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

The epigenome and three-dimensional (3D) –genomic architecture are emerging as key factors in the dynamic regulation of different transcriptional programs required for neuronal functions. Here we utilize an activity-dependent tagging system in mice to determine the epigenetic state, 3D-genome architecture, and transcriptional landscape of engram cells over the lifespan of memory formation and recall. Our findings reveal that memory encoding leads to an epigenetic priming event, marked by increased accessibility of enhancers without corresponding transcriptional changes. Memory consolidation subsequently results in spatial reorganization of large chromatin segments and promoter-enhancer interactions. Finally, with reactivation, engram neurons utilize a subset of de novo long-range interactions, where primed enhancers were brought in contact with their respective promoters to up-regulate genes involved in local protein translation in synaptic compartments. Collectively, our work elucidates the comprehensive transcriptional and epigenomic landscape across the lifespan of memory formation and recall in the hippocampal engram ensemble. The formation and preservation of long-term memories depends on coordinated gene expression and synthesis of synaptic proteins(1). These molecular processes act within a specific population of neurons, referred to as engram cells(2–4). Recent approaches using activity-dependent expression of reporters, provided a framework for exploring the engram ensemble(5–8), but the molecular mechanisms that govern memory storage and retrieval remain poorly understood. Specifically, epigenetic modifications and 3D -genomic architecture are emerging as a key factors in dynamic regulation of gene expression(9–17), and there is an increasing appreciation of their importance in neuronal function, development and disease(14,16,18) Here, we utilized the Targeted Recombination in Active Populations (TRAP) mouse model(5,6), in which activated neurons expressing the Activity Regulated Cytoskeleton Associated Protein, (Arc) gene, are permanently tagged in an inducible manner. Activated neurons during memory encoding, consolidation and recall were sorted and subjected to nuclear RNA sequencing (nRNA-seq), Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and chromosome conformation capture (Hi-C). Our data demonstrates that memory encoding leads to a genome-wide increase in chromatin accessibility, without expected changes in gene expression. Furthermore, we demonstrate that late phase of memory consolidation was associated with re-localization of large chromatin segments (sub-compartments) from inactive to permissive environments, and reorganization of the promoter-enhancer interaction landscape. Finally, reactivation of the neurons during memory recall is associated with de novo promoter-enhancer interactions, utilizing a large subset of the enhancers that were primed during memory encoding. These promoter-enhancer interactions are associated with a robust change in the expression of genes involved in local protein synthesis and synaptic morphogenesis.