Genome-scale CRISPR/Cas9 screening of the deubiquitinase subfamily identifies USP49 as a protein stabilizer of PAX9 and MSX1 regulating odontogenesis
Deubiquitination of proteins by DUBs has emerged as a key regulator of protein stability and has been shown to be critically involved in signal transduction cascades. However, the function and regulation of individual DUBs are not well understood at the molecular level. Several screening approaches to identify DUBs and their target protein substrates in diverse cellular functions have been reported previously. For example, shRNA/siRNA libraries targeting different DUBs or overexpression of ectopically introduced DUBs have been used to investigate the impact of DUB function on target substrate protein stability. These conventional methods have several limitations, such as the limitation of RNAi or shRNA-based methods to transcripts, and these techniques often result in partial repression or restoration of gene functions in a few generations, with temporary effects. The results obtained from these methods may not be accurate and might lead to misinterpretations of the data. To overcome this, we developed a loss-of-function study based on individually generated single cell-derived biallelic DUB knockout clones in mammalian cells and provide an alternate and improved model to explore the molecular mechanism of DUBs. In this study, we used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) mediated DUB knockout screening kit based on genome-scale knockout of the entire set of genes that encode human ubiquitin-specific proteases (USPs), which are the largest subfamily of DUBs (>50 genes), and then we systematically applied the panel to screen DUB candidates that might regulate PAX9 and MSX1 proteins. PAX9 and MSX1 are critical for the transition in odontogenic potential from epithelium to mesenchyme. Although genetic mutations in PAX9 and MSX1 cause tooth agenesis, the regulation of PAX9 and MSX1 protein levels by deubiquitinating enzymes (DUBs) might be a crucial factor during tooth development. Our screening system identified USP49 as a key regulator of PAX9 and MSX1 protein stability. USP49 interacts and deubiquitinates PAX9 and MSX1 protein and upregulates several odontogenic transcription factors in human dental pulp stem cells (hDPSCs). In animal models, USP49 depletion was correlated with downregulation of Pax9 and Msx1 protein expression resulting in several morphological defects in calcified tissues such as reduced dentin growth, reduced enamel space, abnormal enamel formation, and irregular mineralization. Thus, the depletion of USP49 leads to an inadequate transition from bud to cap stage in the early stages of tooth development. This novel finding suggests that the USP49-PAX9-MSX1 axis could serve as a prognostic marker and novel regulatory target in odontogenesis and can serve as a catalyst for bioengineering tooth implant research to improve clinical care.