PROJECT SUMMARY Atrial fibrillation (AF) is the most common sustained arrhythmia in the United States with a 25% lifetime risk, and accounts for one-third of all cardiovascular diseases. Treatment and care associated with AF costs roughly $26 billion/year in the US alone and accounts for 10% of all Medicare spending. Debilitating complications linked to AF include heart failure and stroke. Evidence suggests AF underlies most ischemic and cryptogenic strokes, with women at a higher risk than men. In addition to stroke, several risk factors, outcomes, and treatments of AF demonstrate gender differences for which there is limited mechanistic understanding. Common variation at the non-coding 4q25 locus is resoundingly linked to AF risk, implicating cis-regulatory elements of the paired-like homeodomain transcription factor, PITX2. To understand the molecular drivers of AF, it is imperative to understand both the upstream regulation and downstream actions of PITX2. PITX2 is expressed in the left atrial (LA) and pulmonary vein (PV) myocardium of humans and mice. While work has implicated PITX2 in AF in the LA myocardium, it has also been suggested to underlie AF originating from the PV, the most common trigger site for arrhythmia in human patients. Although animal models of Pitx2 are susceptible to AF, the direct molecular mechanisms regulated by Pitx2 remain undetermined. Using Pitx2 CRISPR-edited mice, the role of Pitx2 in LA and PV myocardium will be interrogated while addressing underlying gender differences in Pitx2 regulation. The central hypothesis is that gender-specific differences in gene regulatory networks underlie differences in AF-susceptibility in men and women. To address this hypothesis, the first aim proposes to examine the transcriptional basis for gender bias in Pitx2 expression and AF-risk. Motif analysis identifies multiple, highly- conserved hormone-receptor binding sites, including androgen- and estrogen-receptor, at a 4q25-associated regulatory element and the Pitx2 promoter. CRISPR-mediated deletion of the 4q25-associated regulatory element confers male-specific reduction in Pitx2 and increased risk of AF by cardiac pacing. Therefore, this aim will ask whether sex-hormone dependent expression influence AF burden and Pitx2 expression through direct transcriptional regulation. In the second aim, the downstream targets of PITX2 will be interrogated to discover whether epigenomic differences between the genders influence the transcription factor milieu in the LA and PV myocardium. Despite the strong association between PITX2 and AF, the function of PITX2 in LA/PV myocardium is poorly understood. Furthermore, significant differences between men and women are present in AF and other cardiovascular diseases; however, few epigenetic or genomic studies of the cardiovascular system examine gender. This aim is designed to illuminate the role of PITX2 in AF while taking a progressive approach to incorporating and leveraging gender differ...