Project Summary/Abstract Nervous system function requires the integration of diverse neuronal subtypes into neural circuits that elicit thought and behavior. Despite the extensive diversity of neuronal subtypes, all neurons share key features, including chemical synapses. Current models indicate that shared neuronal genes, including pan-synaptic genes, are independently regulated by different combinations of transcription factors in distinct neuronal subtypes, whereas subtype-specific synaptic genes are regulated by specific transcription factors called terminal selectors. Our data demonstrate that pan-neuronal and subtype-specific synaptic genes are temporally coordinated during synaptogenesis. We have identified a candidate gene regulatory network (GRN) that includes two known pioneer factors, and propose a model in which positive and negative regulation of chromatin accessibility underlie the coordinated regulation of synaptic gene expression to promote synapse formation. In Aim 1, we propose genomic and in vivo functional validation to test the model that two of our candidate transcription factors, GA-rich motif binding factors CLAMP and GAGA factor, bind to the same promoters of synaptic genes to exert opposite effects on chromatin accessibility and transcription. In Aim 2, we propose a comprehensive and unbiased approach to identifying the GRNs underlying coordinated synaptic gene expression through single-nucleus RNA- and ATAC-Seq. Successful completion of these studies will advance our understanding of how the development of shared neuronal traits is coordinated with cell fate acquisition, and may inform neuronal reprogramming of stem cells.