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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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Detection of Hoxa2-ChIP-seq peaks in the IIBA. (A) Whole-mount ISH on E11.5 wild-type embryos, using Hoxa2 probe (left). Magnification of the same picture shows the branchial area (right); the dotted line delimitates the IIBA. (B) ChIP–qPCR validation of FDR < 10 Hoxa2-bound regions. Fold enrichment of Hoxa2 over IgG negative control antibody (Neg Ab) is shown for each Hoxa2-bound region. Values represent the average of duplicate samples and are adjusted to the average enrichment of Pou6f2 from two independent experiments. Itih4 is a negative control (unbound region). For each sample the standard error of the mean is indicated by error bars. The dotted line represents a threshold of 2-fold or higher significant enrichment. (C) Distribution of Hoxa2 summit regions relative to Reference Sequence (RefSeq) gene structures. The pie charts indicate the proportion of reads for each gene structure; the corresponding numeric values are included. For each pie chart, an enlargement of the boxed area is shown above. CEAS detects significant enrichment of proximal promoter in Hoxa2-bound regions compared to random regions (asterisks).
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gkr1240-F1: Detection of Hoxa2-ChIP-seq peaks in the IIBA. (A) Whole-mount ISH on E11.5 wild-type embryos, using Hoxa2 probe (left). Magnification of the same picture shows the branchial area (right); the dotted line delimitates the IIBA. (B) ChIP–qPCR validation of FDR < 10 Hoxa2-bound regions. Fold enrichment of Hoxa2 over IgG negative control antibody (Neg Ab) is shown for each Hoxa2-bound region. Values represent the average of duplicate samples and are adjusted to the average enrichment of Pou6f2 from two independent experiments. Itih4 is a negative control (unbound region). For each sample the standard error of the mean is indicated by error bars. The dotted line represents a threshold of 2-fold or higher significant enrichment. (C) Distribution of Hoxa2 summit regions relative to Reference Sequence (RefSeq) gene structures. The pie charts indicate the proportion of reads for each gene structure; the corresponding numeric values are included. For each pie chart, an enlargement of the boxed area is shown above. CEAS detects significant enrichment of proximal promoter in Hoxa2-bound regions compared to random regions (asterisks).

Mentions: The second branchial arch (IIBA) is the embryonic area most affected by inactivation of Hoxa2 in mouse. In the absence of Hoxa2, the IIBA generates skeletal elements typical of the more anterior first branchial arch (IBA). This abnormal skeletal development disrupts formation of the middle ear. Hoxa2 expression appears in the IIBA at E8.5 (28), following migration of Hoxa2-positive cranial neural crest cells from the hindbrain, and it is maintained throughout development of the IIBA. Hoxa2-positive cells are still detected in IIBA derivatives at later stages (E13.0). To define the genomic targets of Hoxa2, we performed Hoxa2 ChIP-seq on IIBAs collected at E11.5. Wild-type and Hoxa2 mutant embryos are visually indistinguishable at this stage; shortly after E11.5, the wild-type IIBA undergoes profound morphological changes to produce its final derivatives. Most importantly, Hoxa2 is strongly expressed in E11.5 IIBAs and it is still required for IIBA development at this stage (29) (Figure 1A).Figure 1.


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)

Detection of Hoxa2-ChIP-seq peaks in the IIBA. (A) Whole-mount ISH on E11.5 wild-type embryos, using Hoxa2 probe (left). Magnification of the same picture shows the branchial area (right); the dotted line delimitates the IIBA. (B) ChIP–qPCR validation of FDR < 10 Hoxa2-bound regions. Fold enrichment of Hoxa2 over IgG negative control antibody (Neg Ab) is shown for each Hoxa2-bound region. Values represent the average of duplicate samples and are adjusted to the average enrichment of Pou6f2 from two independent experiments. Itih4 is a negative control (unbound region). For each sample the standard error of the mean is indicated by error bars. The dotted line represents a threshold of 2-fold or higher significant enrichment. (C) Distribution of Hoxa2 summit regions relative to Reference Sequence (RefSeq) gene structures. The pie charts indicate the proportion of reads for each gene structure; the corresponding numeric values are included. For each pie chart, an enlargement of the boxed area is shown above. CEAS detects significant enrichment of proximal promoter in Hoxa2-bound regions compared to random regions (asterisks).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3351182&req=5

gkr1240-F1: Detection of Hoxa2-ChIP-seq peaks in the IIBA. (A) Whole-mount ISH on E11.5 wild-type embryos, using Hoxa2 probe (left). Magnification of the same picture shows the branchial area (right); the dotted line delimitates the IIBA. (B) ChIP–qPCR validation of FDR < 10 Hoxa2-bound regions. Fold enrichment of Hoxa2 over IgG negative control antibody (Neg Ab) is shown for each Hoxa2-bound region. Values represent the average of duplicate samples and are adjusted to the average enrichment of Pou6f2 from two independent experiments. Itih4 is a negative control (unbound region). For each sample the standard error of the mean is indicated by error bars. The dotted line represents a threshold of 2-fold or higher significant enrichment. (C) Distribution of Hoxa2 summit regions relative to Reference Sequence (RefSeq) gene structures. The pie charts indicate the proportion of reads for each gene structure; the corresponding numeric values are included. For each pie chart, an enlargement of the boxed area is shown above. CEAS detects significant enrichment of proximal promoter in Hoxa2-bound regions compared to random regions (asterisks).
Mentions: The second branchial arch (IIBA) is the embryonic area most affected by inactivation of Hoxa2 in mouse. In the absence of Hoxa2, the IIBA generates skeletal elements typical of the more anterior first branchial arch (IBA). This abnormal skeletal development disrupts formation of the middle ear. Hoxa2 expression appears in the IIBA at E8.5 (28), following migration of Hoxa2-positive cranial neural crest cells from the hindbrain, and it is maintained throughout development of the IIBA. Hoxa2-positive cells are still detected in IIBA derivatives at later stages (E13.0). To define the genomic targets of Hoxa2, we performed Hoxa2 ChIP-seq on IIBAs collected at E11.5. Wild-type and Hoxa2 mutant embryos are visually indistinguishable at this stage; shortly after E11.5, the wild-type IIBA undergoes profound morphological changes to produce its final derivatives. Most importantly, Hoxa2 is strongly expressed in E11.5 IIBAs and it is still required for IIBA development at this stage (29) (Figure 1A).Figure 1.

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
Related in: MedlinePlus