Question:

Enhancers are non-coding regulatory elements modulating target gene expression via long-range chromatin interactions. Here we decode enhancer-promoter interactions in human cardiac myocytes and show their disease association and regulatory function.

Methods:

We use FACS-sorted cardiac myocyte nuclei from non-diseased human left ventricles (LV-CM) for Hi-C to detect chromatin interactions. Enhancers and gene promoters were annotated using whole genome ChIP-seq data published previously (Gilsbach et al., Nat. commun. 2018)¹. Virtual 4C viewpoints were used to detect significant interactions between genes and their putative enhancers as well as overlapping disease-associated variants on a genome wide scale. The functional relevance of promoter-enhancer interactions was assessed in vitro in induced pluripotent stem cell-derived ventricular cardiac myocytes (iPSC-CM) by silencing regulatory elements using a RNA programmable nuclease-deficient Cas9 fused to a repressor domain (CRISPRi).

Results:

Our deep Hi-C data (> 1.2 billion reads) of human left ventricular cardiac myocytes (LV-CM; n=3) revealed 174,742 highly significant promoter interactions (p < 1x10^-8). In average, one promoter contacts 6 genomic regions. 36 % of promoter interactions overlap with promoters and 72 % overlap with enhancers. Notably, 51 % of non-coding variants associated with cardiovascular diseases contact cardiac myocyte promoters. A significant enrichment was found for strong enhancers containing genetic variants associated with cardiac arrhythmia (p = 3.2x10^-3), including enhancers within the KCNJ2 locus. To show the functional relevance of disease-associated enhancers, we used CRISPRi. Remarkably, silencing of two disease-associated enhancers in iPSC-CM decreased KCNJ2 expression to 25 % and 60 % of control.

Conclusions:

Our genome-wide Hi-C data provides a profound base to understand spatial genome organization in human cardiac myocytes, including promoter-enhancer interactions. The strong enrichment of cardiovascular disease-associated genetic variants within promoter-enhancer interactions underpins the disease relevance of cardiac myocyte enhancers. Representatively for KCNJ2, we show that these promoter enhancer interactions are important for gene expression homeostasis.

Key words: promoter-enhancer interactome, disease-relevant SNPs, functional interactions

References
[1] Gilsbach R., Schwaderer, M. et al. 2018, Distinct epigenetic programs regulate cardiac myocyte development and disease in the human heart in vivo, Nat. commun., volume 9, 381