One of the most remarkable features of the adult brain is its capacity to undergo structural and functional plasticity in response to sensory experience to learn new information and to adaptively respond to new demands posed by the environment. The cellular mechanisms that underlie this neural plasticity require activity-dependent gene transcription. Underscoring the biological significance of this experience-dependent gene expression to the human brain are the clear genetic associations between the molecular components of inducible gene expression and human developmental disorders of cognition, such as autism. A fundamental aspect of activity-regulated gene expression is the selective deactivation of the immediate early genes (IEGs) that are initially and rapidly induced by neuronal activity. This is in contrast to the delayed, prolonged transcription of delayed response genes (DRGs). The selective deactivation of IEGs is thought to be critically important for shaping cellular plasticity, as the precise levels of IEG protein products, such as ARC, determine the strength of synaptic connections within neural networks. We intend to uncover the mechanisms of this IEG-specific deactivation by taking advantage of the recent comprehensive identification of neural activity-dependent enhancers. There are conflicting models concerning whether the kinetic transcriptional differences between IEGs and DRGs are encoded in cis by the enhancers, the promoters, or both. Thus, we are well positioned to test each of these predictions. We will accomplish this via three key approaches: In Aim 1, we will use ChIP-seq and eRNA-sequencing to monitor genome-wide enhancer activity over a time course of neural activity to identify subsets of enhancers that are deactivated in the continued presence of activity. In Aim 2, we will isolate each enhancer sequence of interest and study its intrinsic activity in a global and unbiased reporter assay to identify those specific enhancer sequences that inactivate under continued activity and may therefore coordinate IEG negative regulation. In Aim 3, we will evaluate at representative genomic loci whether endogenous enhancer landscapes are sufficient to encode IEG-specific deactivation, independent of the core promoter sequences. We will accomplish Aim 3 by using genomic engineering techniques to exchange core promoters of two IEGs and two DRGs to determine whether IEG enhancers possess intrinsic negative regulatory activity that down-regulates the transcription of target promoters under prolonged depolarization conditions. This work will identify cis regulatory sequences that coordinate IEG negative regulation and will provide fundamental insight into the mechanisms of activity-regulated enhancer functioning and the encoded inducible-regulatory logic that differentiates promoters and enhancers. It will advance our pursuit of the molecular mechanisms governing IEG deactivation and has the potential to identify novel molecular targets that could aid in the development of therapeutics to treat diseases of cognition or memory decline.