Phosphorylation of RPT6 controls its ability to bind DNA and regulate gene expression in the hippocampus during memory formation

We used RNA-sequencing along with novel genetic approaches and biochemical, molecular, and behavioral assays to test the hypothesis that pRPT6-S120 functions independently of the proteasome to bind DNA and regulate gene expression during memory formation. RNA-sequencing following siRNA-mediated knockdown of free RPT6 revealed 46 gene targets in the dorsal hippocampus of male rats following fear conditioning, where RPT6 was involved in transcriptional activation and repression. Through CRISPR-dCas9-mediated artificial placement of RPT6 at a target gene promoter, we found that RPT6 DNA binding alone was sufficient for altering gene expression following learning. Further, CRISPR-dCas13-mediated conversion of S120 to glycine on RPT6 revealed that phosphorylation at S120 is necessary for RPT6 to bind DNA and properly regulate transcription during memory formation. Together, we reveal a novel function for phosphorylation of RPT6 in controlling gene transcription during memory formation. Overall design: Male Sprague-Dawley rats were injected into the dorsal hippocampus CA1 region with Accell siRNA against Psmc5 or control (Scr-siRNA). Five days later animals were trained to contextual fear conditioning and the CA1 region (dorsal) was dissected, RNA collected via columns and used for RNA-seq analysis