Tri-Modal Single-Cell Atlas of Developing Human Brain

Epigenetic modifications, such as DNA methylation, evolve throughout development and aging, providing remarkable regulatory diversity and contributing to disease etiology, including neuropsychiatric disorders. However, how these regulatory layers interact across human brain development remains unclear. Leveraging ME-seq, we simultaneously mapped gene expression, chromatin accessibility, and DNA methylation in over 340,000 single nuclei from 40 human brain specimens spanning fetal development to adolescence. Chromatin accessibility and DNA methylation captured partially overlapping cis-regulatory associations. Developmental trajectories revealed distinct temporal kinetics: DNA methylation continued remodeling beyond major transcriptional and accessibility transitions, while developmental clocks yielded modality-dependent age estimates. Modality-specific regulatory networks converged on developmental transcription factors, whereas methylation-defined regulatory regions provided complementary annotations of neuropsychiatric genetic risk. Together, these findings establish DNA methylation as a persistent and complementary dimension of developmental regulation, linking prolonged remodeling to human brain maturation and disease susceptibility.