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Genome-wide occupancy links Hoxa2 to Wnt-β-catenin signaling in mouse embryonic development.

Donaldson IJ, Amin S, Hensman JJ, Kutejova E, Rattray M, Lawrence N, Hayes A, Ward CM, Bobola N - Nucleic Acids Res. (2012)

Bottom Line: Examination of the binding targets of Hoxa2 faithfully captures the processes regulated by Hoxa2 during embryonic development; in addition, it uncovers a large cluster of potential targets involved in the Wnt-signaling pathway.In vivo examination of canonical Wnt-β-catenin signaling reveals activity specifically in Hoxa2 domain of expression, and this is undetectable in Hoxa2 mutant embryos.The comprehensive mapping of Hoxa2-binding sites provides a framework to study Hox regulatory networks in vertebrate developmental processes.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
The regulation of gene expression is central to developmental programs and largely depends on the binding of sequence-specific transcription factors with cis-regulatory elements in the genome. Hox transcription factors specify the spatial coordinates of the body axis in all animals with bilateral symmetry, but a detailed knowledge of their molecular function in instructing cell fates is lacking. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to identify Hoxa2 genomic locations in a time and space when it is actively instructing embryonic development in mouse. Our data reveals that Hoxa2 has large genome coverage and potentially regulates thousands of genes. Sequence analysis of Hoxa2-bound regions identifies high occurrence of two main classes of motifs, corresponding to Hox and Pbx-Hox recognition sequences. Examination of the binding targets of Hoxa2 faithfully captures the processes regulated by Hoxa2 during embryonic development; in addition, it uncovers a large cluster of potential targets involved in the Wnt-signaling pathway. In vivo examination of canonical Wnt-β-catenin signaling reveals activity specifically in Hoxa2 domain of expression, and this is undetectable in Hoxa2 mutant embryos. The comprehensive mapping of Hoxa2-binding sites provides a framework to study Hox regulatory networks in vertebrate developmental processes.

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Intersection between ChIP-seq data and gene-expression analysis. (A) Venn diagram showing the overlap between the genes associated with Hoxa2-bound regions (blue) and those revealed by expression arrays. (B) Average number of Hoxa2-bound regions per gene in the entire ChIP-seq dataset (grey), Hoxa2-bound regions associated with down- (green) and up- (red) regulated genes. On average down- and upregulated genes are associated with a higher number of Hoxa2-bound regions (P = 2.2e–16 and 0.001, respectively). (C) Distribution of Hoxa2 summit regions relative to Reference Sequence (RefSeq) gene structures: promoter (10-kb upstream of the TSS), transcript, and downstream (10-kb downstream of the TTS). Hoxa2-summit regions associated with down and upregulated genes occur more frequently in promoters compared to the entire ChIP-seq dataset (P = 0.05 and 0.005, respectively); the color code is as in (B). (D) Analysis of the distribution of Hox and Pbx–Hox motifs in Hoxa2-summit regions associated with down- and upregulated genes [color code as in (B)]. TAAT is significantly enriched in Hoxa2-bound regions associated with upregulated genes. The numbers on top of each column refer to the percentage of Hoxa2-summit regions containing the motif. (E) Distribution of Hoxa2-bound regions containing Hox (red), Hox-Pbx (purple), Hox and Hox-Pbx (blue), and no motif (turquoise) in regulated genes. Numbers indicate the contribution of each class to the total of Hoxa2-bound regions associated to upregulated genes (inner ring), downregulated genes (middle ring) and the entire Hoxa2 data set (outer ring). (F) Extension of the TAAT motif in down- and upregulated genes. Plots of the density of each nucleotide around each TAAT motif contained in Hoxa2-summit regions associated with up and downregulated genes.
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gkr1240-F4: Intersection between ChIP-seq data and gene-expression analysis. (A) Venn diagram showing the overlap between the genes associated with Hoxa2-bound regions (blue) and those revealed by expression arrays. (B) Average number of Hoxa2-bound regions per gene in the entire ChIP-seq dataset (grey), Hoxa2-bound regions associated with down- (green) and up- (red) regulated genes. On average down- and upregulated genes are associated with a higher number of Hoxa2-bound regions (P = 2.2e–16 and 0.001, respectively). (C) Distribution of Hoxa2 summit regions relative to Reference Sequence (RefSeq) gene structures: promoter (10-kb upstream of the TSS), transcript, and downstream (10-kb downstream of the TTS). Hoxa2-summit regions associated with down and upregulated genes occur more frequently in promoters compared to the entire ChIP-seq dataset (P = 0.05 and 0.005, respectively); the color code is as in (B). (D) Analysis of the distribution of Hox and Pbx–Hox motifs in Hoxa2-summit regions associated with down- and upregulated genes [color code as in (B)]. TAAT is significantly enriched in Hoxa2-bound regions associated with upregulated genes. The numbers on top of each column refer to the percentage of Hoxa2-summit regions containing the motif. (E) Distribution of Hoxa2-bound regions containing Hox (red), Hox-Pbx (purple), Hox and Hox-Pbx (blue), and no motif (turquoise) in regulated genes. Numbers indicate the contribution of each class to the total of Hoxa2-bound regions associated to upregulated genes (inner ring), downregulated genes (middle ring) and the entire Hoxa2 data set (outer ring). (F) Extension of the TAAT motif in down- and upregulated genes. Plots of the density of each nucleotide around each TAAT motif contained in Hoxa2-summit regions associated with up and downregulated genes.

Mentions: To assess the effect of Hoxa2 DNA binding on gene expression, we used microarrays in the same embryonic populations as those interrogated by ChIP-seq. By comparing E11.5 wild-type and Hoxa2- mutant branchial arches, we identified 489 differentially expressed genes (fold difference > 1.3; P < 0.05) of which 359 and 130 were down and upregulated in mutant embryos, respectively. If Hoxa2-bound regions are functionally active, we should expect a marked enrichment of ChIP-seq genes in the genes dysregulated in Hoxa2 mutant branchial arches. We found that 48% (237/489) of Hoxa2-regulated genes had at least one Hoxa2-bound region assigned to them, which represents a highly significant enrichment compared with all genes (P = 1.1e–106) (Figure 4A). Upon separating Hoxa2-regulated genes into down and upregulated, we found that 50% and 42% of the genes down and upregulated in the Hoxa2 mutant, respectively, had at least one Hoxa2-bound region assigned to them (P = 8.9e–87 and 4.2e–23, respectively) (Supplementary Table S6). These results suggest that nearly half of the genes that are dysregulated in the Hoxa2 mutant are directly regulated (either positively or negatively) by Hoxa2.Figure 4.


Genome-wide occupancy links Hoxa2 to Wnt-β-catenin signaling in mouse embryonic development.

Donaldson IJ, Amin S, Hensman JJ, Kutejova E, Rattray M, Lawrence N, Hayes A, Ward CM, Bobola N - Nucleic Acids Res. (2012)

Intersection between ChIP-seq data and gene-expression analysis. (A) Venn diagram showing the overlap between the genes associated with Hoxa2-bound regions (blue) and those revealed by expression arrays. (B) Average number of Hoxa2-bound regions per gene in the entire ChIP-seq dataset (grey), Hoxa2-bound regions associated with down- (green) and up- (red) regulated genes. On average down- and upregulated genes are associated with a higher number of Hoxa2-bound regions (P = 2.2e–16 and 0.001, respectively). (C) Distribution of Hoxa2 summit regions relative to Reference Sequence (RefSeq) gene structures: promoter (10-kb upstream of the TSS), transcript, and downstream (10-kb downstream of the TTS). Hoxa2-summit regions associated with down and upregulated genes occur more frequently in promoters compared to the entire ChIP-seq dataset (P = 0.05 and 0.005, respectively); the color code is as in (B). (D) Analysis of the distribution of Hox and Pbx–Hox motifs in Hoxa2-summit regions associated with down- and upregulated genes [color code as in (B)]. TAAT is significantly enriched in Hoxa2-bound regions associated with upregulated genes. The numbers on top of each column refer to the percentage of Hoxa2-summit regions containing the motif. (E) Distribution of Hoxa2-bound regions containing Hox (red), Hox-Pbx (purple), Hox and Hox-Pbx (blue), and no motif (turquoise) in regulated genes. Numbers indicate the contribution of each class to the total of Hoxa2-bound regions associated to upregulated genes (inner ring), downregulated genes (middle ring) and the entire Hoxa2 data set (outer ring). (F) Extension of the TAAT motif in down- and upregulated genes. Plots of the density of each nucleotide around each TAAT motif contained in Hoxa2-summit regions associated with up and downregulated genes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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gkr1240-F4: Intersection between ChIP-seq data and gene-expression analysis. (A) Venn diagram showing the overlap between the genes associated with Hoxa2-bound regions (blue) and those revealed by expression arrays. (B) Average number of Hoxa2-bound regions per gene in the entire ChIP-seq dataset (grey), Hoxa2-bound regions associated with down- (green) and up- (red) regulated genes. On average down- and upregulated genes are associated with a higher number of Hoxa2-bound regions (P = 2.2e–16 and 0.001, respectively). (C) Distribution of Hoxa2 summit regions relative to Reference Sequence (RefSeq) gene structures: promoter (10-kb upstream of the TSS), transcript, and downstream (10-kb downstream of the TTS). Hoxa2-summit regions associated with down and upregulated genes occur more frequently in promoters compared to the entire ChIP-seq dataset (P = 0.05 and 0.005, respectively); the color code is as in (B). (D) Analysis of the distribution of Hox and Pbx–Hox motifs in Hoxa2-summit regions associated with down- and upregulated genes [color code as in (B)]. TAAT is significantly enriched in Hoxa2-bound regions associated with upregulated genes. The numbers on top of each column refer to the percentage of Hoxa2-summit regions containing the motif. (E) Distribution of Hoxa2-bound regions containing Hox (red), Hox-Pbx (purple), Hox and Hox-Pbx (blue), and no motif (turquoise) in regulated genes. Numbers indicate the contribution of each class to the total of Hoxa2-bound regions associated to upregulated genes (inner ring), downregulated genes (middle ring) and the entire Hoxa2 data set (outer ring). (F) Extension of the TAAT motif in down- and upregulated genes. Plots of the density of each nucleotide around each TAAT motif contained in Hoxa2-summit regions associated with up and downregulated genes.
Mentions: To assess the effect of Hoxa2 DNA binding on gene expression, we used microarrays in the same embryonic populations as those interrogated by ChIP-seq. By comparing E11.5 wild-type and Hoxa2- mutant branchial arches, we identified 489 differentially expressed genes (fold difference > 1.3; P < 0.05) of which 359 and 130 were down and upregulated in mutant embryos, respectively. If Hoxa2-bound regions are functionally active, we should expect a marked enrichment of ChIP-seq genes in the genes dysregulated in Hoxa2 mutant branchial arches. We found that 48% (237/489) of Hoxa2-regulated genes had at least one Hoxa2-bound region assigned to them, which represents a highly significant enrichment compared with all genes (P = 1.1e–106) (Figure 4A). Upon separating Hoxa2-regulated genes into down and upregulated, we found that 50% and 42% of the genes down and upregulated in the Hoxa2 mutant, respectively, had at least one Hoxa2-bound region assigned to them (P = 8.9e–87 and 4.2e–23, respectively) (Supplementary Table S6). These results suggest that nearly half of the genes that are dysregulated in the Hoxa2 mutant are directly regulated (either positively or negatively) by Hoxa2.Figure 4.

Bottom Line: Examination of the binding targets of Hoxa2 faithfully captures the processes regulated by Hoxa2 during embryonic development; in addition, it uncovers a large cluster of potential targets involved in the Wnt-signaling pathway.In vivo examination of canonical Wnt-β-catenin signaling reveals activity specifically in Hoxa2 domain of expression, and this is undetectable in Hoxa2 mutant embryos.The comprehensive mapping of Hoxa2-binding sites provides a framework to study Hox regulatory networks in vertebrate developmental processes.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
The regulation of gene expression is central to developmental programs and largely depends on the binding of sequence-specific transcription factors with cis-regulatory elements in the genome. Hox transcription factors specify the spatial coordinates of the body axis in all animals with bilateral symmetry, but a detailed knowledge of their molecular function in instructing cell fates is lacking. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to identify Hoxa2 genomic locations in a time and space when it is actively instructing embryonic development in mouse. Our data reveals that Hoxa2 has large genome coverage and potentially regulates thousands of genes. Sequence analysis of Hoxa2-bound regions identifies high occurrence of two main classes of motifs, corresponding to Hox and Pbx-Hox recognition sequences. Examination of the binding targets of Hoxa2 faithfully captures the processes regulated by Hoxa2 during embryonic development; in addition, it uncovers a large cluster of potential targets involved in the Wnt-signaling pathway. In vivo examination of canonical Wnt-β-catenin signaling reveals activity specifically in Hoxa2 domain of expression, and this is undetectable in Hoxa2 mutant embryos. The comprehensive mapping of Hoxa2-binding sites provides a framework to study Hox regulatory networks in vertebrate developmental processes.

Show MeSH