Abstract Post-mitotic neurons in the mammalian brain form synapses that dynamically remodel throughout an individual’s lifetime to encode short- and long-term memories. Synaptic plasticity involves spatiotemporal fine- tuning of gene expression levels in response to environmental stimuli, including rapid transcription of immediate early genes on the time scale of minutes and longer-term global chromatin remodeling. The cis- acting genetic and epigenetic elements that govern activity-dependent expression are of outstanding interest toward understanding how experiences sculpt the brain. Here, we submit a proposal entitled ‘Elucidating the 3- D epigenetic determinants of activity-dependent gene expression in mammalian neurons’. We have assembled an interdisciplinary team with critical expertise in genome folding, epigenetics, chromatin engineering, neurobiology, synaptogenesis, electrophysiology, and computational biology. We aim to elucidate the causal link among long-range looping interactions, epigenetic modifications on the linear genome, expression of their spatial target genes, and the activity of mammalian neurons. We hypothesize that immediate early genes will functionally engage in singular short-range loops to rapidly activate expression on the time scale of seconds to minutes in response to the environmental stimulus of neuronal activation. By contrast, we posit that secondary response genes will spatially connect via architectural proteins into complex, long-range, pre-existing topological configurations to poise the genome for a second wave of expression on the order of hours to days in response to neuronal firing. To test our hypotheses, we will create high-resolution genome folding maps using the Hi-C during a time course of activation in mouse hippocampal neurons. We will identify activity- dependent enhancers and gene expression genome-wide and determine their temporal profile with respect pre-formed and activity-dependent loops. We will formulate mathematical models to predict activity-dependent expression of immediate early genes and secondary response genes from the timing of enhancer activation and looping contacts. By integrating single nucleotide variants linked to autism, schizophrenia, bipolar disorder, addiction, and attention-deficit/hyperactivity disorder with our models, we will predict the specific target genes and potential pathways involved in neurological disease. Finally, we will dissect the functional role for loops and enhancer activity in regulating the activity-dependent transcription of Bdnf and c-fos using CRISPR genome editing of architectural protein binding motifs and CRISPRi inhibition of specific enhancers. Our work wi...