synthetic construct Raw sequence reads. synthetic construct

Heart-specific enhancers drive expression of genes specifically in heart tissues. We find that low-affinity ETS transcription factor binding sites are necessary for the FoxF enhancer in Ciona and the GATA4-G9 enhancer in mice. To determine if higher affinity sites would result in gain-of-function activity, we tested the human GATA4-G9 enhancer and 2 variants with optimized ETS sites in human iPSC-cardiomyocytes, using a reporter assay. We discovered that both variants with optimized ETS sites drove gain-of-function activity and that just a single nucleotide variant within a human GATA4 enhancer increases ETS binding affinity and causes gain-of-function enhancer activity. The prevalence of suboptimal-affinity sites within enhancers creates a vulnerability whereby affinity-optimizing SNVs can lead to gain-of-function gene expression, changes in cellular identity, and organismal-level phenotypes that could contribute to the evolution of novel traits or diseases.