Distal enhancer elements in DNA enable higher-order chromatin interactions that facilitate gene expression programs and thus contribute to cellular phenotype and function. Enhancers regulate numerous aspects of cell and tissue-specific gene expression patterns in the developing and adult brain, and have been highly implicated in mental health and disease. In neuronal systems, enhancer elements are subject to widespread, bidirectional transcription that yields non-coding enhancer RNA (eRNA). However, the precise function of eRNAs is still unclear, with different models proposing unique regulatory functions. Emerging evidence suggests that eRNAs function to modify epigenetic states at gene regulatory elements, which are critical for long-term behavioral and neuronal plasticity. Specific Aim 1 of this proposal seeks to utilize whole-genome sequencing approaches to define transcriptional and epigenetic signatures of neuronal activity at enhancers. In addition to revealing new therapeutic candidates, this aim will employ novel targeted eRNA delivery strategies based on CRISPR technology to allow manipulation of eRNAs at specific enhancers to determine their function. Specific Aim 2 will examine interactions between eRNAs and epigenetic profiles at enhancers and promoters of linked genes, which will establish the hierarchical relationships between molecular interactions at enhancers. Specific Aim 3 will examine the contributions of eRNAs to neuronal function and memory formation in the adult brain using hippocampus-dependent contextual fear conditioning, a robust assay of learning and memory that requires activity-dependent gene transcription. This novel approach will demonstrate the necessity of unique eRNAs at specific genes for neuronal activity and behavior, and also enable the first investigation of whether modulation of eRNAs is sufficient to alter memory function. In addition to revealing the exact nature and scope of eRNAs in neuronal gene regulation, this proposal will pave the way for future experiments that will explore how manipulation of enhancers could be used to reprogram circuits that have become maladaptive in mental illness and cognitive disease states.