Non-canonical K27 polyubiquitination is a sex-specific regulator of contextual fear memory in the hippocampus but not the amygdala

Polyubiquitination is a process whereby multiple ubiquitin proteins link to each other on a target substrate, marking that substate for various cellular processes of which protein degradation via the proteasome is the most common. Recently, evidence has emerged suggesting that the most common forms of proteasome-dependent (K48) and proteasome-independent (K63, M1) polyubiquitination have sex-specific roles in contextual fear memory formation in the amygdala and hippocampus. However, there are 8 different linkage sites at which polyubiquitin chains can form, most of which have not been studied in the brain. Lysine 27 (K27) polyubiquitination is a less common, non-canonical mark that has not been well studied and may be connected to the protein degradation process. To date, K27 polyubiquitination has never been examined in the brain under any condition. Here, we found that K27 polyubiquitination was selectively increased in the hippocampus after contextual fear conditioning in female, but not male, rats, though neither sex showed changes in this polyubiquitin mark in the amygdala. Consistent with this, CRISPR-dCas13-mediated knockdown of K27 polyubiquitination in the hippocampus selectively impaired contextual fear memory retention in the hippocampus of females, but not males. Proteomic analyses revealed ACAT1 as a target of K27 polyubiquitination in the female hippocampus following fear conditioning, though this mark was not associated with degradation of the target protein. Together, these data suggest that K27 polyubiquitination has a sex-selective role in fear memory formation in the hippocampus. These findings advance our understanding of molecular mechanisms of fear memory formation and the importance of sex as a biological variable in this process • K27 polyubiquitination levels increased in the female hippocampus, but not amygdala, following contextual fear contextual fear conditioning • K27 polyubiquitination expression levels remained unchanged in the male hippocampus and amygdala following fear conditioning. • Knockdown of K27 polyubiquitination in the hippocampus impaired contextual fear memory in females, but not males • ACAT1 was the only target of K27 polyubiquitination in the female hippocampus following contextual fear conditioning