Evolution of gene regulatory networks during human cardiogenesis

Here, we show through comparative epigenomic analysis the identification of enhancers and promoters that have gained activity in humans during cardiogenesis. These cis-regulatory elements (CREs) are associated with genes involved in heart development and function, and may account for species-specific differences between human and mouse hearts. Supporting these findings, genetic variants that are associated with human cardiac phenotypic/disease traits, particularly those differing between human and mouse, are enriched in human-gained CREs. During early stages of human cardiogenesis, these CREs are also gained within genomic loci of transcriptional regulators, potentially expanding their role in human heart development. In particular, we discovered that gained enhancers in the locus of the early human developmental regulator ZIC3 are selectively accessible within a subpopulation of mesoderm cells which exhibits cardiogenic potential, thus possibly extending the function of ZIC3 beyond its conserved left-right asymmetry role. Genetic deletion of these enhancers identified a human gained enhancer that was required for not only ZIC3 and early cardiac gene expression at the mesoderm stage but also cardiomyocyte differentiation. Overall, our results illuminate how human gained CREs may contribute to human-specific cardiac attributes, and provide insight into how transcriptional regulators may gain developmental roles through the evolutionary acquisition of enhancers. Overall design: Mesoderm cells from three independent differentiations were dissociated with Accutase (STEMCELL) at 37ºC for 10 min and processed fresh for single cell combinatorial indexing (sci)ATAC-seq6,7. Dissociated cells were stained with DAPI (Thermo Fisher Scientific). 500,000 viable cells (DAPI negative) were sorted using a SH800 sorter (Sony) and processed for snATAC-seq