Mapping the epigenomic and transcriptomic interplay during memory formation and recall in the hippocampal engram ensemble

Purpose: we utilized a mouse model that permanently labels neurons activated throughout a specific experience to decipher the interplay between chromatin accessibility, 3D-chromatin architecture and transcriptional changes across different memory phases. Non activated (basal) and activated neurons during memory encoding (early), consolidation (late) and recall (reactivated) were sorted and subjected to nuclear RNA sequencing (nRNA-seq) to determine gene expression, ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to assess chromatin accessibility, chromosome conformation capture (Hi-C) to identify global 3D -genome architecture and promoter capture Hi-C to identify the long range promoter-enhancer interactions. Results: We have found that during each memory phase, the same promoters interact more frequently with a distinct subset of enhancers (i.e. unique). We also identified a smaller subset of interactions in which the promoters were interacting with the same enhancers across different memory phases (i.e. common). Furthermore, Reactivated neurons presented significantly stronger interaction scores (as calculated by CHiCAGO). Hence, although the number of unique interactions was similar across early, late and reactivated states, stronger interaction scores indicate that specific promoter- enhancer interactions occur more frequently during memory recall. By utilizing promoter-capture Hi-C (pc-HiC) technique, we studied the precise changes that occur in long-range promoter-enhancers interactions during the process of memory encoding, consolidation and recall. For this study, we used custom-designed collection of 10,000 biotinylated RNA “baits” targeting ~5000 promoters. To obtain 10,000 nuclei per condition, we pooled the hippocampal tissue from multiple mice (10-12) and isolated each group of cells via fluorescence-activated cell sorting (FACS). Collected nuclei were subjected to pc-HiC library preparation in 3-5 biological replicates.