Single-cell chromatin tracing reveals multimodal molecular programs during memory formation

Experience-dependent activity is converted to coordinated molecular programs in neuronal ensembles during memory formation. However, due to the sparsity of the ensembles and the transience of immediate early gene (IEG) expression, it is unclear how IEGs engage downstream secondary response genes (SRGs) to regulate learning-specific neuroplasticity. Here, we generated a single-cell multiomic atlas of aversive learning and developed ChromTRAP, which retrospectively identifies recently activated neuronal ensembles from AP-1 (FOS/JUN)-centred chromatin traces. We integrated transcription, chromatin accessibility, histone modifications, and FOS occupancy across the amygdala, hippocampus, and prefrontal cortex. This revealed regulatory programs of learning-associated genes (LAGs), defined as SRGs preferentially induced by associative learning relative to baseline activity or independent stimulus exposure. These programs followed a brain-region- and cell-type-specific proximal-distal regulatory logic: gene-proximal Polycomb-associated H3K27me3 remodeling and AP-1-bound H3K27ac-marked distal enhancers. LAGs were further associated with enhanced intercellular signaling and MEF-family activity. Our findings establish a single-cell multiomic framework for linking learning experience to layered epigenetic regulation during activity-dependent neuroplasticity.