Dosage-sensitive transcription factors (TFs) underlie altered gene regulation in human developmental disorders, and cell-type specific gene regulation is linked to the reorganization of 3D chromatin during cellular differentiation. Here, we show dose-dependent regulation of chromatin organization by the congenital heart disease (CHD)- linked, lineage-restricted TF TBX5 in human cardiomyocyte differentiation. Genome organization, including compartments, topologically associated domains, and chromatin loops, are sensitive to reduced TBX5 dosage in a human model of CHD, with variations in response across individual cells. Regions normally bound by TBX5 are especially sensitive, while co-occupancy with CTCF partially protects TBX5-bound TAD boundaries and loop anchors. These results highlight the importance of lineage-restricted TF dosage in cell-type specific 3D chromatin dynamics, suggesting a new mechanism for TF-dependent disease.

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect, but mechanisms for patterning the IVS are largely unknown. Here we show that a Tbx5 ⁺ /Mef2cAHF ⁺ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first and second heart field interface. Ablation of Tbx5 ⁺ /Mef2cAHF ⁺ progenitors causes IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the congenital heart defect transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects and patterning defects, including misexpression of Slit2 and Ntn1 , which encode guidance cues. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes ventricular septation defects and perturbed septal lineage distributions. Thus, we identify Tbx5 as a candidate selector gene, directing progenitors and regulating essential cues, to pattern a compartment boundary for proper cardiac septation, revealing mechanisms for cardiac birth defects.

Dosage-sensitive transcription factors (TFs) underlie altered gene regulation in human developmental disorders, and cell type–specific gene regulation is linked to the reorganization of three-dimensional (3D) chromatin during cellular differentiation. In this work, we show dose-dependent regulation of chromatin organization by the congenital heart disease (CHD)–linked, lineage-restricted TF TBX5 in human cardiomyocyte differentiation. Genome organization, including compartments, topologically associated domains, and chromatin loops, was sensitive to reduced TBX5 dosage in a human model of CHD, with variations in response across individual cells. Cohesin binding was reduced at TBX5-bound enhancer elements in a TBX5 dose-dependent manner, providing a potential mechanism for disrupted loop formation. These results highlight the importance of lineage-restricted TF dosage in cell type–specific 3D chromatin dynamics, suggesting a mechanism for TF-dependent disease.

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect (CHD), but mechanisms for patterning the IVS are largely unknown. Here, we show that a Tbx5 ⁺ /Mef2cAHF ⁺ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first- and second heart field interface. Ablation of Tbx5 ⁺ /Mef2cAHF ⁺ progenitors cause IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the CHD transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects (VSDs) and patterning defects, including misexpression of Slit2 and Ntn1 , which encode guidance cues. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes VSDs and perturbed septal lineage distributions. Thus, we identify Tbx5 as a candidate selector gene, directing progenitors and regulating essential cues, to pattern a compartment boundary for proper cardiac septation, revealing mechanisms for cardiac birth defects.