In Vivo Dissection of Chamber-Selective Enhancers Reveals Estrogen-Related Receptor as a Regulator of Ventricular Cardiomyocyte Identity

Circulation. 2023 Mar 14;147(11):881-896. doi: 10.1161/CIRCULATIONAHA.122.061955. Epub 2023 Jan 27.


Background: Cardiac chamber-selective transcriptional programs underpin the structural and functional differences between atrial and ventricular cardiomyocytes (aCMs and vCMs). The mechanisms responsible for these chamber-selective transcriptional programs remain largely undefined.

Methods: We nominated candidate chamber-selective enhancers (CSEs) by determining the genome-wide occupancy of 7 key cardiac transcription factors (GATA4, MEF2A, MEF2C, NKX2-5, SRF, TBX5, TEAD1) and transcriptional coactivator P300 in atria and ventricles. Candidate enhancers were tested using an adeno-associated virus-mediated massively parallel reporter assay. Chromatin features of CSEs were evaluated by performing assay of transposase accessible chromatin sequencing and acetylation of histone H3 at lysine 27-HiChIP on aCMs and vCMs. CSE sequence requirements were determined by systematic tiling mutagenesis of 29 CSEs at 5 bp resolution. Estrogen-related receptor (ERR) function in cardiomyocytes was evaluated by Cre-loxP-mediated inactivation of ERRα and ERRγ in cardiomyocytes.

Results: We identified 134 066 and 97 506 regions reproducibly occupied by at least 1 transcription factor or P300, in atria or ventricles, respectively. Enhancer activities of 2639 regions bound by transcription factors or P300 were tested in aCMs and vCMs by adeno-associated virus-mediated massively parallel reporter assay. This identified 1092 active enhancers in aCMs or vCMs. Several overlapped loci associated with cardiovascular disease through genome-wide association studies, and 229 exhibited chamber-selective activity in aCMs or vCMs. Many CSEs exhibited differential chromatin accessibility between aCMs and vCMs, and CSEs were enriched for aCM- or vCM-selective acetylation of histone H3 at lysine 27-anchored loops. Tiling mutagenesis of 29 CSEs identified the binding motif of ERRα/γ as important for ventricular enhancer activity. The requirement of ERRα/γ to activate ventricular CSEs and promote vCM identity was confirmed by loss of the vCM gene profile in ERRα/γ knockout vCMs.

Conclusions: We identified 229 CSEs that could be useful research tools or direct therapeutic gene expression. We showed that chamber-selective multi-transcription factor, P300 occupancy, open chromatin, and chromatin looping are predictive features of CSEs. We found that ERRα/γ are essential for maintenance of ventricular identity. Finally, our gene expression, epigenetic, 3-dimensional genome, and enhancer activity atlas provide key resources for future studies of chamber-selective gene regulation.

Keywords: ERRalpha estrogen-related receptor; enhancer elements, genetic; epigenomics; gene expression regulation; heart atria; heart ventricles.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Chromatin / genetics
  • Chromatin / metabolism
  • Enhancer Elements, Genetic / genetics
  • Estrogens
  • Genome-Wide Association Study
  • Histones* / genetics
  • Histones* / metabolism
  • Humans
  • Lysine / genetics
  • Lysine / metabolism
  • Myocytes, Cardiac* / metabolism
  • Promoter Regions, Genetic
  • Transcription Factors / genetics
  • Transcription Factors / metabolism


  • Histones
  • Lysine
  • Transcription Factors
  • Chromatin
  • Estrogens