Abstract Atrial fibrillation (AF) is the most common sustained arrhythmia, affecting 59 million individuals worldwide. Impairment of atrial cardiomyocyte (aCM) gene regulatory mechanisms predisposes to atrial fibrillation. The transcription factor TBX5 is essential for normal atrial rhythm, and its inactivation causes loss of aCM enhancer accessibility, looping, and transcriptional identity. Here we investigated the mechanisms by which TBX5 regulates chromatin organization.