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bioRxiv · 10.1101/2025.03.27.645641

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

Mouren, J.-C.·Torres, M.·van Ouwerkerk, A.·Manosalva, I.·Gallardo, F.·Spicuglia, S.·Ballester, B.
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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 EEs 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 EEs in the cis-regulation of host and distal gene expression. Through large-scale cancer genome analyses, we reveal that EE mutations correlate with dysregulated target-gene expression and clinical outcomes, highlighting their potential relevance in disease. Evolutionary comparisons show that EEs exhibit both strong sequence constraint and lineage-specific plasticity, suggesting that they serve ancient regulatory functions while also contributing to species divergence. Our findings redefine the landscape of functional elements by establishing EEs as a component of gene regulation, while revealing how coding regions can simultaneously fulfil both protein-coding and cis-regulatory roles.

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Mouren, J.-C., Torres, M., van Ouwerkerk, A., Manosalva, I., Gallardo, F., Spicuglia, S., Ballester, B.. 2025-04-01. Exonic enhancers are a widespread class of dual-function regulatory elements. https://doi.org/10.1101/2025.03.27.645641

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