Abstract
Transcriptional enhancers direct precise gene expression patterns during development and harbor the majority of variants associated with phenotypic diversity, evolutionary adaptations, and disease. Pinpointing which enhancer variants contribute to changes in gene expression and phenotypes is a major challenge. Here, we find that suboptimal or low-affinity binding sites are necessary for precise gene expression during heart development. Single-nucleotide variants (SNVs) can optimize the affinity of ETS binding sites, causing gain-of-function (GOF) gene expression, cell migration defects, and phenotypes as severe as extra beating hearts in the marine chordate Ciona robusta. In human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a SNV within a human GATA4 enhancer increases ETS binding affinity and causes GOF enhancer activity. The prevalence of suboptimal-affinity sites within enhancers creates a vulnerability whereby affinity-optimizing SNVs can lead to GOF gene expression, changes in cellular identity, and organismal-level phenotypes that could contribute to the evolution of novel traits or diseases.