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>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

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