Functional evolution, modification, and derivatization of mammalian developmental enhancers

The second study subjects the same five enhancers to intensive sequence perturbation, assaying over 35,000 variants organized into four classes: dense multi-size tiling, multi-hit saturation mutagenesis, model-guided compaction, and TFBS-anchored sequence derivatization including synthetic thripsis. This dissection revealed sharp non-additivity between fragment size and activity, a spectrum of mutational robustness, rare but consequential inter-TFBS epistasis, and a strong influence of background sequence on enhancer output independent of TFBS arrangement.

Together, these datasets provide a comprehensive resource for understanding how enhancer sequences encode function, robustness, and evolvability across the mammalian phylogeny.