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Foxf genes integrate tbx5 and hedgehog pathways in the second heart field for cardiac septation.

Hoffmann AD, Yang XH, Burnicka-Turek O, Bosman JD, Ren X, Steimle JD, Vokes SA, McMahon AP, Kalinichenko VV, Moskowitz IP - PLoS Genet. (2014)

Bottom Line: GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro.This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps.Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics, Pathology, and Human Genetics, The University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The Second Heart Field (SHF) has been implicated in several forms of congenital heart disease (CHD), including atrioventricular septal defects (AVSDs). Identifying the SHF gene regulatory networks required for atrioventricular septation is therefore an essential goal for understanding the molecular basis of AVSDs. We defined a SHF Hedgehog-dependent gene regulatory network using whole genome transcriptional profiling and GLI-chromatin interaction studies. The Forkhead box transcription factors Foxf1a and Foxf2 were identified as SHF Hedgehog targets. Compound haploinsufficiency for Foxf1a and Foxf2 caused atrioventricular septal defects, demonstrating the biological relevance of this regulatory network. We identified a Foxf1a cis-regulatory element that bound the Hedgehog transcriptional regulators GLI1 and GLI3 and the T-box transcription factor TBX5 in vivo. GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro. This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps. Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

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Integration of Hedgehog and Tbx5 activity on an enhancer at Foxf1a.(A) Integration of Hedgehog and Tbx5 activity on an enhancer at Foxf1a. ChIP-seq for GLI3 (Figure 2) and TBX5 [35] identified a candidate Foxf1a enhancer. (B) ChIP-PCR from microdissected pSHF for GLI3, GLI1 and TBX5 demonstrated in vivo binding of each factor to the candidate enhancer. (C) Luciferase assays demonstrated that GLI1 and TBX5 individually and together synergistically activated the enhancer. Activation of enhancer with mutated GLI binding sites was significantly reduced by GLI1; however, synergistic GLI1/TBX5 activity is largely maintained. Activation of enhancer with mutated TBX binding sites was reduced cells transfected with TBX5 alone, but activation in cells transfected with both GLI1 and TBX5 was still relatively high. (D) Representative images of the enhancer activated specific posterior SHF expression of lacZ in transient transgenic embryos at E9.5. Atria: At; Ventricle: V. P-values:, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.
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pgen-1004604-g005: Integration of Hedgehog and Tbx5 activity on an enhancer at Foxf1a.(A) Integration of Hedgehog and Tbx5 activity on an enhancer at Foxf1a. ChIP-seq for GLI3 (Figure 2) and TBX5 [35] identified a candidate Foxf1a enhancer. (B) ChIP-PCR from microdissected pSHF for GLI3, GLI1 and TBX5 demonstrated in vivo binding of each factor to the candidate enhancer. (C) Luciferase assays demonstrated that GLI1 and TBX5 individually and together synergistically activated the enhancer. Activation of enhancer with mutated GLI binding sites was significantly reduced by GLI1; however, synergistic GLI1/TBX5 activity is largely maintained. Activation of enhancer with mutated TBX binding sites was reduced cells transfected with TBX5 alone, but activation in cells transfected with both GLI1 and TBX5 was still relatively high. (D) Representative images of the enhancer activated specific posterior SHF expression of lacZ in transient transgenic embryos at E9.5. Atria: At; Ventricle: V. P-values:, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.

Mentions: We hypothesized that Foxf1a may represent a direct downstream target of Hedgehog signaling and/or Tbx5 in the SHF. We identified Foxf1a as a candidate direct target based on unbiased interrogation of GLI3T and TBX5 transcription factor chromatin interaction and transcriptional profiling data sets. We intersected our SHF GLI3T ChIP data set (Figure 2B) with a published TBX5 ChIP-seq data set generated from HL-1 cardiomyocytes [33] to define regions with potential co-occupancy of both transcription factors. The intersection of the ChIP-seq datasets identified a single overlapping interaction peak for Gli3T (in the SHF (Figure 2B)) and TBX5 (in HL-1 cardiomyocytes) [33] located approximately 90 kb upstream of the Foxf1a transcription start site (Figure 5A and Figure S3). The Foxf1a transcriptional start site is the closest protein-coding gene to the described peak. The transcriptional start site for a non-coding RNA is located approximately 1.3 kbp upstream of Foxf1a, oriented in the opposite direction [34]. Closer interrogation of the sequence underlying the interaction domains revealed a conserved canonical T-box binding site (AGGTGTGG; chr 8, nucleotides 123,517,714–721, NCBI137/mm9 assembly) and a conserved canonical Gli binding site (GGACCACCCAGC; chr 8, nucleotides 123,517,754–762, NCBI137/mm9 assembly) within 30 base pairs of one another (Figure 5A). We evaluated the sequence information content for these sites from our SHF Gli3 ChIP-seq experiment and found close agreement with published binding sites for Gli3 [23], [33]. This chromatin interaction data in combination with the Tbx5 and Hedgehog signaling-dependent Foxf1a SHF expression (Figure 3) identified this conserved region (mouse chromosome 8, nucleotides 123,517,714–762) as a candidate Foxf1a cis-regulatory element.


Foxf genes integrate tbx5 and hedgehog pathways in the second heart field for cardiac septation.

Hoffmann AD, Yang XH, Burnicka-Turek O, Bosman JD, Ren X, Steimle JD, Vokes SA, McMahon AP, Kalinichenko VV, Moskowitz IP - PLoS Genet. (2014)

Integration of Hedgehog and Tbx5 activity on an enhancer at Foxf1a.(A) Integration of Hedgehog and Tbx5 activity on an enhancer at Foxf1a. ChIP-seq for GLI3 (Figure 2) and TBX5 [35] identified a candidate Foxf1a enhancer. (B) ChIP-PCR from microdissected pSHF for GLI3, GLI1 and TBX5 demonstrated in vivo binding of each factor to the candidate enhancer. (C) Luciferase assays demonstrated that GLI1 and TBX5 individually and together synergistically activated the enhancer. Activation of enhancer with mutated GLI binding sites was significantly reduced by GLI1; however, synergistic GLI1/TBX5 activity is largely maintained. Activation of enhancer with mutated TBX binding sites was reduced cells transfected with TBX5 alone, but activation in cells transfected with both GLI1 and TBX5 was still relatively high. (D) Representative images of the enhancer activated specific posterior SHF expression of lacZ in transient transgenic embryos at E9.5. Atria: At; Ventricle: V. P-values:, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214600&req=5

pgen-1004604-g005: Integration of Hedgehog and Tbx5 activity on an enhancer at Foxf1a.(A) Integration of Hedgehog and Tbx5 activity on an enhancer at Foxf1a. ChIP-seq for GLI3 (Figure 2) and TBX5 [35] identified a candidate Foxf1a enhancer. (B) ChIP-PCR from microdissected pSHF for GLI3, GLI1 and TBX5 demonstrated in vivo binding of each factor to the candidate enhancer. (C) Luciferase assays demonstrated that GLI1 and TBX5 individually and together synergistically activated the enhancer. Activation of enhancer with mutated GLI binding sites was significantly reduced by GLI1; however, synergistic GLI1/TBX5 activity is largely maintained. Activation of enhancer with mutated TBX binding sites was reduced cells transfected with TBX5 alone, but activation in cells transfected with both GLI1 and TBX5 was still relatively high. (D) Representative images of the enhancer activated specific posterior SHF expression of lacZ in transient transgenic embryos at E9.5. Atria: At; Ventricle: V. P-values:, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.
Mentions: We hypothesized that Foxf1a may represent a direct downstream target of Hedgehog signaling and/or Tbx5 in the SHF. We identified Foxf1a as a candidate direct target based on unbiased interrogation of GLI3T and TBX5 transcription factor chromatin interaction and transcriptional profiling data sets. We intersected our SHF GLI3T ChIP data set (Figure 2B) with a published TBX5 ChIP-seq data set generated from HL-1 cardiomyocytes [33] to define regions with potential co-occupancy of both transcription factors. The intersection of the ChIP-seq datasets identified a single overlapping interaction peak for Gli3T (in the SHF (Figure 2B)) and TBX5 (in HL-1 cardiomyocytes) [33] located approximately 90 kb upstream of the Foxf1a transcription start site (Figure 5A and Figure S3). The Foxf1a transcriptional start site is the closest protein-coding gene to the described peak. The transcriptional start site for a non-coding RNA is located approximately 1.3 kbp upstream of Foxf1a, oriented in the opposite direction [34]. Closer interrogation of the sequence underlying the interaction domains revealed a conserved canonical T-box binding site (AGGTGTGG; chr 8, nucleotides 123,517,714–721, NCBI137/mm9 assembly) and a conserved canonical Gli binding site (GGACCACCCAGC; chr 8, nucleotides 123,517,754–762, NCBI137/mm9 assembly) within 30 base pairs of one another (Figure 5A). We evaluated the sequence information content for these sites from our SHF Gli3 ChIP-seq experiment and found close agreement with published binding sites for Gli3 [23], [33]. This chromatin interaction data in combination with the Tbx5 and Hedgehog signaling-dependent Foxf1a SHF expression (Figure 3) identified this conserved region (mouse chromosome 8, nucleotides 123,517,714–762) as a candidate Foxf1a cis-regulatory element.

Bottom Line: GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro.This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps.Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics, Pathology, and Human Genetics, The University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
The Second Heart Field (SHF) has been implicated in several forms of congenital heart disease (CHD), including atrioventricular septal defects (AVSDs). Identifying the SHF gene regulatory networks required for atrioventricular septation is therefore an essential goal for understanding the molecular basis of AVSDs. We defined a SHF Hedgehog-dependent gene regulatory network using whole genome transcriptional profiling and GLI-chromatin interaction studies. The Forkhead box transcription factors Foxf1a and Foxf2 were identified as SHF Hedgehog targets. Compound haploinsufficiency for Foxf1a and Foxf2 caused atrioventricular septal defects, demonstrating the biological relevance of this regulatory network. We identified a Foxf1a cis-regulatory element that bound the Hedgehog transcriptional regulators GLI1 and GLI3 and the T-box transcription factor TBX5 in vivo. GLI1 and TBX5 synergistically activated transcription from this cis-regulatory element in vitro. This enhancer drove reproducible expression in vivo in the posterior SHF, the only region where Gli1 and Tbx5 expression overlaps. Our findings implicate Foxf genes in atrioventricular septation, describe the molecular underpinnings of the genetic interaction between Hedgehog signaling and Tbx5, and establish a molecular model for the selection of the SHF gene regulatory network for cardiac septation.

Show MeSH
Related in: MedlinePlus