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: our data demonstrates that the initial phase of memory formation alters the chromatin accessibility landscape of activated neurons, with long lasting stable changes occurring predominantly within enhancer regions. Moreover, many of these enhancers did not return to their baseline state after stimulation ceased, but remain accessible and stable throughout all memory phases. We measured genome-wide changes of chromatin accessibility across different phases of memory. 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 ATAC-seq library preparation in 3 biological replicates.