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

Abstract

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-highly integrative chromatin immunoprecipitation 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 152 832 and 54 824 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.

Novel Pan-ERR Agonists Ameliorate Heart Failure Through Enhancing Cardiac Fatty Acid Metabolism and Mitochondrial Function

“…These observations are consistent with the well-established ERR function in transcriptional activation of FAO and mitochondrial genes and its recently identified role in muscle contraction. 13,43,69,70 Among the large number of downregulated DEGs, we found marked enrichment in pathways that are essential for cell proliferation and developmental processes, such as cell cycle, focal adhesion, extracellular matrix-receptor interaction, DNA replication, and Hippo signaling. 71 Reactivation of cell cycle and fetal gene expression has been causally linked to the development of cardiomyopathy and HF [72][73][74] ; thus, suppression of fetal gene expression could be beneficial for HF.…”

Tbx5maintains atrial identity by regulating an atrial enhancer network

“…Recent work aimed at identifying important regulators of chamber identity uncovered the TBOX motif as an enriched motif in atrial-specific enhancers and demonstrated elevated expression of Tbx5 in aCMs compared to vCMs 3 . Other studies demonstrated that TBX5 is required for expression of atrialspecific genes, including Nppa and Bmp10 25 .…”

“…To better understand changes in the atrial gene expression profile, we examined bulk RNA-seq of atrial tissue from a previous study of adult ubiquitous Tbx5 knockout 17 . We compared differentially expressed left atrial genes to sets of genes with aCM-selective or vCM-selective expression as defined by RNA sequencing of purified cardiomyocytes from each chamber (|fold change| > 1.5, Padj < 0.05) 3 . Gene set enrichment analysis with aCM-selective genes revealed statistically significant reduction in Tbx5 knockout atria (Fig.…”

“…Atrial and ventricular cardiomyocytes (aCMs and vCMs) are distinct cardiomyocyte subtypes specialized for distinct physiological functions [3][4][5] . The mechanisms that specify and maintain these differences are only beginning to be elucidated.…”

Spatiotemporal transcriptome atlas of human embryos after gastrulation

“…As one of the initial solid organs in human embryo, the heart displayed a remarkable level of the regulon activity of ESRRG, which is tied to the maturation of cardiac myocytes and maintenance of ventricular identity 38,39 , between PCW3-6 (Fig. 2M-N).…”