Kajiwara, E. A. N. | Search bioRxiv

bioRxiv2026

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

Gene expression during mammalian development is orchestrated by non-coding cis-regulatory DNA elements (CREs) such as distal enhancers1-3. Despite their fundamental importance, and notwithstanding recent progress in predictive modeling4-9, many high-level properties of enhancer grammar remain unresolved. How does the length of an autonomously active CRE constrain its activity? How robust are CREs to mutations or rearrangements of transcription factor binding sites (TFBSs)? And how much epistasis exists among these sites? As predictive models solely trained on endogenous CREs are unlikely to resolve these questions10, 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.

genomics↗

bioRxiv2026

Retracing and rewriting the evolutionary trajectories of mammalian developmental enhancers

Cis-regulatory elements (CREs) such as enhancers play a central role in orchestrating mammalian development, yet how they have gained, lost, maintained or changed function over the course of mammalian evolution remains poorly understood. To address this gap, we densely mapped the functional evolution of five mouse developmental enhancers by testing orthologous sequences from 480 extant and ancestrally reconstructed mammalian genomes (Zoonomia1, Cactus2) with massively parallel reporter assays (MPRAs). This phylogenetic dissection revealed diverse modes of evolution, from lineage-restricted activity to deep functional conservation despite extensive sequence divergence. To pinpoint causal changes, we developed a model-driven reconstitution strategy that uses deep learning-based predictions of chromatin accessibility to re-introduce a succession of mutations into ancestral orthologs; this revealed critical transcription factor binding site (TFBS) changes and pervasive context-dependent epistasis, including instances where mutational effects were strongly contingent on the order of their introduction. When we extended this strategy to tune the activity of extant orthologs, we found that ablation of enhancer function required as few as one to seven mutations, whereas enhancement was constrained by element-specific activity ceilings--a striking asymmetry in the predictability of model-guided enhancer editing. Together, these results shed light on how the plasticity of mammalian enhancers intersects with their evolution, and advance a framework for reprogramming the activity of endogenous CREs at nucleotide resolution.

molecular biology↗