Abstract
Cell differentiation is orchestrated by transcription factors (TFs) binding to enhancers, shaping gene regulatory networks that drive neuronal lineage specification. Deciphering these enhancer-driven networks in human forebrain development is essential for understanding the genetic basis of neurodevelopmental disorders. Through integrative epigenomic and transcriptomic analyses of human forebrain organoids derived from 10 individuals with autism spectrum disorder (ASD) and their neurotypical fathers, we constructed a comprehensive enhancer-driven gene regulatory network (GRN) of early neurodevelopment. This GRN revealed hierarchical regulatory transitions guiding neuronal differentiation and was experimentally validated via CRISPR interference (CRISPRi) and loss-of-function analyses. A subnetwork linked ASD-associated transcriptomic alterations to dysregulated TF activity, implicating FOXG1, BHLHE22, EOMES, and NEUROD2 as key regulators of excitatory neuron specification in macrocephalic ASD. These findings suggest that ASD disrupts enhancer-driven regulatory frameworks, altering neuronal cell fate decisions in the developing fetal brain.