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Erg is a crucial regulator of endocardial-mesenchymal transformation during cardiac valve morphogenesis.

Vijayaraj P, Le Bras A, Mitchell N, Kondo M, Juliao S, Wasserman M, Beeler D, Spokes K, Aird WC, Baldwin HS, Oettgen P - Development (2012)

Bottom Line: Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes.The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells.We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
During murine embryogenesis, the Ets factor Erg is highly expressed in endothelial cells of the developing vasculature and in articular chondrocytes of developing bone. We identified seven isoforms for the mouse Erg gene. Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes. The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells. Homozygous Erg(ΔEx3/ΔEx3) knockout mice are viable, fertile and do not display any overt phenotype. By contrast, homozygous Erg(ΔEx4/ΔEx4) knockout mice are embryonic lethal, which is associated with a marked reduction in endocardial-mesenchymal transformation (EnMT) during cardiac valve morphogenesis. We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

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Conserved Ets binding sites in the Snail1 promoter and Snail2 intronic regions. (A) The mouse Snail1 promoter region showing conserved Ets binding sites (EBS, vertical bars). The Snail1 promoter sequences from mouse, rat, human, orangutan and dog are aligned. Highly conserved Ets DNA binding sites were identified within the red box at −650 as analyzed using the UCSC genome browser. (B) The mouse Snail2 promoter region showing conserved Ets binding sites. The Snail2 promoter sequences from mouse, rat, human, orangutan, dog and horse are aligned. Highly conserved Ets DNA binding sites were identified within the red box at +157 using the UCSC genome browser. (C) ChIP analysis of Erg binding at the Snail1 −650 site by qPCR. (D) Quantitative ChIP analysis of Erg binding at the Snail2 +157 site using real-time PCR. (C,D) The input DNA control sample (1:200) was used to normalize the quantification of each PCR product. Rabbit IgG and hearts from ErgΔEx4/ΔEx4 embryos were employed as negative controls.
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Figure 13: Conserved Ets binding sites in the Snail1 promoter and Snail2 intronic regions. (A) The mouse Snail1 promoter region showing conserved Ets binding sites (EBS, vertical bars). The Snail1 promoter sequences from mouse, rat, human, orangutan and dog are aligned. Highly conserved Ets DNA binding sites were identified within the red box at −650 as analyzed using the UCSC genome browser. (B) The mouse Snail2 promoter region showing conserved Ets binding sites. The Snail2 promoter sequences from mouse, rat, human, orangutan, dog and horse are aligned. Highly conserved Ets DNA binding sites were identified within the red box at +157 using the UCSC genome browser. (C) ChIP analysis of Erg binding at the Snail1 −650 site by qPCR. (D) Quantitative ChIP analysis of Erg binding at the Snail2 +157 site using real-time PCR. (C,D) The input DNA control sample (1:200) was used to normalize the quantification of each PCR product. Rabbit IgG and hearts from ErgΔEx4/ΔEx4 embryos were employed as negative controls.

Mentions: To examine whether Snail1 and Snail2 are direct targets of Erg, we first analyzed the proximal promoter and intronic regions of Snail1 and Snail2 for highly conserved Ets binding sites. We identified one highly conserved Ets binding site at −650 in the proximal promoter of Snail1 (Fig. 13A). Similarly, the intronic region of Snail2 at +157 contained a highly conserved Ets binding domain (Fig. 13B). To evaluate whether Erg binds to these regions in vivo, we conducted ChIP assays from mouse embryo hearts at E10.5 using an Erg antibody. Hearts from ErgΔEx4/ΔEx4 embryos were employed as negative controls. From the genomic DNA fragments immunoprecipitated with Erg antibody, amplification of the regions spanning the Ets binding sites were analyzed by qPCR (Fig. 13C,D; supplementary material Table S1). A significant amplification of the region spanning the Ets binding sites was observed in the Erg+/+ heart samples compared with the IgG or negative controls. It is imperative to note that the input was diluted 1:200 because whole embryo hearts were used for the assay and it is likely that Erg may only regulate Snail1 and Snail2 in a subset of cells, and therefore the signal might not be highly enriched over the input. Overall, these results indicate that Erg binds specifically to regulatory regions in the Snail1 and Snail2 genes in E10.5 heart.


Erg is a crucial regulator of endocardial-mesenchymal transformation during cardiac valve morphogenesis.

Vijayaraj P, Le Bras A, Mitchell N, Kondo M, Juliao S, Wasserman M, Beeler D, Spokes K, Aird WC, Baldwin HS, Oettgen P - Development (2012)

Conserved Ets binding sites in the Snail1 promoter and Snail2 intronic regions. (A) The mouse Snail1 promoter region showing conserved Ets binding sites (EBS, vertical bars). The Snail1 promoter sequences from mouse, rat, human, orangutan and dog are aligned. Highly conserved Ets DNA binding sites were identified within the red box at −650 as analyzed using the UCSC genome browser. (B) The mouse Snail2 promoter region showing conserved Ets binding sites. The Snail2 promoter sequences from mouse, rat, human, orangutan, dog and horse are aligned. Highly conserved Ets DNA binding sites were identified within the red box at +157 using the UCSC genome browser. (C) ChIP analysis of Erg binding at the Snail1 −650 site by qPCR. (D) Quantitative ChIP analysis of Erg binding at the Snail2 +157 site using real-time PCR. (C,D) The input DNA control sample (1:200) was used to normalize the quantification of each PCR product. Rabbit IgG and hearts from ErgΔEx4/ΔEx4 embryos were employed as negative controls.
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Figure 13: Conserved Ets binding sites in the Snail1 promoter and Snail2 intronic regions. (A) The mouse Snail1 promoter region showing conserved Ets binding sites (EBS, vertical bars). The Snail1 promoter sequences from mouse, rat, human, orangutan and dog are aligned. Highly conserved Ets DNA binding sites were identified within the red box at −650 as analyzed using the UCSC genome browser. (B) The mouse Snail2 promoter region showing conserved Ets binding sites. The Snail2 promoter sequences from mouse, rat, human, orangutan, dog and horse are aligned. Highly conserved Ets DNA binding sites were identified within the red box at +157 using the UCSC genome browser. (C) ChIP analysis of Erg binding at the Snail1 −650 site by qPCR. (D) Quantitative ChIP analysis of Erg binding at the Snail2 +157 site using real-time PCR. (C,D) The input DNA control sample (1:200) was used to normalize the quantification of each PCR product. Rabbit IgG and hearts from ErgΔEx4/ΔEx4 embryos were employed as negative controls.
Mentions: To examine whether Snail1 and Snail2 are direct targets of Erg, we first analyzed the proximal promoter and intronic regions of Snail1 and Snail2 for highly conserved Ets binding sites. We identified one highly conserved Ets binding site at −650 in the proximal promoter of Snail1 (Fig. 13A). Similarly, the intronic region of Snail2 at +157 contained a highly conserved Ets binding domain (Fig. 13B). To evaluate whether Erg binds to these regions in vivo, we conducted ChIP assays from mouse embryo hearts at E10.5 using an Erg antibody. Hearts from ErgΔEx4/ΔEx4 embryos were employed as negative controls. From the genomic DNA fragments immunoprecipitated with Erg antibody, amplification of the regions spanning the Ets binding sites were analyzed by qPCR (Fig. 13C,D; supplementary material Table S1). A significant amplification of the region spanning the Ets binding sites was observed in the Erg+/+ heart samples compared with the IgG or negative controls. It is imperative to note that the input was diluted 1:200 because whole embryo hearts were used for the assay and it is likely that Erg may only regulate Snail1 and Snail2 in a subset of cells, and therefore the signal might not be highly enriched over the input. Overall, these results indicate that Erg binds specifically to regulatory regions in the Snail1 and Snail2 genes in E10.5 heart.

Bottom Line: Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes.The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells.We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
During murine embryogenesis, the Ets factor Erg is highly expressed in endothelial cells of the developing vasculature and in articular chondrocytes of developing bone. We identified seven isoforms for the mouse Erg gene. Four share a common translational start site encoded by exon 3 (Ex3) and are enriched in chondrocytes. The other three have a separate translational start site encoded by Ex4 and are enriched in endothelial cells. Homozygous Erg(ΔEx3/ΔEx3) knockout mice are viable, fertile and do not display any overt phenotype. By contrast, homozygous Erg(ΔEx4/ΔEx4) knockout mice are embryonic lethal, which is associated with a marked reduction in endocardial-mesenchymal transformation (EnMT) during cardiac valve morphogenesis. We show that Erg is required for the maintenance of the core EnMT regulatory factors that include Snail1 and Snail2 by binding to their promoter and intronic regions.

Show MeSH
Related in: MedlinePlus