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HOXA13 Is essential for placental vascular patterning and labyrinth endothelial specification.

Shaut CA, Keene DR, Sorensen LK, Li DY, Stadler HS - PLoS Genet. (2008)

Bottom Line: Notably, pro-vascular genes, including Tie2 and Foxf1, exhibited reduced expression in the mutant endothelia, which also exhibited elevated expression of genes normally expressed in lymphatic or sinusoidal endothelia.ChIP analysis of HOXA13-DNA complexes in the placenta confirmed that HOXA13 binds the Tie2 and Foxf1 promoters in vivo.Taken together, these findings demonstrate that HOXA13 directly regulates Tie2 and Foxf1 in the placental labyrinth endothelia, providing a functional explanation for the mid-gestational lethality exhibited by Hoxa13 mutant embryos as well as a novel transcriptional program necessary for the specification of the labyrinth vascular endothelia.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, Oregon, United States of America.

ABSTRACT
In eutherian mammals, embryonic growth and survival is dependent on the formation of the placenta, an organ that facilitates the efficient exchange of oxygen, nutrients, and metabolic waste between the maternal and fetal blood supplies. Key to the placenta's function is the formation of its vascular labyrinth, a series of finely branched vessels whose molecular ontogeny remains largely undefined. In this report, we demonstrate that HOXA13 plays an essential role in labyrinth vessel formation. In the absence of HOXA13 function, placental endothelial cell morphology is altered, causing a loss in vessel wall integrity, edema of the embryonic blood vessels, and mid-gestational lethality. Microarray analysis of wild-type and mutant placentas revealed significant changes in endothelial gene expression profiles. Notably, pro-vascular genes, including Tie2 and Foxf1, exhibited reduced expression in the mutant endothelia, which also exhibited elevated expression of genes normally expressed in lymphatic or sinusoidal endothelia. ChIP analysis of HOXA13-DNA complexes in the placenta confirmed that HOXA13 binds the Tie2 and Foxf1 promoters in vivo. In vitro, HOXA13 binds sequences present in the Tie2 and Foxf1 promoters with high affinity (K(d) = 27-42 nM) and HOXA13 can use these bound promoter regions to direct gene expression. Taken together, these findings demonstrate that HOXA13 directly regulates Tie2 and Foxf1 in the placental labyrinth endothelia, providing a functional explanation for the mid-gestational lethality exhibited by Hoxa13 mutant embryos as well as a novel transcriptional program necessary for the specification of the labyrinth vascular endothelia.

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HOXA13 binds to gene-regulatory regions in Tie2 and Foxf1 to facilitate gene expression.(A). Left panel: Chromatin immunoprecipitation (ChIP) of heterozygous control placental chromatin using a HOXA13 antibody identified DNA sequences present in the Tie2 and Foxf1 promoter regions and confirms that HOXA13 associates with these sequences in vivo. Right panel: Parallel assays using Hoxa13 homozygous mutant placentas failed to enrich for the same DNA regions, suggesting that HOXA13's DNA binding function, which is absent in the homozygous mutant protein, is necessary for in vivo association at the Tie2 and Foxf1 loci. Input indicates positive control confirming the presence of the Tie2 and Foxf1 DNA elements in both heterozygous and homozygous mutant chromatin samples. ChIP = + HOXA13 antibody; No-AB = no antibody negative control; Pre-Im = IgG control rabbit pre-immune sera; Water = no DNA PCR control (B). Sequence analysis revealed HOXA13 binding sites (red text) in the ChIP-positive PCR amplification products for both Foxf1 and Tie2. (C) EMSA analysis of the ChIP-positive PCR amplification products confirms HOXA13 can bind these sites in a concentration-dependent manner. A13-DBD = HOXA13 DNA binding domain peptide; Cold Comp.  = the Tie2 or Foxf1 binding sites lacking radioactive labeling. (D) The HOXA13 DNA binding domain binds the DNA sequences present in the ChIP-positive region with high affinity exhibiting a Kd of 48±4 nM for the Foxf1 region and 27±1.4 nM and 22±1.6 nM for the two sequences present in Tie2. Error bars denote the standard error for the averaged millipolarization values at each HOXA13-DBD concentration. The DNA sequences of the Tie2 or Foxf1 promoter regions used in the assay are denoted by the color text. (E) Conversion of the Tie2 or Foxf1 binding sites to the sequence TGAC ablates the affinity of the HOXA13 DNA binding domain for these gene-specific promoter sequences. Error bars denote the standard error for the averaged millipolarization values at each HOXA13-DBD concentration. (F) In vitro assessment of the 140 bp Tie2 and 121 bp Foxf1 ChIP fragments confirms that HOXA13 can use these minimal sites to direct gene expression, confirming their capacity to function as gene-regulatory elements. Values represent average detected luciferase activity after normalization for transfection with a Renilla luciferase reporter±standard error.
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pgen-1000073-g010: HOXA13 binds to gene-regulatory regions in Tie2 and Foxf1 to facilitate gene expression.(A). Left panel: Chromatin immunoprecipitation (ChIP) of heterozygous control placental chromatin using a HOXA13 antibody identified DNA sequences present in the Tie2 and Foxf1 promoter regions and confirms that HOXA13 associates with these sequences in vivo. Right panel: Parallel assays using Hoxa13 homozygous mutant placentas failed to enrich for the same DNA regions, suggesting that HOXA13's DNA binding function, which is absent in the homozygous mutant protein, is necessary for in vivo association at the Tie2 and Foxf1 loci. Input indicates positive control confirming the presence of the Tie2 and Foxf1 DNA elements in both heterozygous and homozygous mutant chromatin samples. ChIP = + HOXA13 antibody; No-AB = no antibody negative control; Pre-Im = IgG control rabbit pre-immune sera; Water = no DNA PCR control (B). Sequence analysis revealed HOXA13 binding sites (red text) in the ChIP-positive PCR amplification products for both Foxf1 and Tie2. (C) EMSA analysis of the ChIP-positive PCR amplification products confirms HOXA13 can bind these sites in a concentration-dependent manner. A13-DBD = HOXA13 DNA binding domain peptide; Cold Comp.  = the Tie2 or Foxf1 binding sites lacking radioactive labeling. (D) The HOXA13 DNA binding domain binds the DNA sequences present in the ChIP-positive region with high affinity exhibiting a Kd of 48±4 nM for the Foxf1 region and 27±1.4 nM and 22±1.6 nM for the two sequences present in Tie2. Error bars denote the standard error for the averaged millipolarization values at each HOXA13-DBD concentration. The DNA sequences of the Tie2 or Foxf1 promoter regions used in the assay are denoted by the color text. (E) Conversion of the Tie2 or Foxf1 binding sites to the sequence TGAC ablates the affinity of the HOXA13 DNA binding domain for these gene-specific promoter sequences. Error bars denote the standard error for the averaged millipolarization values at each HOXA13-DBD concentration. (F) In vitro assessment of the 140 bp Tie2 and 121 bp Foxf1 ChIP fragments confirms that HOXA13 can use these minimal sites to direct gene expression, confirming their capacity to function as gene-regulatory elements. Values represent average detected luciferase activity after normalization for transfection with a Renilla luciferase reporter±standard error.

Mentions: Because Tie2 and Foxf1 are strongly expressed in the placental labyrinth and mice lacking TIE2 or FOXF1 exhibit vascular defects most similar to those present in Hoxa13 mutant labyrinths, we hypothesized that HOXA13 directly regulates Tie2 or Foxf1 to facilitate labyrinth vascular development [8], [39], [64]–[70]. Testing this hypothesis, we first examined whether HOXA13 binds the promoters for Tie2 or Foxf1 in vivo using a HOXA13 antibody to immunoprecipitate HOXA13-DNA complexes present in labyrinth chromatin (Figure 10A). Previous characterization of the HOXA13 antibody confirmed that it can bind both wild type and mutant HOXA13 proteins and facilitate chromatin immunoprecipitation (ChIP) of HOXA13-bound gene regulatory elements [71]–[73]. PCR analysis of the first 3000 base-pairs (bp) (−3000 to +1) of the Tie2 and Foxf1 promoters revealed a single region in each locus bound by HOXA13 in wild type (not shown) and heterozygous mutant placental labyrinths (Figure 10A and 10B). In contrast, the homozygous mutant HOXA13 protein, which lacks its DNA binding domain, failed to associate with the same promoter regions (Figure 10A). Parallel ChIP assays did not detect HOXA13 association with the promoters of other pro-vascular genes mis-expressed in the mutant labyrinth including CD36, Caveolin-1, and Neuropilin-1 (data not shown).


HOXA13 Is essential for placental vascular patterning and labyrinth endothelial specification.

Shaut CA, Keene DR, Sorensen LK, Li DY, Stadler HS - PLoS Genet. (2008)

HOXA13 binds to gene-regulatory regions in Tie2 and Foxf1 to facilitate gene expression.(A). Left panel: Chromatin immunoprecipitation (ChIP) of heterozygous control placental chromatin using a HOXA13 antibody identified DNA sequences present in the Tie2 and Foxf1 promoter regions and confirms that HOXA13 associates with these sequences in vivo. Right panel: Parallel assays using Hoxa13 homozygous mutant placentas failed to enrich for the same DNA regions, suggesting that HOXA13's DNA binding function, which is absent in the homozygous mutant protein, is necessary for in vivo association at the Tie2 and Foxf1 loci. Input indicates positive control confirming the presence of the Tie2 and Foxf1 DNA elements in both heterozygous and homozygous mutant chromatin samples. ChIP = + HOXA13 antibody; No-AB = no antibody negative control; Pre-Im = IgG control rabbit pre-immune sera; Water = no DNA PCR control (B). Sequence analysis revealed HOXA13 binding sites (red text) in the ChIP-positive PCR amplification products for both Foxf1 and Tie2. (C) EMSA analysis of the ChIP-positive PCR amplification products confirms HOXA13 can bind these sites in a concentration-dependent manner. A13-DBD = HOXA13 DNA binding domain peptide; Cold Comp.  = the Tie2 or Foxf1 binding sites lacking radioactive labeling. (D) The HOXA13 DNA binding domain binds the DNA sequences present in the ChIP-positive region with high affinity exhibiting a Kd of 48±4 nM for the Foxf1 region and 27±1.4 nM and 22±1.6 nM for the two sequences present in Tie2. Error bars denote the standard error for the averaged millipolarization values at each HOXA13-DBD concentration. The DNA sequences of the Tie2 or Foxf1 promoter regions used in the assay are denoted by the color text. (E) Conversion of the Tie2 or Foxf1 binding sites to the sequence TGAC ablates the affinity of the HOXA13 DNA binding domain for these gene-specific promoter sequences. Error bars denote the standard error for the averaged millipolarization values at each HOXA13-DBD concentration. (F) In vitro assessment of the 140 bp Tie2 and 121 bp Foxf1 ChIP fragments confirms that HOXA13 can use these minimal sites to direct gene expression, confirming their capacity to function as gene-regulatory elements. Values represent average detected luciferase activity after normalization for transfection with a Renilla luciferase reporter±standard error.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2367452&req=5

pgen-1000073-g010: HOXA13 binds to gene-regulatory regions in Tie2 and Foxf1 to facilitate gene expression.(A). Left panel: Chromatin immunoprecipitation (ChIP) of heterozygous control placental chromatin using a HOXA13 antibody identified DNA sequences present in the Tie2 and Foxf1 promoter regions and confirms that HOXA13 associates with these sequences in vivo. Right panel: Parallel assays using Hoxa13 homozygous mutant placentas failed to enrich for the same DNA regions, suggesting that HOXA13's DNA binding function, which is absent in the homozygous mutant protein, is necessary for in vivo association at the Tie2 and Foxf1 loci. Input indicates positive control confirming the presence of the Tie2 and Foxf1 DNA elements in both heterozygous and homozygous mutant chromatin samples. ChIP = + HOXA13 antibody; No-AB = no antibody negative control; Pre-Im = IgG control rabbit pre-immune sera; Water = no DNA PCR control (B). Sequence analysis revealed HOXA13 binding sites (red text) in the ChIP-positive PCR amplification products for both Foxf1 and Tie2. (C) EMSA analysis of the ChIP-positive PCR amplification products confirms HOXA13 can bind these sites in a concentration-dependent manner. A13-DBD = HOXA13 DNA binding domain peptide; Cold Comp.  = the Tie2 or Foxf1 binding sites lacking radioactive labeling. (D) The HOXA13 DNA binding domain binds the DNA sequences present in the ChIP-positive region with high affinity exhibiting a Kd of 48±4 nM for the Foxf1 region and 27±1.4 nM and 22±1.6 nM for the two sequences present in Tie2. Error bars denote the standard error for the averaged millipolarization values at each HOXA13-DBD concentration. The DNA sequences of the Tie2 or Foxf1 promoter regions used in the assay are denoted by the color text. (E) Conversion of the Tie2 or Foxf1 binding sites to the sequence TGAC ablates the affinity of the HOXA13 DNA binding domain for these gene-specific promoter sequences. Error bars denote the standard error for the averaged millipolarization values at each HOXA13-DBD concentration. (F) In vitro assessment of the 140 bp Tie2 and 121 bp Foxf1 ChIP fragments confirms that HOXA13 can use these minimal sites to direct gene expression, confirming their capacity to function as gene-regulatory elements. Values represent average detected luciferase activity after normalization for transfection with a Renilla luciferase reporter±standard error.
Mentions: Because Tie2 and Foxf1 are strongly expressed in the placental labyrinth and mice lacking TIE2 or FOXF1 exhibit vascular defects most similar to those present in Hoxa13 mutant labyrinths, we hypothesized that HOXA13 directly regulates Tie2 or Foxf1 to facilitate labyrinth vascular development [8], [39], [64]–[70]. Testing this hypothesis, we first examined whether HOXA13 binds the promoters for Tie2 or Foxf1 in vivo using a HOXA13 antibody to immunoprecipitate HOXA13-DNA complexes present in labyrinth chromatin (Figure 10A). Previous characterization of the HOXA13 antibody confirmed that it can bind both wild type and mutant HOXA13 proteins and facilitate chromatin immunoprecipitation (ChIP) of HOXA13-bound gene regulatory elements [71]–[73]. PCR analysis of the first 3000 base-pairs (bp) (−3000 to +1) of the Tie2 and Foxf1 promoters revealed a single region in each locus bound by HOXA13 in wild type (not shown) and heterozygous mutant placental labyrinths (Figure 10A and 10B). In contrast, the homozygous mutant HOXA13 protein, which lacks its DNA binding domain, failed to associate with the same promoter regions (Figure 10A). Parallel ChIP assays did not detect HOXA13 association with the promoters of other pro-vascular genes mis-expressed in the mutant labyrinth including CD36, Caveolin-1, and Neuropilin-1 (data not shown).

Bottom Line: Notably, pro-vascular genes, including Tie2 and Foxf1, exhibited reduced expression in the mutant endothelia, which also exhibited elevated expression of genes normally expressed in lymphatic or sinusoidal endothelia.ChIP analysis of HOXA13-DNA complexes in the placenta confirmed that HOXA13 binds the Tie2 and Foxf1 promoters in vivo.Taken together, these findings demonstrate that HOXA13 directly regulates Tie2 and Foxf1 in the placental labyrinth endothelia, providing a functional explanation for the mid-gestational lethality exhibited by Hoxa13 mutant embryos as well as a novel transcriptional program necessary for the specification of the labyrinth vascular endothelia.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, Oregon, United States of America.

ABSTRACT
In eutherian mammals, embryonic growth and survival is dependent on the formation of the placenta, an organ that facilitates the efficient exchange of oxygen, nutrients, and metabolic waste between the maternal and fetal blood supplies. Key to the placenta's function is the formation of its vascular labyrinth, a series of finely branched vessels whose molecular ontogeny remains largely undefined. In this report, we demonstrate that HOXA13 plays an essential role in labyrinth vessel formation. In the absence of HOXA13 function, placental endothelial cell morphology is altered, causing a loss in vessel wall integrity, edema of the embryonic blood vessels, and mid-gestational lethality. Microarray analysis of wild-type and mutant placentas revealed significant changes in endothelial gene expression profiles. Notably, pro-vascular genes, including Tie2 and Foxf1, exhibited reduced expression in the mutant endothelia, which also exhibited elevated expression of genes normally expressed in lymphatic or sinusoidal endothelia. ChIP analysis of HOXA13-DNA complexes in the placenta confirmed that HOXA13 binds the Tie2 and Foxf1 promoters in vivo. In vitro, HOXA13 binds sequences present in the Tie2 and Foxf1 promoters with high affinity (K(d) = 27-42 nM) and HOXA13 can use these bound promoter regions to direct gene expression. Taken together, these findings demonstrate that HOXA13 directly regulates Tie2 and Foxf1 in the placental labyrinth endothelia, providing a functional explanation for the mid-gestational lethality exhibited by Hoxa13 mutant embryos as well as a novel transcriptional program necessary for the specification of the labyrinth vascular endothelia.

Show MeSH
Related in: MedlinePlus