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Genomic targets of Brachyury (T) in differentiating mouse embryonic stem cells.

Evans AL, Faial T, Gilchrist MJ, Down T, Vallier L, Pedersen RA, Wardle FC, Smith JC - PLoS ONE (2012)

Bottom Line: The T-box transcription factor Brachyury (T) is essential for formation of the posterior mesoderm and the notochord in vertebrate embryos.Here we use chromatin immunoprecipitation and mouse promoter microarrays to identify targets of Brachyury in embryoid bodies formed from differentiating mouse ES cells.Rather, we have identified an (AC)(n) repeat sequence, which is conserved in the rat but not in human, zebrafish or Xenopus.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Background: The T-box transcription factor Brachyury (T) is essential for formation of the posterior mesoderm and the notochord in vertebrate embryos. Work in the frog and the zebrafish has identified some direct genomic targets of Brachyury, but little is known about Brachyury targets in the mouse.

Methodology/principal findings: Here we use chromatin immunoprecipitation and mouse promoter microarrays to identify targets of Brachyury in embryoid bodies formed from differentiating mouse ES cells. The targets we identify are enriched for sequence-specific DNA binding proteins and include components of signal transduction pathways that direct cell fate in the primitive streak and tailbud of the early embryo. Expression of some of these targets, such as Axin2, Fgf8 and Wnt3a, is down regulated in Brachyury mutant embryos and we demonstrate that they are also Brachyury targets in the human. Surprisingly, we do not observe enrichment of the canonical T-domain DNA binding sequence 5'-TCACACCT-3' in the vicinity of most Brachyury target genes. Rather, we have identified an (AC)(n) repeat sequence, which is conserved in the rat but not in human, zebrafish or Xenopus. We do not understand the significance of this sequence, but speculate that it enhances transcription factor binding in the regulatory regions of Brachyury target genes in rodents.

Conclusions/significance: Our work identifies the genomic targets of a key regulator of mesoderm formation in the early mouse embryo, thereby providing insights into the Brachyury-driven genetic regulatory network and allowing us to compare the function of Brachyury in different species.

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Analysis of Axin2, a negative regulator of the Wnt pathway.(A) Location analysis of Axin2. For details see legend to Fig. 4. (B) Quantitative RT-PCR expression profile for Axin2 during ES cell differentiation, expressed relative to beta actin. (C, D) Expression of Axin2 studied by in situ hybridisation; in each, the top image shows a dorsal view, and the bottom image a lateral view. (C) Phenotypically wild type (+/+ or +/T) embryo at E8.5–8.75 and (D) a mutant (T/T) embryo, both derived from crosses of Brachyury heterozygous mutant mice. Axin2 expression is detected with NBT/BCIP (purple) and the insets show a lateral view after double staining for Brachyury detected with INT/BCIP (orange brown). Note that in the wild type embryo Axin2 is expressed in tailbud, paraxial mesoderm and lateral margin of the neural folds. In the mutant embryo expression of Axin2 is greatly reduced (n = 9). Scale bars are 250 µm.
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pone-0033346-g005: Analysis of Axin2, a negative regulator of the Wnt pathway.(A) Location analysis of Axin2. For details see legend to Fig. 4. (B) Quantitative RT-PCR expression profile for Axin2 during ES cell differentiation, expressed relative to beta actin. (C, D) Expression of Axin2 studied by in situ hybridisation; in each, the top image shows a dorsal view, and the bottom image a lateral view. (C) Phenotypically wild type (+/+ or +/T) embryo at E8.5–8.75 and (D) a mutant (T/T) embryo, both derived from crosses of Brachyury heterozygous mutant mice. Axin2 expression is detected with NBT/BCIP (purple) and the insets show a lateral view after double staining for Brachyury detected with INT/BCIP (orange brown). Note that in the wild type embryo Axin2 is expressed in tailbud, paraxial mesoderm and lateral margin of the neural folds. In the mutant embryo expression of Axin2 is greatly reduced (n = 9). Scale bars are 250 µm.

Mentions: Amongst the identified Brachyury targets are many genes encoding positive and negative regulators of the Wnt signalling pathway (Fig. 3A). Enrichment peaks in the promoter regions of Dkk1, Ctnnb1/β-catenin, Dvl3, and γ-catenin/Jup show Brachyury binding (Fig. 3B, C, D and E) and also reveal the presence of AC repeats (green bars) and imperfect T-binding sites (blue bars). Of these Wnt-related genes, Wnt3a and Axin2 both show strong Brachyury binding peaks around their transcription start sites in our ChIP-chip analyses (Figs. 4A, 5A), and their temporal expression patterns both resemble that of Brachyury in our embryoid body system (Figs. 4B, 5B). For Wnt3a, a variant Brachyury site is positioned close to an AC repeat sequence in the first intron, and a canonical TCACACCT Brachyury site is upstream of the transcription start site (Fig. 4A). In the case of Axin2, a canonical Brachyury site is positioned close to a variant site and to an AC repeat (Fig. 5A).


Genomic targets of Brachyury (T) in differentiating mouse embryonic stem cells.

Evans AL, Faial T, Gilchrist MJ, Down T, Vallier L, Pedersen RA, Wardle FC, Smith JC - PLoS ONE (2012)

Analysis of Axin2, a negative regulator of the Wnt pathway.(A) Location analysis of Axin2. For details see legend to Fig. 4. (B) Quantitative RT-PCR expression profile for Axin2 during ES cell differentiation, expressed relative to beta actin. (C, D) Expression of Axin2 studied by in situ hybridisation; in each, the top image shows a dorsal view, and the bottom image a lateral view. (C) Phenotypically wild type (+/+ or +/T) embryo at E8.5–8.75 and (D) a mutant (T/T) embryo, both derived from crosses of Brachyury heterozygous mutant mice. Axin2 expression is detected with NBT/BCIP (purple) and the insets show a lateral view after double staining for Brachyury detected with INT/BCIP (orange brown). Note that in the wild type embryo Axin2 is expressed in tailbud, paraxial mesoderm and lateral margin of the neural folds. In the mutant embryo expression of Axin2 is greatly reduced (n = 9). Scale bars are 250 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316570&req=5

pone-0033346-g005: Analysis of Axin2, a negative regulator of the Wnt pathway.(A) Location analysis of Axin2. For details see legend to Fig. 4. (B) Quantitative RT-PCR expression profile for Axin2 during ES cell differentiation, expressed relative to beta actin. (C, D) Expression of Axin2 studied by in situ hybridisation; in each, the top image shows a dorsal view, and the bottom image a lateral view. (C) Phenotypically wild type (+/+ or +/T) embryo at E8.5–8.75 and (D) a mutant (T/T) embryo, both derived from crosses of Brachyury heterozygous mutant mice. Axin2 expression is detected with NBT/BCIP (purple) and the insets show a lateral view after double staining for Brachyury detected with INT/BCIP (orange brown). Note that in the wild type embryo Axin2 is expressed in tailbud, paraxial mesoderm and lateral margin of the neural folds. In the mutant embryo expression of Axin2 is greatly reduced (n = 9). Scale bars are 250 µm.
Mentions: Amongst the identified Brachyury targets are many genes encoding positive and negative regulators of the Wnt signalling pathway (Fig. 3A). Enrichment peaks in the promoter regions of Dkk1, Ctnnb1/β-catenin, Dvl3, and γ-catenin/Jup show Brachyury binding (Fig. 3B, C, D and E) and also reveal the presence of AC repeats (green bars) and imperfect T-binding sites (blue bars). Of these Wnt-related genes, Wnt3a and Axin2 both show strong Brachyury binding peaks around their transcription start sites in our ChIP-chip analyses (Figs. 4A, 5A), and their temporal expression patterns both resemble that of Brachyury in our embryoid body system (Figs. 4B, 5B). For Wnt3a, a variant Brachyury site is positioned close to an AC repeat sequence in the first intron, and a canonical TCACACCT Brachyury site is upstream of the transcription start site (Fig. 4A). In the case of Axin2, a canonical Brachyury site is positioned close to a variant site and to an AC repeat (Fig. 5A).

Bottom Line: The T-box transcription factor Brachyury (T) is essential for formation of the posterior mesoderm and the notochord in vertebrate embryos.Here we use chromatin immunoprecipitation and mouse promoter microarrays to identify targets of Brachyury in embryoid bodies formed from differentiating mouse ES cells.Rather, we have identified an (AC)(n) repeat sequence, which is conserved in the rat but not in human, zebrafish or Xenopus.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT

Background: The T-box transcription factor Brachyury (T) is essential for formation of the posterior mesoderm and the notochord in vertebrate embryos. Work in the frog and the zebrafish has identified some direct genomic targets of Brachyury, but little is known about Brachyury targets in the mouse.

Methodology/principal findings: Here we use chromatin immunoprecipitation and mouse promoter microarrays to identify targets of Brachyury in embryoid bodies formed from differentiating mouse ES cells. The targets we identify are enriched for sequence-specific DNA binding proteins and include components of signal transduction pathways that direct cell fate in the primitive streak and tailbud of the early embryo. Expression of some of these targets, such as Axin2, Fgf8 and Wnt3a, is down regulated in Brachyury mutant embryos and we demonstrate that they are also Brachyury targets in the human. Surprisingly, we do not observe enrichment of the canonical T-domain DNA binding sequence 5'-TCACACCT-3' in the vicinity of most Brachyury target genes. Rather, we have identified an (AC)(n) repeat sequence, which is conserved in the rat but not in human, zebrafish or Xenopus. We do not understand the significance of this sequence, but speculate that it enhances transcription factor binding in the regulatory regions of Brachyury target genes in rodents.

Conclusions/significance: Our work identifies the genomic targets of a key regulator of mesoderm formation in the early mouse embryo, thereby providing insights into the Brachyury-driven genetic regulatory network and allowing us to compare the function of Brachyury in different species.

Show MeSH
Related in: MedlinePlus