Limits...
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.

Show MeSH
Conservation of BRACHYURY binding in the human genome.(A) ChIP-qPCR performed on samples from differentiated hECSs using a specific anti-BRACHYURY IgG and a non-specific control IgG. Graph shows enrichment for regulatory regions of Brachyury targets (AXIN2, FGF8, JUP, WNT3A) and a negative control region (NCAPD2). Results are expressed relative to input chromatin divided by the enrichment for the non-specific control antibody. (B) BRACHYURY binding in the human genome. The short red lines below the chromosomal coordinates (hg19) depict the position of the PCR amplicons relative to the beginning of the human genes (blue). The three bottom tracks show the genome sequence conservation between human and mouse, zebrafish and vertebrate genomes (Genome Browser, http://genome.ucsc.edu/).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3316570&req=5

pone-0033346-g007: Conservation of BRACHYURY binding in the human genome.(A) ChIP-qPCR performed on samples from differentiated hECSs using a specific anti-BRACHYURY IgG and a non-specific control IgG. Graph shows enrichment for regulatory regions of Brachyury targets (AXIN2, FGF8, JUP, WNT3A) and a negative control region (NCAPD2). Results are expressed relative to input chromatin divided by the enrichment for the non-specific control antibody. (B) BRACHYURY binding in the human genome. The short red lines below the chromosomal coordinates (hg19) depict the position of the PCR amplicons relative to the beginning of the human genes (blue). The three bottom tracks show the genome sequence conservation between human and mouse, zebrafish and vertebrate genomes (Genome Browser, http://genome.ucsc.edu/).

Mentions: We therefore made use of this newly developed in vitro differentiation system to ask if BRACHYURY binds the homologous human regulatory regions of some key mouse targets: AXIN2, FGF8, JUP and WNT3A. As in the mouse, these regions contain imperfect T-binding motifs (data not shown). Our experiments involving ChIP-qPCR with hESC-derived mesoderm cells indeed detected a strong enrichment for these sequences, thus indicating that BRACHYURY binds to the same genomic regions in the human (Fig. 7A).


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)

Conservation of BRACHYURY binding in the human genome.(A) ChIP-qPCR performed on samples from differentiated hECSs using a specific anti-BRACHYURY IgG and a non-specific control IgG. Graph shows enrichment for regulatory regions of Brachyury targets (AXIN2, FGF8, JUP, WNT3A) and a negative control region (NCAPD2). Results are expressed relative to input chromatin divided by the enrichment for the non-specific control antibody. (B) BRACHYURY binding in the human genome. The short red lines below the chromosomal coordinates (hg19) depict the position of the PCR amplicons relative to the beginning of the human genes (blue). The three bottom tracks show the genome sequence conservation between human and mouse, zebrafish and vertebrate genomes (Genome Browser, http://genome.ucsc.edu/).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316570&req=5

pone-0033346-g007: Conservation of BRACHYURY binding in the human genome.(A) ChIP-qPCR performed on samples from differentiated hECSs using a specific anti-BRACHYURY IgG and a non-specific control IgG. Graph shows enrichment for regulatory regions of Brachyury targets (AXIN2, FGF8, JUP, WNT3A) and a negative control region (NCAPD2). Results are expressed relative to input chromatin divided by the enrichment for the non-specific control antibody. (B) BRACHYURY binding in the human genome. The short red lines below the chromosomal coordinates (hg19) depict the position of the PCR amplicons relative to the beginning of the human genes (blue). The three bottom tracks show the genome sequence conservation between human and mouse, zebrafish and vertebrate genomes (Genome Browser, http://genome.ucsc.edu/).
Mentions: We therefore made use of this newly developed in vitro differentiation system to ask if BRACHYURY binds the homologous human regulatory regions of some key mouse targets: AXIN2, FGF8, JUP and WNT3A. As in the mouse, these regions contain imperfect T-binding motifs (data not shown). Our experiments involving ChIP-qPCR with hESC-derived mesoderm cells indeed detected a strong enrichment for these sequences, thus indicating that BRACHYURY binds to the same genomic regions in the human (Fig. 7A).

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