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Functional evolution, modification, and derivatization of mammalian developmental enhancers

ABSTRACT: This dataset encompasses two complementary massively parallel reporter assay (MPRA) studies that together dissect the functional architecture and evolutionary dynamics of mammalian cis-regulatory elements (CREs) at nucleotide resolution, using five parietal endoderm enhancers as a model system. The first study maps the functional evolution of these enhancers by testing orthologous sequences from 480 extant and ancestrally reconstructed mammalian genomes (Zoonomia/Cactus). Using a model-driven reconstitution strategy guided by deep learning predictions of chromatin accessibility, we traced causal transcription factor binding site (TFBS) changes across lineages, revealing pervasive context-dependent epistasis and diverse modes of evolutionary divergence. Targeted enhancer editing further demonstrated a striking asymmetry: ablation of enhancer activity required as few as one to seven mutations, whereas activity enhancement was constrained by element-specific ceilings. 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE328309 | GEO | 2026/04/20

REPOSITORIES: GEO

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Functional evolution, modification, and derivatization of mammalian developmental enhancers

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