EVOlutionary NEUronal enHANCEr grammar

Objective

The human brain’s expansion is a hallmark of evolution, yet the molecular mechanisms underlying species-specific traits remain poorly understood. Regulatory DNA elements like enhancers are key drivers of neuronal gene expression, but how multiple sequence differences accumulated across species shape enhancer activity remains largely unknown. EvoNeuHance (EVOlutionary NEUronal enHANCEr grammar) aims to decode the cis-regulatory logic of enhancer evolution (sequence changes in DNA) and its modulation by the trans environment (availability of TFs and cofactors) during primate neurogenesis. Using massively parallel reporter assays in two relevant cell types across four primate species, I will generate the first cross-species atlas of enhancer activity during early brain development. First, I will quantify the activity of 40,000 orthologous enhancers across primates and train predictive models linking sequence divergence with activity. Next, I will reconstruct enhancer evolution using synthetic variant libraries, including stepwise “species-morphed” gradients to identify sequence features driving cell- and species-specific activity. Finally, I will perturb candidate trans-acting factors with CRISPRi to test their influence on the activity of synthetic enhancer variants and apply genome engineering to revert endogenous enhancers to ancestral or synthetic states, assessing how cis- and trans-changes reshape regulatory outcomes and 3D chromatin architecture. This interdisciplinary approach delivers the most comprehensive dissection of cis-trans enhancer logic across primates to date, combining functional genomics, synthetic biology, and computational modeling. Results will reveal how sequence divergence (cis) and cellular context (trans) jointly shape enhancer activity, uncovering principles of primate brain evolution and providing a blueprint for rational design of synthetic enhancers with translational potential in regenerative medicine and gene therapy.