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Although identification of substrates for an enzyme is a key step in elucidation of its biological functions, detection of the interaction between enzymes and substrates remains challenging. We recently developed a new approach, termed differential proteomics-based identification of ubiquitylation substrates (DiPIUS), for the discovery of substrates of ubiquitin ligases. We have now applied this approach to Fbxw7, the F-box protein component of an Skp1-Cul1-F-box protein-type ubiquitin ligase and, thereby, identified two similar transcription factors, old astrocyte specifically induced substance (OASIS) and BBF2 human homolog on chromosome 7 (BBF2H7), as candidate substrates. Coimmunoprecipitation analysis confirmed that the and isoforms of Fbxw7 interact with OASIS and BBF2H7 in vivo. Sustained overexpression of Fbxw7 resulted in marked down-regulation of OASIS and BBF2H7, whereas RNAi-mediated Fbxw7 depletion stabilized both proteins. Mutation of a putative Cdc4 phosphodegron in OASIS and BBF2H7 attenuated their association with Fbxw7 and resulted in their stabilization. Depletion of Fbxw7 promoted the differentiation of mouse C2C12 mesenchymal cells into osteoblasts in association with the accumulation of OASIS. Conversely, overexpression of Fbxw7 in C2C12 cells resulted in down-regulation of Col1A1 mRNA, a target of OASIS. Conditional ablation of Fbxw7 in primary mouse mesenchymal cells promoted chondrogenesis in association with up-regulation of BBF2H7, whereas overexpression of Fbxw7 inhibited chondrogenesis in ATDC5 cells. Collectively, our results suggest that OASIS and BBF2H7 are bona fide substrates of Fbxw7 and that Fbxw7 controls osteogenesis and chondrogenesis by targeting OASIS and BBF2H7, respectively, for degradation.