Viral-mediated fluorescent labelling of activated hippocampal memory engrams to study epigenetic dynamics associated with gene expression

• vkTRAP enables selective labeling and tracing of hippocampal engram cells during fear memory. • vkTRAP exhibits fear memory, assessed by behavior and electrophysiology. • vkTRAP represents a versatile approach for efficient FANS of activated neuronal nuclei. • vkTRAP enables cell-specific epigenetic profiling of hippocampal engram cells. Memory formation activates a relatively sparse population of engram cells that store long-term memories. Changes in the epigenetic landscape and 3D chromatin architecture have been proposed as key candidate regulators of transcriptional waves that control gene expression in engram cells; however, isolating chromatin efficiently from engram cells has remained challenging. Double-transgenic T argeted R ecombination in A ctive P opulations (dTRAP) mice have enabled indelible EYFP labeling of hippocampal engram cells expressing the immediate-early gene (IEG) Arc when ArcCreER T2 mice are crossed with R26R- STOP-floxed-EYFP mice and exposed to learning paradigms. A major limitation of dTRAP mice is that labeling of activated hippocampal Arc + neurons with soluble EYFP compromises the efficiency of fluorescence-activated nuclear sorting (FANS) of engram nuclei, and hence isolation of chromatin. Here, we used viral-mediated delivery of GFP-KASH (AAV-PHP.eB-FLEX-EGFP-KASH) to ArcCreER T2 mice −generating vkTRAP mice- to enable precise and robust endogenous perinuclear fluorescent tagging of activated hippocampal neurons following contextual fear conditioning (CFC). At 24 h post-CFC (24 h-CFC), vkTRAP mice exhibited a robust freezing behavior. Electrophysiological recordings in CA1 hippocampal slices showed occluded long-term potentiation (LTP). Efficient FANS-based isolation of hippocampal engram nuclei enabled chromatin immunoprecipitation (ChIP) assays (detecting H3K4me3, H3K9ac and H3K27ac) at promoters of immediate-early ( Egr1 ) and plasticity-related ( Dlg4 /PSD95) genes. Expression peaks of both Egr1 and Dlg4/ PSD95 transcripts during memory acquisition (1 h-CFC) and consolidation (24 h-CFC) were accompanied by active epigenetic histone mark profiles. We conclude that vkTRAP provides a robust model to study epigenomic regulation in engram cells.