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 differentially expressed gene (DEG) analysis revealed relatively low amount of changes in the early phase, when compared to basal-state neurons (Basal vs. Early). In contrast, neurons from late phase presented higher numbers of DEGs despite there being more similarity in chromatin accessibility at these time points (Early vs. Late; Late vs. Reactivated). Our studies illuminate for the first time the unique transcriptional landscape of reactivated engram cells where we observed up-regulation of genes involved in mRNA transport and local protein translation in synaptic compartments. These changes corresponded to morphological and functional changes in those neurons. To elucidate how changes in chromatin accessibility and promoter-enhancer interactions were affecting gene expression, we performed nuclear RNA-seq (nRNA-seq) from all the neuronal sub-populations. 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 nRNA-seq library preparation (SmartSeq2 protocol) in 3 biological replicates.