Project Summary/Abstract The broad goal of this proposal is to understand how fear memories are formed and stored in the brain. Post- traumatic stress disorder (PTSD) affects nearly 5% of the world population, however, current treatments have limited efficacy in reversing the symptoms of this disorder. Furthermore, females are more likely than males to develop PTSD, though the mechanisms controlling this sex-dependent predisposition remain equivocal. Recently, work from our group and others have implicated protein degradation mediated by the ubiquitin- proteasome system (UPS) in fear memory formation in the amygdala, the primary brain region involved in emotional processing. However, the functional role of protein degradation in fear memory formation has yet to be identified. Furthermore, previous studies on UPS-mediated protein degradation have focused exclusively on males, so little is known about whether similar UPS mechanisms control the formation of fear memories in females. In our preliminary studies, we found that in males protein degradation was localized to the nucleus of amygdala cells following fear learning, suggesting a potential role in transcriptional control, though this has never been directed tested. Surprisingly, females did not show any changes in UPS-mediated protein degradation in the amygdala following fear learning though did have elevated baseline ubiquitin-proteasome activity in comparison with males, which was associated with increased DNA 5-hydroxymethylation (5-hmc) of Uba52, one of the ubiquitin coding genes. This suggests that elevated baseline UPS activity in females could be regulated by altered DNA methylation of Uba52 and may be sufficient for them to acquire fear memories. The work in this proposal is designed to answer these important questions about sex-dependent differences in the role of nuclear protein degradation in transcriptional control during fear memory formation. Using a combination of pharmacology and sophisticated CRISPR-dCas9 manipulations of proteasome activity in combination with mass spectrometry and whole genome next generation RNA-seq technology, Aim 1 will test whether nuclear protein degradation is involved in transcriptional control during fear memory formation and whether this varies between males and females. Aim 2 will use cutting-edge CRISPR-dCas9 technology to control the methylation state of Uba52 and will test how this effects baseline differences in ubiquitin- proteasome activity in the amygdala of males and females. Finally, in Aim 3 we will using CRISPR-dCas9 technology to test whether increased baseline protein degradation in the amygdala of females primes them to acquire fear memories. Collectively, this study will answer important questions about sex-dependent differences in the role of protein degradation in fear memory formation. The results obtained from this project could have important implications for understanding the etiology of sex-related differences in fear memory ...