Project Summary The sex-steroid hormone 17-estradiol (E2) has a well-established role in mediating neuronal physiology, altering gene expression, and facilitating memory formation. Despite decades of research on E2’s neuromodulatory effects, much remains unknown regarding which specific gene targets regulated by E2 contribute to its effects on neuronal function and memory. This is in part due to a lack of experimental feasibility, as the tools necessary for examining cell-type specific transcriptional regulation by E2, and targeted manipulation of specific E2-sensitive genes, were not previously available. Here, I propose to overcome these hurdles by using single-nuclei RNA- and ATAC-sequencing, which will allow for comprehensive and unbiased identification of gene targets regulated by E2 in both sexes at the single-cell level (Aim 1). This approach is advantageous to the few existing studies examining transcriptomic regulation of sex-steroid hormones, which use brain homogenates containing a variety of cell types for sequencing experiments. Although such work has been informative, the receptors through which E2 orchestrates transcriptional control are expressed in a wide variety of distinct cell types. As such, higher resolution approaches such 10X snRNA-seq and snATAC-seq at the individual cell level will be necessary to understand the full myriad of estrogenic regulation of gene expression. Another limitation of previous studies has been the inability to directly manipulate E2-sensitive gene targets to empirically test the requirement of such changes in expression on memory formation. This proposal will overcome such limitations by using cutting-edge CRISPR/dCas9-based epigenetic editing approaches to test the requirement of E2 action at specific gene loci in mediating its beneficial mnemonic effects (Aim 2). Finally, although circulating levels of E2 have been the focus of much neuroendocrinology research, E2 biosynthesis also occurs locally in the adult brain of both sexes, via a process regulated by the enzyme aromatase (Cyp19a1). Aim 3 will use a CRISPR/dCas9 approach to bidirectionally regulate aromatase expression specifically in neurons, to test the role of neuron-specific E2 synthesis in memory formation. Collectively, these experiments will provide much needed insight into how E2 acts as a key modulator of neuronal function and memory in both sexes.