Single-cell multiomics across nine mammals reveals cell-type-specific regulatory conservation in the brain

Understanding gene regulation in the brain is essential for defining the genetic basis of neurologic disease. Cis-regulatory elements (CREs) regulate gene expression, but their function and evolution depend on sequence and cis- and trans-regulatory context. We generated single-nucleus RNA- and ATAC-seq data from cortex across nine mammalian species and identified cell-type-specific candidate CREs. We developed a multidimensional conservation framework integrating sequence, chromatin accessibility, and enhancer-gene associations. Massively parallel reporter assays in human neural progenitor cells and neurons measured activity of conserved and human-specific CREs, while CRISPR interference validated enhancer function at conserved loci, including FAM181B. Motif enrichment identified transcription factors distinguishing conserved from evolved CREs. Linkage disequilibrium score regression showed that conserved and human-specific CREs were enriched for neuropsychiatric GWAS risk, whereas neurodegenerative risk was restricted to conserved elements. These findings define functional dimensions of enhancer conservation and reveal how regulatory evolution shapes brain biology and disease susceptibility.