Enhancer-driven regulatory network of forebrain human development provides insights into autism

Cell differentiation involves shifts in chromatin organization allowing transcription factors (TFs) to bind enhancer elements and modulate gene expression. The TF-enhancer-gene regulatory interactions that control the formation of neuronal lineages have yet to be charted in humans. Here, we mapped enhancer elements and conducted an integrative analysis of epigenomic and transcriptomic profiles across 60 days of differentiation of human forebrain organoids derived from 10 individuals with autism spectrum disorder (ASD) and their neurotypical fathers. This multi-omics profiling allowed us to build an enhancer-driven gene regulatory network (GRN) of early neural development. We validated the GRN by performing a loss-of-function experiment with FOXG1 – one of the master TFs in the development of the mammalian brain. Analysis of the constructed GRN identified regulatory hierarchies driving the specification of neuronal cell types. By analyzing differential gene expression in ASD through the GRN hierarchy, we associated the ASD transcriptomic signatures to altered activity of key TFs. We found that macrocephalic ASD was principally driven by an increased activity of BHLHE22, FOXG1, EOMES, and NEUROD2, which are major regulators of excitatory neuron fate. Normocephalic ASD, on the contrary, was driven by decreased activity of those same TFs and by an increased activity of LMX1B and FOXB1 – two upstream TF repressors of FOXG1. These findings suggest that ASD is characterized by an altered early gene regulatory program that specifies neuronal cell lineages in the fetal brain. Thus, constructing a GRN of early brain development modeled in organoids provides insights into the etiology of ASD and can guide future experimental approaches to establish its genetic causes and treatment strategies. ### Competing Interest Statement The authors have declared no competing interest. This study did not generate new unique reagents or DNA constructs. Primary cell lines and iPSC lines are shared via the Infinity BiologiX LLC repository (). Datasets reported in this study are available through the NIMH Data Archive (NDA). The scRNA-seq and scATAC-seq data are under collection #C3957, url: ; the bulk RNA-seq data are under collection #C2424, url: .