Functional architecture of cardiac TF regulatory landscapes in control of mammalian heart development

Congenital heart disease (CHD), the most common human birth defect, often results from disruptions in gene regulatory networks (GRNs) that control cardiac lineage specification and cell type identity during heart development. A conserved core set of cardiac transcription factors (TFs) orchestrates these processes through combinatorial interactions that are cell type-specific and tightly regulated across space and time. However, the genomic enhancer architecture that integrates upstream effectors to establish precise cardiac TF dosage and downstream transcriptional output remains largely unresolved. Here, we assessed the functional necessity of five developmental heart enhancer modules previously linked to the regulation of <i>Gata4</i> and <i>Hand2</i>, core cardiac TFs exhibiting overlapping roles in myocardial and endocardial development. While individual enhancer deletions in mouse embryonic hearts revealed a surprising degree of transcriptional resilience, a subset of <i>Gata4</i> enhancers proved indispensable for embryonic progression in a genetically compromised background. To achieve higher precision in cardiac cell type-specific enhancer prediction, we applied single-nucleus multiome profiling, enabling the delineation of cardiac cistromes underlying heart morphogenesis. By integrating this resource with deep learning applications, site-directed transgenesis, and chromatin conformation modeling, we mapped the cardiac enhancer repertoire and regulatory signatures that orchestrate <i>Hand2</i> dynamics across distinct cardiac compartments and lineages. Genome editing further revealed an essential role for the <i>Hand2</i>-upstream regulatory interval (<i>H2</i>-URI) in transcriptional control of endocardial lineage effectors and, consequently, trabecular network formation and cardiac cushion patterning. Together our findings highlight substantial resilience in the <i>cis</i>-regulatory architectures governing cardiac TF dynamics and demonstrate that combinatorial integration of upstream lineage identities across modular enhancer landscapes establishes the cardiac cell type-specific programs driving heart morphogenesis. These results advance the reconstruction of cardiac GRNs and enhance the functional interpretation of CHD-associated variants.