Exonic enhancers are a widespread class of dual-function regulatory elements.

Abstract

Exonic enhancers (EEs) occupy an under-appreciated niche in gene regulation. By integrating transcription factor binding, chromatin accessibility, and high-throughput enhancer-reporter assays, we demonstrate that many protein-coding exons possess enhancer activity across species. These candidate EEs (cEEs) exhibit characteristic epigenomic signatures, form long-range interactions with gene promoters, and can be altered by both nonsynonymous and synonymous variants. CRISPR-mediated inactivation demonstrated the involvement of cEEs in the cis-regulation of host and distal gene expression. Through large-scale cancer genome analyses, we reveal that cEE mutations correlate with dysregulated target-gene expression and clinical outcomes, highlighting their potential relevance in disease. Evolutionary comparisons show that cEEs exhibit both strong sequence constraint and lineage-specific plasticity, suggesting that they serve ancient regulatory functions while also contributing to species divergence. Our findings expand the landscape of functional elements by establishing cEEs as a component of gene regulation, while revealing how coding regions can simultaneously fulfil both protein-coding and cis-regulatory roles.