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.由去泛素化酶（Deubiquitinating enzymes，DUBs）介导的蛋白质去泛素化，已成为蛋白质稳定性的关键调控机制，并被证实广泛参与信号转导级联反应。然而，目前在分子层面，人们对单个DUB的功能及其调控机制仍缺乏深入了解。此前已有多项研究报道了多种筛选策略，用于在不同细胞功能中鉴定DUB及其靶蛋白底物。例如，靶向不同DUB的shRNA/siRNA文库，或异位过表达外源引入的DUB，曾被用于探究DUB功能对靶底物蛋白稳定性的影响。 这类传统方法存在诸多局限：基于RNAi或shRNA的技术仅能作用于转录本层面，且往往仅能在数代细胞内实现基因功能的部分抑制或恢复，效应具有暂时性。由此获得的实验结果可能并不准确，甚至会导致对数据的误读。为克服上述缺陷，我们构建了基于哺乳动物细胞中单细胞来源的双等位基因DUB敲除克隆的功能丧失性研究体系，并提供了一种可用于探究DUB分子机制的替代且更优化的研究模型。 本研究依托全基因组范围敲除人类泛素特异性蛋白酶（ubiquitin-specific proteases，USPs）——该家族是DUBs中最大的亚家族（包含超过50个基因）——的全部编码基因，使用了成簇规律间隔短回文重复序列/CRISPR相关蛋白（clustered regularly interspaced short palindromic repeats/CRISPR-associated，CRISPR/Cas9）介导的DUB敲除筛选试剂盒，随后系统性地运用该筛选体系，对可能调控PAX9与MSX1蛋白的DUB候选物进行筛选。 PAX9与MSX1对于牙源性潜能从上皮向间充质的转化至关重要。尽管PAX9与MSX1的遗传突变会导致牙齿发育不全，但去泛素化酶（DUBs）对PAX9与MSX1蛋白水平的调控，可能是牙齿发育过程中的关键影响因素。 我们的筛选体系将USP49鉴定为调控PAX9与MSX1蛋白稳定性的关键调控因子。USP49可与PAX9、MSX1蛋白相互作用并介导其去泛素化，同时在人类牙髓干细胞（human dental pulp stem cells，hDPSCs）中上调多种牙源性转录因子的表达。 在动物模型中，USP49的敲低会导致Pax9与Msx1蛋白表达下调，进而引发钙化组织的多种形态学缺陷，如牙本质生长受限、釉质间隙缩小、釉质形成异常以及矿化不规则。因此，USP49的缺失会导致牙齿发育早期阶段从蕾状期向帽状期的转化不充分。 这一新发现表明，USP49-PAX9-MSX1调控轴可作为牙发生过程中的预后标志物与新型调控靶点，同时可为生物工程牙齿植入研究提供新思路，以助力临床诊疗水平的提升。