs from de novo motif search of sequences under β-peaks. Note the identification of consensus TCF/LEF binding but also other known transcription factor binding motifs. Statistical significance (e-values) and the number of β-peaks are indicated below each motif logo. The analysis of motif distribution shows central enrichment of motifs within β-peak regions (500 bp window).
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DEV131664F1: β-catenin ChIP-seq analysis of Xenopus early gastrulae. (A) Experimental design of β-catenin ChIP-seq analysis. Early gastrulae were collected and fixed. Following chromatin shearing, β-catenin antibodies were used to selectively precipitate DNA fragments bound by β-catenin-containing protein complexes. Subsequently, the precipitated DNA fragments were sequenced. (B) Genome view of example β-catenin target gene hoxd1. Note the clear β-catenin ChIP-seq peaks (β-peaks) downstream (to the left) of the hoxd1 locus. (C) Scatter plot combining peak calling analysis by SPP [considering signal strength, applying false discovery rate (FDR)≤0.1] and MACS2 (considering fold change, applying P≤0.01) software, with black dots indicating 10,638 β-peaks reproducibly called [applying an irreproducible discovery rate (IDR)≤0.01]. (D,E) β-peaks are associated with sequences throughout the genome (D) but enriched close to and just upstream (putative promoter) of the transcriptional start site (TSS) of nearby genes (E; analysed in 500 bp bins). Pie chart (D) shows the percentage of β-peaks according to their location relative to TSS (within 1 kb, 1-5 kb, 5-10 kb, 10-50 kb, over 50 kb upstream or downstream of TSS). (F) Heat map illustrating genome-wide association of β-peaks with histone modifications and transcription co-factor binding sites indicative of cis-regulatory modules (CRMs; such as promoters and enhancers) in patterns that can be clustered into ten groups. Each horizontal line represents the 5 kb downstream and upstream regions of ChIP-seq data around a β-peak. (G) Enriched motifs from de novo motif search of sequences under β-peaks. Note the identification of consensus TCF/LEF binding but also other known transcription factor binding motifs. Statistical significance (e-values) and the number of β-peaks are indicated below each motif logo. The analysis of motif distribution shows central enrichment of motifs within β-peak regions (500 bp window).

Mentions: We developed a reliable β-catenin ChIP protocol for analysis at the early gastrula stage (stage 10.25, Fig. 1A, Fig. S2) by optimising first chromatin shearing conditions for fragments of ∼200 bp (Fig. S2A), then the immunoprecipitation of chromatin-associated β-catenin protein with two different β-catenin antibodies, as well as IgG as a negative control (see Materials and Methods). Specific binding of β-catenin by the antibodies was validated by western blotting and also by β-catenin ChIP-qPCR (Fig. S2B-D). In the ChIP-qPCR validation, we analysed known WREs in genes known to be Wnt regulated at this stage [hoxd1 (Janssens et al., 2010) and msgn1 (Wang et al., 2007)] as positive controls, and genomic regions not containing WREs (from odc1 and hoxd1) as negative controls. ChIP DNA samples and input control DNA samples were each pooled from three validated ChIP experiments and sequenced.Fig. 1.


Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules.

Nakamura Y, de Paiva Alves E, Veenstra GJ, Hoppler S - Development (2016)

β-catenin ChIP-seq analysis of Xenopus early gastrulae. (A) Experimental design of β-catenin ChIP-seq analysis. Early gastrulae were collected and fixed. Following chromatin shearing, β-catenin antibodies were used to selectively precipitate DNA fragments bound by β-catenin-containing protein complexes. Subsequently, the precipitated DNA fragments were sequenced. (B) Genome view of example β-catenin target gene hoxd1. Note the clear β-catenin ChIP-seq peaks (β-peaks) downstream (to the left) of the hoxd1 locus. (C) Scatter plot combining peak calling analysis by SPP [considering signal strength, applying false discovery rate (FDR)≤0.1] and MACS2 (considering fold change, applying P≤0.01) software, with black dots indicating 10,638 β-peaks reproducibly called [applying an irreproducible discovery rate (IDR)≤0.01]. (D,E) β-peaks are associated with sequences throughout the genome (D) but enriched close to and just upstream (putative promoter) of the transcriptional start site (TSS) of nearby genes (E; analysed in 500 bp bins). Pie chart (D) shows the percentage of β-peaks according to their location relative to TSS (within 1 kb, 1-5 kb, 5-10 kb, 10-50 kb, over 50 kb upstream or downstream of TSS). (F) Heat map illustrating genome-wide association of β-peaks with histone modifications and transcription co-factor binding sites indicative of cis-regulatory modules (CRMs; such as promoters and enhancers) in patterns that can be clustered into ten groups. Each horizontal line represents the 5 kb downstream and upstream regions of ChIP-seq data around a β-peak. (G) Enriched motifs from de novo motif search of sequences under β-peaks. Note the identification of consensus TCF/LEF binding but also other known transcription factor binding motifs. Statistical significance (e-values) and the number of β-peaks are indicated below each motif logo. The analysis of motif distribution shows central enrichment of motifs within β-peak regions (500 bp window).
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Related In: Results  -  Collection

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DEV131664F1: β-catenin ChIP-seq analysis of Xenopus early gastrulae. (A) Experimental design of β-catenin ChIP-seq analysis. Early gastrulae were collected and fixed. Following chromatin shearing, β-catenin antibodies were used to selectively precipitate DNA fragments bound by β-catenin-containing protein complexes. Subsequently, the precipitated DNA fragments were sequenced. (B) Genome view of example β-catenin target gene hoxd1. Note the clear β-catenin ChIP-seq peaks (β-peaks) downstream (to the left) of the hoxd1 locus. (C) Scatter plot combining peak calling analysis by SPP [considering signal strength, applying false discovery rate (FDR)≤0.1] and MACS2 (considering fold change, applying P≤0.01) software, with black dots indicating 10,638 β-peaks reproducibly called [applying an irreproducible discovery rate (IDR)≤0.01]. (D,E) β-peaks are associated with sequences throughout the genome (D) but enriched close to and just upstream (putative promoter) of the transcriptional start site (TSS) of nearby genes (E; analysed in 500 bp bins). Pie chart (D) shows the percentage of β-peaks according to their location relative to TSS (within 1 kb, 1-5 kb, 5-10 kb, 10-50 kb, over 50 kb upstream or downstream of TSS). (F) Heat map illustrating genome-wide association of β-peaks with histone modifications and transcription co-factor binding sites indicative of cis-regulatory modules (CRMs; such as promoters and enhancers) in patterns that can be clustered into ten groups. Each horizontal line represents the 5 kb downstream and upstream regions of ChIP-seq data around a β-peak. (G) Enriched motifs from de novo motif search of sequences under β-peaks. Note the identification of consensus TCF/LEF binding but also other known transcription factor binding motifs. Statistical significance (e-values) and the number of β-peaks are indicated below each motif logo. The analysis of motif distribution shows central enrichment of motifs within β-peak regions (500 bp window).
Mentions: We developed a reliable β-catenin ChIP protocol for analysis at the early gastrula stage (stage 10.25, Fig. 1A, Fig. S2) by optimising first chromatin shearing conditions for fragments of ∼200 bp (Fig. S2A), then the immunoprecipitation of chromatin-associated β-catenin protein with two different β-catenin antibodies, as well as IgG as a negative control (see Materials and Methods). Specific binding of β-catenin by the antibodies was validated by western blotting and also by β-catenin ChIP-qPCR (Fig. S2B-D). In the ChIP-qPCR validation, we analysed known WREs in genes known to be Wnt regulated at this stage [hoxd1 (Janssens et al., 2010) and msgn1 (Wang et al., 2007)] as positive controls, and genomic regions not containing WREs (from odc1 and hoxd1) as negative controls. ChIP DNA samples and input control DNA samples were each pooled from three validated ChIP experiments and sequenced.Fig. 1.

Bottom Line: Here, a direct comparison of genome-wide occupancy of β-catenin with a stage-matched Wnt-regulated transcriptome reveals that only a subset of β-catenin-bound genomic loci are transcriptionally regulated by Wnt signalling.The transcriptional response to Wnt signalling depends on additional mechanisms, such as BMP or FGF signalling for the particular genes we investigated, which do not influence β-catenin recruitment.Chromatin association of β-catenin, even to functional Wnt-response elements, can no longer be considered a proxy for identifying transcriptionally Wnt-regulated genes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, Foresterhill Health Campus, University of Aberdeen, Aberdeen AB25 2ZD, UK.

No MeSH data available.


Related in: MedlinePlus