The Cardiac TBX5 Interactome Reveals a Chromatin Remodeling Network Essential for Cardiac Septation

Human mutations in the cardiac transcription factor gene TBX5 cause Congenital Heart Disease (CHD), however the underlying mechanism is unknown. We report characterization of the endogenous TBX5 cardiac interactome and demonstrate that TBX5, long considered a transcriptional activator, interacts biochemically and genetically with the Nucleosome Remodeling and Deacetylase (NuRD) repressor complex. Incompatible gene programs are repressed by TBX5 in the developing heart. CHD missense mutations that disrupt the TBX5-NuRD interaction cause depression of a subset of repressed genes. Furthermore, the TBX5-NuRD interaction is required for heart development. Phylogenetic analysis showed that the TBX5-NuRD interaction domain evolved during early diversification of vertebrates, simultaneous with the evolution of cardiac septation. Collectively, this work defines a TBX5-NuRD interaction essential to cardiac development and the evolution of the mammalian heart, and when altered may contribute to human CHD

<i>De Novo</i> and Rare Variants at Multiple Loci Support the Oligogenic Origins of Atrioventricular Septal Heart Defects

Congenital heart disease (CHD) has a complex genetic etiology, and recent studies suggest that high penetrance de novo mutations may account for only a small fraction of disease. In a multi-institutional cohort surveyed by exome sequencing, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons) we observe variation at novel and known loci related to a specific cardiac malformation the atrioventricular septal defect (AVSD). In a primary analysis, by combining developmental coexpression networks with inheritance modeling, we identify a de novo mutation in the DNA binding domain of NR1D2 (p.R175W). We show that p.R175W changes the transcriptional activity of Nr1d2 using an in vitro transactivation model in HUVEC cells. Finally, we demonstrate previously unrecognized cardiovascular malformations in the Nr1d2tm1-Dgen knockout mouse. In secondary analyses we map genetic variation to protein-interaction networks suggesting a role for two collagen genes in AVSD, which we corroborate by burden testing in a second replication cohort of 100 AVSDs and 533 controls (p = 8.37e-08). Finally, we apply a rare-disease inheritance model to identify variation in genes previously associated with CHD (ZFPM2, NSD1, NOTCH1, VCAN, and MYH6), cardiac malformations in mouse models (ADAM17, CHRD, IFT140, PTPRJ, RYR1 and ATE1), and hypomorphic alleles of genes causing syndromic CHD (EHMT1, SRCAP, BBS2, NOTCH2, and KMT2D) in 14 of 59 trios, greatly exceeding variation in control trios without CHD (p = 9.60e-06). In total, 32% of trios carried at least one putatively disease-associated variant across 19 loci,suggesting that inherited and de novo variation across a heterogeneous group of loci may contribute to disease risk

<i>Foxf</i> Genes Integrate <i>Tbx5</i> and Hedgehog Pathways in the Second Heart Field for Cardiac Septation

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.</p

Tbx5 drives <i>Aaldh1a2</i> expression to regulate a ra-hedgehog-wnt gene regulatory network coordinating cardiopulmonary development

The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. Here using Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA) signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary (CP) development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing CP evolution and birth defects

ETV2 primes hematoendothelial gene enhancers prior to hematoendothelial fate commitment

Summary: Mechanisms underlying distinct specification, commitment, and differentiation phases of cell fate determination remain undefined due to difficulties capturing these processes. Here, we interrogate the activity of ETV2, a transcription factor necessary and sufficient for hematoendothelial differentiation, within isolated fate intermediates. We observe transcriptional upregulation of Etv2 and opening of ETV2-binding sites, indicating new ETV2 binding, in a common cardiac-hematoendothelial progenitor population. Accessible ETV2-binding sites are active at the Etv2 locus but not at other hematoendothelial regulator genes. Hematoendothelial commitment coincides with the activation of a small repertoire of previously accessible ETV2-binding sites at hematoendothelial regulators. Hematoendothelial differentiation accompanies activation of a large repertoire of new ETV2-binding sites and upregulation of hematopoietic and endothelial gene regulatory networks. This work distinguishes specification, commitment, and sublineage differentiation phases of ETV2-dependent transcription and suggests that the shift from ETV2 binding to ETV2-bound enhancer activation, not ETV2 binding to target enhancers, drives hematoendothelial fate commitment

Transcriptional profiling of SHF from <i>shh^−/−</i> embryos.

<p>(A) Microdissection for isolation of SHF tissues. E9.5 embryos were isolated (I). Thoracic tissues including the heart were removed from head and tail, kept for genotyping or non-cardiac controls (II). Neural tube was removed (III). SHF tissue was bisected and separated from the heart (IV). Microdissected tissue was kept as anterior SHF (Va), posterior SHF (Vb) or heart (Vc). (B) RT-PCR demonstrates decreased expression of <i>Shh</i>, <i>Gli1</i> and <i>Ptch1</i> in <i>shh</i> mutant SHF tissues isolated for transcriptional profiling (C) Gene Ontology biological processes (GOBPs) enriched in the transcriptional profile analysis of SHF tissue from wild-type and <i>Shh</i> mutant embryos identifies developmental terms. (D) 13 genes identified in the transcriptional profile were verified as Shh-dependent using RT-qPCR (relative quantitation, RQ). * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.</p

Analysis of ChIP-Seq data and its intersection with transcriptional profiling data.

<p>(A) Distribution of ChIP-seq peaks highlighted the modeled GLI3-binding centered in peak regions, using MACS2 software. (B) GLI3 ChIP-seq revealed 1316 peaks defining potential binding sites in the mouse genome. Intersection with <i>shh</i>-dependent transcriptional profiling identified 112 candidate direct Hedgehog-dependent target genes. (C) Summary of <i>de novo</i> and known motifs enriched in <i>shh</i>-dependent GLI3-bound regions (Top 2 sub-panels) compared with similar known GLI motifs from literature and TRANSFAC database (Bottom 3 sub-panels). (D) Among the 112 genes, 26 are transcription factors or regulators of transcription, a significant over-representation. (E) Among the 112 genes, 4 are FOX family transcription factors, a significant over-representation.</p

Model for Hedgehog/Tbx5 interaction.

<p>(A) Intersection of <i>Tbx5</i> expression, restricted to the posterior SHF and heart, and <i>Gli1</i> expression, broadly expressed in axial mesenchyme and brain but excluded from the heart, is the posterior SHF. Activation of TBX5/GLI1 responsive enhancer is observed principally in the overlap between the <i>Tbx5</i> and <i>Gli1</i> expression domains. (B) In the presence of GLI activator (GLIA) alone, the enhancer is weakly active. In the presence of both GLIA and TBX5 is transcription from the enhancer strongly activated. When the GLI binding site is mutated, GLIA alone is insufficient to activate strong expression, but GlLIA may interact with TBX5 to activate expression more strongly than TBX5 alone.</p

Integration of Hedgehog and Tbx5 activity on an enhancer at <i>Foxf1a</i>.

<p>(A) Integration of Hedgehog and Tbx5 activity on an enhancer at <i>Foxf1a</i>. ChIP-seq for GLI3 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004604#pgen-1004604-g002" target="_blank">Figure 2</a>) and TBX5 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004604#pgen.1004604-Kothary1" target="_blank">[35]</a> identified a candidate <i>Foxf1a</i> enhancer. (B) ChIP-PCR from microdissected pSHF for GLI3, GLI1 and TBX5 demonstrated <i>in vivo</i> 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 <i>lacZ</i> 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.</p