Genome-wide expression profiling of USP9X/Y knockdown in the DU145 cell culture model

Public transcriptomics studies have shown that several genes display pronounced gender differences in their expression in the human brain, and these differences may influence the clinical manifestations and risk for neuronal disorders. While disease relevant implications have already been proposed for gender differences in hormone levels, life style and genetic diversity, a systems level analysis of brain gene expression disparities between the genders in the context of brain disorders like Alzheimer’s disease (AD) has not yet been conducted. Here we apply a transcriptome-wide analysis to discover genes with gender-specific expression and significant alterations in public post mortem brain tissue from AD patients compared to unaffected controls. We identify the sex-linked ubiquitin specific peptidase 9 (USP9) as an outstanding candidate gene with one of the largest ratios of male to female median expression levels during adulthood and a highly significant underexpression of the Y-chromosomal form of the gene (USP9Y) in male AD patients compared to controls. Previous studies have shown that USP9 can modulate the phosphorylation of the microtubule-associated protein tau (MAPT), whose dysregulation in AD is thought to play a key role in disease. Here, after observing a high positive correlation between USP9Y and MAPT expression in public transcriptomics data from AD case/control studies, we show that USP9 knockdown results in significantly decreased MAPT expression in the in vitro DU145 cell culture model. From the analysis of transcriptomics data for the knockdown in DU145 cells and prior knowledge from the literature, we derive a model for a USP9-dependent regulatory mechanism modulating MAPT expression via BACH1 and SMAD4. Overall, the combined transcriptomics analyses and identified functional links between USP9 and MAPT suggest USP9 may partially account for gender differences observed in tauopathies and could provide a new target for intervention strategies to modulate MAPT expression and phosphorylation. To assess the biomolecular effects of knocking down USP9 on a transcriptome-wide scale, the human DU145 prostate cancer cell culture model was chosen, after confirming that both USP9X and USP9Y are robustly expressed in this model. ShRNA constructs were then designed for the knockdown of USP9X and USP9X/Y (i.e. targeting USP9X and USP9Y jointly) to infer potential USP9Y-specific gene alterations indirectly by comparing both knockdowns. As readout, microarray expression profiling using the Affymetrix Human Gene 2.0 platform was performed with triplicate samples for each of five considered conditions (USP9X/Y knockdown, USP9X knockdown, scrambled RNA control, transfection reagent control, and unperturbed control).