DNMT3A Stability Is Maintained By Ubiquitin-Specific Peptidase 11 (USP11) and Sumoylation Countering Degradation

DNA methyltransferase 3A (DNMT3A) regulates hematopoietic stem cell (HSC) differentiation and is frequently mutated in clonal hematopoiesis (CH) and hematologic malignancies. Our recent study indicated that ~30% of CH-related DNMT3A mutations downregulate its expression at the protein level. Using CRISPR screening, this study also revealed that the E3 ubiquitin ligase adaptor DCAF8 induced protein degradation in unstable (downregulated) DNMT3A mutants in 293T cells. Based on these results, we hypothesize that understanding the mechanisms of DNMT3A protein turnover will be useful to develop a new therapeutic approach for hematologic malignancies. However, the mechanisms of DNMT3A protein turnover remain incompletely understood, and it is uncertain whether our findings are generalizable to other cell lines, animal models and clinical applications. First, we repeated CRISPR screening to examine factors that mediate the stability of unstable DNMT3A1W297Del mutant protein in a neuroblastoma cell line (Kelly cells). As in our previous study (293T cells), DCAF8 was enriched as a DNMT3A1W297Del degrader. We also identified several deubiquitinating enzymes (USP10, USP11 and USP41) as potential stabilizers in the screening. Next, using immunoprecipitation mass spectrometry (IP-MS), we analyzed the interactome between DNMT3A1W297del and deubiquitinating enzymes with or without the E1 ubiquitin inhibitor, which resulted in the stabilization of DNMT3A1W297del. Several deubiquitinating enzymes (USP1, USP7, and USP11) increased their interaction with DNMT3A1W297del after DNMT3A1 W297del deubiquitylation. USP11 was identified as a stabilizer in both CRISPR and IP-MS screenings. These data indicate that DCAF8 plays a major role in degradation of DNMT3A1W297Del, and USP11 is one of the strongest candidates for a major DNMT3A deubiquitinating enzyme. We then examined whether USP11 was involved in DNMT3A1 W297del protein expression in the presence or absence of DCAF8. USP11 overexpression increased DNMT3A1W297del and the phenotype was boosted in DCAF8 KO 293T cells. We also assessed whether USP11 was involved in DNMT3A1 and -3A2 protein turnover, performing co-IP with both isoforms. Interestingly, USP11 strongly interacted with the long isoform of DNMT3A (DNMT3A1) and stabilized its protein expression, but not the short isoform (DNMT3A2). Next, we performed co-IP-MS to compare which proteins differentially interact with DNMT3A1WT or DNMT3A1W297del. Interestingly, proteins involved in SUMOylation strongly and preferentially interacted with DNMT3A1WT. We also performed the co-IP-MS with DNMT3A1W297del in the presence or absence of DCAF8; DNMT3A1W297del preferentially interacted with SUMO proteins in DCAF8 KO cells. Overall, the interaction between DNMT3A1 and SUMOylation proteins is essential for the stabilization of DNMT3A1. Next, we also examined whether USP11 is involved in DNMT3A1 SUMOylation. We performed co-IP with DNMT3A1, and we found that USP11 induces DNMT3A1 SUMOylation. We also confirmed that USP11 promotes the binding of multiple E3 SUMO ligases (CBX4 and PIAS1) to DNMT3A1. Furthermore, we treated Kelly and THP-1 cells with TAK-981, an inhibitor of the SUMO activating enzyme; TAK-981 induced DNMT3A1 destabilization. Finally, we examined whether USP11 was involved in the methyltransferase activity on DNMT3A1 using the HOXA5-Snrpn-BFP reporter system. We found that USP11 overexpression enhanced the methyltransferase activity of DNMT3A1. Taken together, these data show that USP11 promotes DNMT3A1 stabilization through SUMOylation. In summary, the deubiquitinating enzyme USP11 SUMOylates DNMT3A1 and maintains steady-state protein levels, counteracting the activity of DCAF8. Using our molecular findings, we will pursue the development of therapeutic strategies to modulate DNMT3A to impact CH and hematologic malignancies.