Enhancer mutational screen in vivo

Abstract

Transcriptional enhancers are key to dynamic gene regulation, but current knowledge on which sequences and motifs are essential for proper enhancer function is still incomplete. This limits our ability to interpret the relevance of enhancer mutations for disease risk. Kosicki et al. decided to mutate human developmental enhancers and investigate their functional sensitivity in vivo. Specifically, they selected seven enhancers, which are active in embryonic brain, heart, limb and face, and derived around 1,700 transgenic mice carrying 260 human enhancer alleles. The mutagenesis approach involved altering 12-bp blocks in each enhancer. This large-scale experiment showed that 69% of all tested blocks are essential for enhancer activity. As expected, most mutations led to a loss of function (60%), whereas only 9% created gain-of-function alleles. In addition, the authors used machine learning to predict the identity of crucial nucleotides at enhancers. Most of the predicted motifs or sites (88%) agreed with the experimental data. Indeed, the model demonstrated good sensitivity and identified 59% of functional blocks. The scale of this in vivo study is impressive and it clearly highlights the degree to which human developmental enhancers are sensitive to perturbation. This analysis contributes substantially to our growing understanding of enhancer structure and function.

Original reference: Nature https://doi.org/10.1038/s41586-025-09182-w (2025)