GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE328nnn/GSE328309/primaryOK200OtherMus musculusOtherhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE328309GEOGSEfalseFunctional evolution, modification, and derivatization of mammalian developmental enhancersThis 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.2026/04/20GSE328309GSM9678486GSM9678485GSM9678484GSM9678483GSM9678489GSM9678488GSM9678487GSM9682835GSM9678482GSM9678481GSM9678491GSM9678480GSM967849030172328309Mus musculus