Recruitment of homodimeric proneural factors by conserved CAT-CAT E-boxes drives major epigenetic reconfiguration in cortical neurogenesis

Proneural factors of the basic helix-loop-helix family coordinate neurogenesis and neurodifferentiation. Among them, NEUROG2 and NEUROD2 subsequently act to specify neurons of the glutamatergic lineage. Disruption of these factors, their target genes and binding DNA motifs has been linked to various neuropsychiatric disorders. Proneural factors bind to specific DNA motifs called E-boxes (hexanucleotides of the form CANNTG, composed of two CAN half sites on opposed strands). While corticogenesis heavily relies on E-box activity, the collaboration of proneural factors on different E-box types and their chromatin remodeling mechanisms remain largely unknown. Here, we conducted a comprehensive analysis using chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for NEUROG2 and NEUROD2, along with time-matched single-cell RNA-seq, ATAC-seq and DNA methylation data from the developing mouse cortex. Our findings show that these factors are highly enriched in transiently active genomic regions during intermediate stages of neuronal differentiation. Although they primarily bind CAG-containing E-boxes, their binding in dynamic regions is notably enriched in CAT-CAT E-boxes (i.e. CATATG, denoted as 5'3' half sites for dimers), which undergo significant DNA demethylation and exhibit the highest levels of evolutionary constraint. Aided by HT-SELEX data reanalysis, structural modeling and DNA footprinting, we propose that these proneural factors exert maximal chromatin remodeling influence during intermediate stages of neurogenesis by binding as homodimers to CAT-CAT motifs. This study provides an in-depth integrative analysis of the dynamic regulation of E-boxes during neuronal development, enhancing our understanding of the mechanisms underlying the binding specificity of critical proneural factors.