Project Summary / Abstract In the hours after learning, the activation of gene expression follows a specific pattern, producing transient waves of expression needed for long-term memory consolidation. These changes require non-genetic (i.e., “epigenetic”) events, including modifications to: DNA-organizing proteins known as histones, the DNA itself, and the accessibility of DNA to proteins. Additionally, the molecular changes necessary for memory require a form of RNA-based regulation. In the absence of such changes, the long-lasting regulation of gene expression during memory storage fails, and this could account for defects in memory that accompany many psychiatric disorders. Our long-term goal is to define novel epigenetic mechanisms underlying memory storage and synaptic plasticity by taking advantage of recent advances in our understanding of histone modifications, in the development of single-cell RNA technology, and in the function of regulatory mechanisms mediated by small noncoding RNAs. During the previous funding period, we defined a novel metabolic source of acetyl-CoA in the nucleus and have obtained preliminary data about new forms of histone acylation and crotonylation associated with spatial learning. We also developed bioinformatic tools to analyze single nuclear RNA sequencing data to identify neurons activated by learning. We further established the reversal of microRNA (miRNA)-mediated mRNA silencing as a novel epigenetic means of regulating activity-dependent translation, linking synaptic activity to translational upregulation of key memory-related targets. These major findings provide the basis of our proposed experiments that we believe will define the next frontiers in our understanding of the epigenetic mechanisms of memory consolidation. In Specific Aim 1, we will examine the impact of a novel histone modification, histone crotonylation, on the epigenetic landscape and gene expression during memory consolidation. In Specific Aim 2, we will define cell type-specific transcriptional signatures of the hippocampal neurons during memory consolidation and retrieval. In Specific Aim 3, we will elucidate the microRNA- dependent mechanisms that regulate long-term memory and synaptic plasticity driven by a microRNA processing complex. An understanding of these key epigenetic regulatory mechanisms involved in the consolidation and storage of long-term memories is expected to ultimately lead to the development of new treatments for the debilitating cognitive deficits associated with psychiatric disorders such as schizophrenia, autism, bipolar disorder, post-traumatic stress disorder and depression.