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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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Hoxa2 is upstream of Wnt–β-catenin signaling. (A) Validation of binding regions linked to genes in the Wnt-signaling pathway by ChIP–qPCR. Percent Input is shown for each Hoxa2-bound region and the corresponding negative antibody control (Neg Ab). Values represent the average of duplicate samples. Art3 is a negative control gene (unbound region). (B) The differentially expressed genes (from the list in Supplementary Table S6) linked to Wnt GO categories are shown together with their corresponding fold changes. (C–F) Expression of Fzd4 and Wnt5a in wild-type (C, E) and mutant (D, F) embryos. In situ hybridization on whole mount E10.5 wild-type and Hoxa2 mutant (C, D) and E12.5 wild-type and Hoxa2 mutant (E, F) using Fzd4 (C, D) and Wnt5a (E, F) probes. Arrow in (C) and (D), and arrowhead in (E) and (F), indicate the embryonic area where Fzd4 and Wnt5a are downregulated in the mutant, respectively. (G and H). Whole mount lacZ staining of E11.5 BAT-Gal (G) and BAT-Gal; Hoxa2−/− (H) embryos. (G) High Wnt canonical activity is detected in the IIBA of E11.0 BAT-Gal transgenic embryos. (H) Wnt canonical activity is lost in the IIBA (arrow) in the absence of Hoxa2. Dotted line separates IIBA from IBA. (I and J) BAT-Gal (I) and wild-type (J) E13.0 embryos. (I) Wnt canonical activity is mostly confined to the pinna of the outer ear (arrowhead). (J) In situ hybridization using Hoxa2 probe shows the pinna remains Hoxa2 positive at later stages (arrowhead). Ea, ear; e, eye.
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gkr1240-F5: Hoxa2 is upstream of Wnt–β-catenin signaling. (A) Validation of binding regions linked to genes in the Wnt-signaling pathway by ChIP–qPCR. Percent Input is shown for each Hoxa2-bound region and the corresponding negative antibody control (Neg Ab). Values represent the average of duplicate samples. Art3 is a negative control gene (unbound region). (B) The differentially expressed genes (from the list in Supplementary Table S6) linked to Wnt GO categories are shown together with their corresponding fold changes. (C–F) Expression of Fzd4 and Wnt5a in wild-type (C, E) and mutant (D, F) embryos. In situ hybridization on whole mount E10.5 wild-type and Hoxa2 mutant (C, D) and E12.5 wild-type and Hoxa2 mutant (E, F) using Fzd4 (C, D) and Wnt5a (E, F) probes. Arrow in (C) and (D), and arrowhead in (E) and (F), indicate the embryonic area where Fzd4 and Wnt5a are downregulated in the mutant, respectively. (G and H). Whole mount lacZ staining of E11.5 BAT-Gal (G) and BAT-Gal; Hoxa2−/− (H) embryos. (G) High Wnt canonical activity is detected in the IIBA of E11.0 BAT-Gal transgenic embryos. (H) Wnt canonical activity is lost in the IIBA (arrow) in the absence of Hoxa2. Dotted line separates IIBA from IBA. (I and J) BAT-Gal (I) and wild-type (J) E13.0 embryos. (I) Wnt canonical activity is mostly confined to the pinna of the outer ear (arrowhead). (J) In situ hybridization using Hoxa2 probe shows the pinna remains Hoxa2 positive at later stages (arrowhead). Ea, ear; e, eye.

Mentions: ChIP–qPCR was performed on a Bio-Rad Chromo4 Real-time PCR system using SYBR green (Dynamo) according to the manufacturer's instructions (50 µl PCR reaction for each primer set was performed using the following program: Step 1: 95°C 15 min; Step 2: 95°C 30 s, 55°C 1 min, 72°C 30 s, repeat 49 times; Step 3: melting curve from 55°C to 95°C). Results were analyzed using Bio-Rad Opticon Monitor 3.1.32. Fold enrichment of each bound region was calculated over a negative antibody control relative to input. In Figure 5A, values are expressed as percent input for each bound region and the corresponding negative antibody control. Primer sequences are listed in Supplementary Table S7.


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)

Hoxa2 is upstream of Wnt–β-catenin signaling. (A) Validation of binding regions linked to genes in the Wnt-signaling pathway by ChIP–qPCR. Percent Input is shown for each Hoxa2-bound region and the corresponding negative antibody control (Neg Ab). Values represent the average of duplicate samples. Art3 is a negative control gene (unbound region). (B) The differentially expressed genes (from the list in Supplementary Table S6) linked to Wnt GO categories are shown together with their corresponding fold changes. (C–F) Expression of Fzd4 and Wnt5a in wild-type (C, E) and mutant (D, F) embryos. In situ hybridization on whole mount E10.5 wild-type and Hoxa2 mutant (C, D) and E12.5 wild-type and Hoxa2 mutant (E, F) using Fzd4 (C, D) and Wnt5a (E, F) probes. Arrow in (C) and (D), and arrowhead in (E) and (F), indicate the embryonic area where Fzd4 and Wnt5a are downregulated in the mutant, respectively. (G and H). Whole mount lacZ staining of E11.5 BAT-Gal (G) and BAT-Gal; Hoxa2−/− (H) embryos. (G) High Wnt canonical activity is detected in the IIBA of E11.0 BAT-Gal transgenic embryos. (H) Wnt canonical activity is lost in the IIBA (arrow) in the absence of Hoxa2. Dotted line separates IIBA from IBA. (I and J) BAT-Gal (I) and wild-type (J) E13.0 embryos. (I) Wnt canonical activity is mostly confined to the pinna of the outer ear (arrowhead). (J) In situ hybridization using Hoxa2 probe shows the pinna remains Hoxa2 positive at later stages (arrowhead). Ea, ear; e, eye.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1240-F5: Hoxa2 is upstream of Wnt–β-catenin signaling. (A) Validation of binding regions linked to genes in the Wnt-signaling pathway by ChIP–qPCR. Percent Input is shown for each Hoxa2-bound region and the corresponding negative antibody control (Neg Ab). Values represent the average of duplicate samples. Art3 is a negative control gene (unbound region). (B) The differentially expressed genes (from the list in Supplementary Table S6) linked to Wnt GO categories are shown together with their corresponding fold changes. (C–F) Expression of Fzd4 and Wnt5a in wild-type (C, E) and mutant (D, F) embryos. In situ hybridization on whole mount E10.5 wild-type and Hoxa2 mutant (C, D) and E12.5 wild-type and Hoxa2 mutant (E, F) using Fzd4 (C, D) and Wnt5a (E, F) probes. Arrow in (C) and (D), and arrowhead in (E) and (F), indicate the embryonic area where Fzd4 and Wnt5a are downregulated in the mutant, respectively. (G and H). Whole mount lacZ staining of E11.5 BAT-Gal (G) and BAT-Gal; Hoxa2−/− (H) embryos. (G) High Wnt canonical activity is detected in the IIBA of E11.0 BAT-Gal transgenic embryos. (H) Wnt canonical activity is lost in the IIBA (arrow) in the absence of Hoxa2. Dotted line separates IIBA from IBA. (I and J) BAT-Gal (I) and wild-type (J) E13.0 embryos. (I) Wnt canonical activity is mostly confined to the pinna of the outer ear (arrowhead). (J) In situ hybridization using Hoxa2 probe shows the pinna remains Hoxa2 positive at later stages (arrowhead). Ea, ear; e, eye.
Mentions: ChIP–qPCR was performed on a Bio-Rad Chromo4 Real-time PCR system using SYBR green (Dynamo) according to the manufacturer's instructions (50 µl PCR reaction for each primer set was performed using the following program: Step 1: 95°C 15 min; Step 2: 95°C 30 s, 55°C 1 min, 72°C 30 s, repeat 49 times; Step 3: melting curve from 55°C to 95°C). Results were analyzed using Bio-Rad Opticon Monitor 3.1.32. Fold enrichment of each bound region was calculated over a negative antibody control relative to input. In Figure 5A, values are expressed as percent input for each bound region and the corresponding negative antibody control. Primer sequences are listed in Supplementary Table S7.

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