Abstract Body: Introduction: During the transition from fetal to adult stages, the heart undergoes dramatic mitochondrial and contractile maturation, and dysregulation of this process may contribute to heart failure. Previously, we identified estrogen-related receptors (ERR) α and γ as key regulators of cardiomyocyte (CM) maturation. Here, we aimed to investigate the mechanisms whereby ERRs coordinately regulate energy metabolic and contractile maturation gene programs and to identify naturally occurring human variants within ERR binding sites.
Methods/Results: Genomic interrogation of ERRγ ChIP-seq datasets generated from human induced pluripotent stem cell-derived CMs revealed that ERRγ-bound sites regulating metabolic genes were enriched in proximal promoter regions (< 5 kbp from the transcription start site), whereas sites linked to genes involved in cardiac-specific processes including contractile function, ion transport, and development were distributed across promoters and enhancers (5 kbp–5 Mbp). CRISPR/Cas9 and PROTAC-mediated depletion of ERRα or γ resulted in downregulated expression of many adult cardiac-specific gene programs. However, canonical ERR metabolic gene targets were not substantially affected unless both ERRα and γ were depleted, suggesting the cardiac-specific gene targets are more vulnerable to loss of ERR function. Intersection analysis of the ERR ChIP-seq datasets with gnomAD identified variants in ERR binding sites. Query of the Children's Hospital of Philadelphia Birth Defects Biorepository and Trans-Omics for Precision Medicine (TOPMed) databases identified rare ERR site variants in probands with congenital heart disease (CHD). Expression of the genes linked to the variants were affected by loss of ERRα/γ. Reference and variant ERR binding site-reporter assays demonstrated that a subset of variants altered ERR-driven transcriptional activation.
Conclusion: ERRα and γ orchestrate metabolic and contractile maturation via topologically defined genomic regions that exhibit differential susceptibility to allelic loss of function. ERR-bound enhancers regulating cardiac-specific processes contain sequence variants that potentially contribute to genetically determined heart diseases such as CHD.