Persistent chromatin loops shape gene expression plasticity upon stimulation and restimulation of human neurons

Persistent molecular correlates of long-term memory storage remain an open question. Here, we stimulate and re-stimulate human neurons and use multi-modal single-nucleus technologies to query DNA methylation, higher-order chromatin folding, and gene expression. We find enduring traces of activity-gained and activity-lost chromatin loops. Genes anchoring persistent activity-gained loops exhibit activity-upregulated expression, whereas persistent activity-lost loops anchor activity-downregulated genes that remain repressed five days post-stimulation. CTCF-bound looped enhancers and promoters are refractory to activity-dynamic DNA methylation. Looped enhancers bound by CTCF can exhibit memory of activity-induced histone modifications and persistent expression of activity-upregulated genes. Upon second stimulation, activity-upregulated genes are robustly re-induced when unlooped but remain nonresponsive at persistent loops akin to habituation. Activity-independent gene expression can be downregulated when unlooped but protected from homeostatic downscaling when anchored in persistent loops. Our data reveal long-term genome folding persistence linked to plasticity of activity-dependent gene expression during recall in human neurons.