An emerging regulatory principle governing enhancers is the use of suboptimal affinity binding sites to encode tissue-specific gene expression. Here we investigate if optimizing single-nucleotide variants that violate this principle can disrupt tissue-specific gene expression and development. The ZRS enhancer mediates expression of Shh in the posterior of the developing limb buds and is critical for limb and digit development. We find that the ZRS contains suboptimal-affinity ETS binding sites. Two human mutations and a synthetic mutation that optimize the affinity of the ETS-A site from 0.15 to 0.25 relative binding affinity cause polydactyly with the same penetrance and severity. Further increasing the affinity of the ETS-A site results in more penetrant and severe phenotypes. The prevalent use of suboptimal affinity binding sites within enhancers to encode tissue-specificity creates a vulnerability within genomes whereby variants that optimize affinity, even subtly, can be pathogenic. This provides a generalizable approach to identify causal variants that underlie enhanceropathies.

SummaryEnhancers direct precise gene expression patterns during development and harbor the majority of variants associated with disease. We find that suboptimal affinity ETS transcription factor binding sites are prevalent within Ciona and human developmental heart enhancers. Here we demonstrate in two diverse systems, Ciona intestinalis and human iPSC-derived cardiomyocytes (iPSC-CMs), that single nucleotide changes can optimize the affinity of ETS binding sites, leading to gain-of-function gene expression associated with heart phenotypes. In Ciona, ETS affinity-optimizing SNVs lead to ectopic expression and phenotypic changes including two beating hearts. In human iPSC-CMs, an affinity-optimizing SNV associated with QRS duration occurs within an SCN5A enhancer and leads to increased enhancer activity. Our mechanistic approach provides a much-needed systematic framework that works across different enhancers, cell types and species to pinpoint causal enhancer variants contributing to enhanceropathies, phenotypic diversity and evolutionary changes.In BriefThe prevalent use of low-affinity ETS sites within developmental heart enhancers creates vulnerability within genomes whereby single nucleotide changes can dramatically increase binding affinity, causing gain-of-function enhancer activity that impacts heart development.HighlightsETS affinity-optimizing SNVs can lead to migration defects and a multi-chambered heart.An ETS affinity-optimizing human SNV within an SCN5A enhancer increases expression and is associated with QRS duration.Searching for ETS affinity-optimizing variants is a systematic and generalizable approach to pinpoint causal enhancer variants.