Sleep-dependent DNA methylation remodeling in basolateral amygdala fear engrams

How sleep consolidates fear memories at the molecular level within individual engram neurons is unknown. Using activity-dependent tagging and sequencing, we profiled the DNA methylome and transcriptome of engram and non-engram nuclei from the basolateral amygdala (BLA) after contextual fear conditioning with and without post-learning sleep deprivation. Fear learning induces coordinated CpG and CpH DNA methylation remodeling specifically in engram neurons, with each showing unique temporal dynamics, revealing CpH methylation as a separate component of the engram's epigenetic signature. Interestingly, sleep deprivation partially reverses learning-induced methylation changes and impairs memory recall, indicating that sleep supports engram-specific epigenetic consolidation. Integrating the engram methylome with single-cell transcriptome profiling, we construct a multi-omic molecular atlas of the BLA fear engram and identify Mid1 as a learning- and sleep-regulated methylation target. BLA-wide and engram-specific Mid1 knockdown establishes its functional requirement for fear memory consolidation, providing an approach to identify behaviorally relevant epigenetic targets.