Defining Gene Regulatory Networks of Cardiac Rhythm

Cardiac rhythm is extremely robust, generating two billion contraction cycles during the average human lifespan. Transcriptional control of cardiac rhythm is poorly understood. We found that removal of the transcription factor gene Tbx5 from the adult mouse caused primary spontaneous and sustained atrial fibrillation (AF). Atrial cardiomyocytes from the Tbx5-mutant mice exhibited action-potential abnormalities, including spontaneous depolarizations, that were rescued by chelating free calcium. We identified a multi-tiered transcriptional network that linked seven previously defined AF risk loci: TBX5 directly activated PITX2, and TBX5 and PITX2 antagonistically regulated membrane effector genes Scn5a, Gja1, Ryr2, Dsp, and Atp2a2. In addition, reduced Tbx5 dose by adult-specific haploinsufficiency caused decreased target gene expression, myocardial automaticity, and AF inducibility, which were all rescued by Pitx2 haploinsufficiency in mice. These results defined a transcriptional architecture for atrial rhythm control organized as an incoherent feed-forward loop, driven by TBX5 and modulated by PITX2. TBX5/PITX2 interplay provides tight control of atrial rhythm effector gene expression, and perturbation of the co-regulated network caused AF susceptibility. This work provides a model for the molecular mechanisms underpinning the genetic implication of multiple AF GWAS loci and will contribute to future efforts to stratify patients for AF risk based on genotype. Transcription factors (TFs) determine context-dependent gene expression by binding and modulating enhancers. Defining functional TF-dependent enhancers from the thousands of TF binding locations genome-wide remains a fundamental challenge for understanding TF-dependent gene regulatory networks. Based on evidence that non-coding RNA (ncRNA) is transcribed from enhancers, we hypothesized that TF-dependent ncRNA transcriptional profiling would identify functional TF-dependent enhancers. We defined TF-dependent ncRNAs for TBX5, a critical cardiac TF, by deep sequencing ncRNAs from wildtype versus Tbx5 mutant mouse atrium. Genome-wide, Tbx5-dependent ncRNAs were enriched for chromatin accessibility and tissue-specific marks of active enhancers. Tbx5-dependent ncRNA-defined enhancers were enriched for TBX5 binding and demonstrated robust Tbx5-dependent activity in-vitro. The direction and magnitude of Tbx5-dependent enhancer transcription correlated positively with that of Tbx5-dependent target gene expression, providing a quantitative metric for Tbx5-dependent enhancer function. Tbx5-dependent atrial ncRNAs identified enhancers required for expression of a calcium-handling network previously associated with Tbx5 function in the atrium, elucidating a physiologically relevant gene regulatory network. Application of TF-dependent enhancer transcription may allow broad elucidation of TF-dependent gene regulatory networks