Additional sex combs like transcriptional regulator 1 (ASXL1) is indispensable for normal development and myeloid differentiation. 1-3 Mutation of ASXL1 is one of the most common mutations among various myeloid malignancies, especially in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia, and is an independent poor prognostic factor among patients with MDS. 4 We previously demonstrated that the expression of C-terminal-truncated ASXL1 inhibited myeloid differentiation and induced an MDS-like disease in mice through the dominantnegative repression of polycomb repressive complex 2 (PRC2) function. 3 Asxl1 KO mice developed similar diseases, 1 suggesting that regulation of ASXL1 expression or turnover of ASXL1 proteins is closely associated with myeloid malignancies. However, how the expression levels of ASXL1 proteins are regulated remains to be elucidated. Furthermore, endogenous ASXL1 proteins are usually difficult to detect, suggesting the fragility of ASXL1. In this study, we demonstrate that ASXL1 is subject to K351 ubiquitination and subsequent degradation, and that interaction with the deubiquitinating enzyme, ubiquitin-specific peptidase 7 (USP7), stabilizes ASXL1.

To identify the modification of additional sex combs like transcriptional regulator 1 (ASXL1) proteins that regulate protein stability, we first performed mass spectrometry. FLAG-tagged wildtype ASXL1 (ASXL1-WT) or C-terminal-truncated ASXL1 (ASXL1-MT) was precipitated from transfected HEK293T cells, followed by incubation with an anti-FLAG Affinity Gel and nano liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) after trypsin digestion. We found that ubiquitination occurred on K351 of ASXL1 (Figure 1a and Supplementary Table 1), suggesting that K351 ubiquitination and the subsequent proteasome-mediated degradation contribute to the difficulty in detecting ASXL1 proteins. In fact, treatment of HEK293T cells and HL60 cells with MG-132, a proteasome inhibitor, enabled the detection of endogenous ASXL1 proteins (Figure 1b). Likewise, retrovirally overexpressed ASXL1-MT (1900-1922del; E635RfsX15), derived from myelodysplastic syndrome (MDS) patients, and ASXL1-WT proteins were stabilized in the presence of MG-132 (Figure 1c and Supplementary Figure S1). Next, to clarify the function of the K351 ubiquitination, we constructed and expressed a mutant of ASXL1-MT1 (1934dupG; G646WfsX12) or ASXL1-MT2 (1900-1922del; E635RfsX15) with an arginine substitution for K351 (K351R) in HEK293T cells and in mouse IL-3-dependent myeloid 32Dcl3 cells. Using western blotting, we found that, as expected, the K351R mutants of ASXL1-MTs exhibited increased stability compared with ASXL1-MTs (Figure 1d). Kim et al. 5 previously performed a systematic and quantitative assessment of the ubiquitin modified proteome (ubiquitinome) and identified thousands of potentially ubiquitinated proteins, including ASXL1 which can be modified at K1437 as well as at K351. Thus, we constructed three ASXL1 mutants: K351R, K1437R and K351R/K1437R. As shown in Figure 1e, K351 appeared to be mainly required for the degradation of the ASXL1 proteins. These experiments strongly indicated that ubiquitination at K351 is associated with ASXL1 stability. We previously reported that