SUMMARY Precise regulation of atrial gene expression is crucial to maintain atrial homeostasis, and disorders or gene mutations that impact atrial gene expression can cause atrial fibrillation (AF), a serious arrhythmia that affects an estimated 33 million people worldwide. However, there remain many gaps in our understanding of atrial gene regulation, including the mechanisms that underlie chamber-selective gene expression. Human genetic studies have shown that sequence variants near the cardiac transcription factor gene TBX5 are associated with greater AF risk, and mice with Tbx5 deficiency develop AF. Although recent work has identified some TBX5-regulated genes that contribute to AF susceptibility, the atrial TBX5-centered transcriptional network is incompletely explored. Elucidation of this network and its sensitivity to TBX5 dose would reveal nodal points in AF pathogenesis and may suggest new approaches to prevent or treat AF. The overarching goal of this re- search proposal is to elucidate the atrial gene regulatory network and how it is perturbed in AF. The proposal buillds on novel reagents and techniques developed in the Pu lab to interrogate transcriptional mechanisms in vivo, including highly sensitive and reproducible cardiac transcription factor ChIP-seq through in vivo biotinyla- tion (bioChIP-seq), massively parallel in vivo measurement of cis-regulatory element (CRE) activity (AAV- MPRA assay), and mosaic gene inactivation strategies to hone in direct, cell autonomous effects of gene inac- tivation. In Aim 1, we use bioChIP-seq and AAV-MPRA to define atrial CREs and to dissect the sequence fea- tures required for their chamber selective activity. In Aim 2, we determine the effect of TBX5 deficiency on the occupancy of other TFs and p300, the activity of CREs, and the expression of atrial genes. We use these data to define the TBX5-centered atrial gene regulatory network, and to determine how this network is perturbed by TBX5 haploinsufficiency or knockout. In Aim 3, we test the hypothesis, suggested by our preliminary data, that TBX5 regulates atrial genes through functional and physical interaction with TEAD1. Successful completion of this proposal will lead to new insights into atrial gene regulation and its perturbation in AF.