Multi-scale dissection, compaction and derivatization of mammalian developmental enhancers

, we subjected several endogenous CREs to intensive sequence-level perturbation. Specifically, we assayed >35,000 variants of 5 parietal endoderm enhancers, with variants organized into four perturbation classes, designed to probe: (i) the functional sufficiency of sub-fragments via dense multi-size tiling, (ii) local epistasis via multi-hit saturation mutagenesis, (iii) activity-size tradeoffs via model-guided compaction, or (iv) functional resilience via sequence derivatization anchored on key TFBSs, including random deposition, reconstitution, and synthetic thripsis. This multi-scale dissection revealed rich phenomena. Sub-tiling uncovered sharp non-additivity between activity and fragment size, highlighting strongly synergistic TFBS clusters. Compaction showed that natural CREs lie far from the activity-size Pareto front, and that model-guided deletions can yield shorter yet stronger elements. Mutational scanning exposed a spectrum of CRE robustness, from tolerant to fragile, together with rare but consequential epistasis between individual TFBSs. Finally, TFBS-anchored derivatization demonstrated that 'background' sequence can influence activity on par with TFBS arrangement. Strikingly, a substantial fraction of CRE derivatives exceeded the activity of their endogenous progenitors. Taken together, these results reveal both 'soft' and 'stiff' directions in regulatory sequence space, advancing a quantitative phenomenology of how enhancer sequences encode function and robustness.