A Novel Non-Canonical Phosphodegron Regulates EZH2 Proteasomal Degradation and H3K27 Trimethylation Activity in Hematopoietic Malignancies

Introduction: Enhancer of zeste homologue 2 (EZH2) is a critical enzymatic subunit of the polycomb repressive complex 2 (PRC2) which trimethylates histone H3 (H3K27) to mediate gene repression. EZH2 is aberrantly activated and overexpressed in several hematologic malignancies and is associated with aggressive clinical behavior. In particular, recurrent mutations targeting Y641 of EZH2 are common in germinal center derived lymphomas. Critically, the cellular machinery and mechanisms that regulate EZH2 by post-translational modification are not well understood. We have previously shown that β-transducin repeat containing protein (βTrCP) is the substrate specific adaptor for ubiquitin mediated degradation of EZH2, however the degron motif by which βTrCP recognizes EZH2 is unknown. In order to better understand the post-translational regulation of EZH2, we sought to identify the non-canonical degron motif that regulates EZH2 stability.

Methods: βTrCP recognizes a phosphorylated consensus degron motif DpSG(X)_2-5pS in its substrates. To investigate the post-translational modifications regulating the half-life of EZH2 protein we generated a series of truncation and site directed mutants of βTrCP including deletion of 7^th WD repeat domain and mutation of R474A. The afore mentioned βTrCP mutants were assessed for their interaction with EZH2. Having established the non-canonical degron (DpS⁶⁰¹KNVpS⁶⁰⁵) in EZH2, to further characterize the role of this degron in EZH2-βTrCP interaction we generated several EZH2 mutants including site-directed deletion of putative degron motif (DSKNVS) or alanine substitution mutations of critical serine residues S601A and S605A alone or in combination. Using wild-type as well as EZH2 mutants we investigated their ability to interact with βTrCP and consequent lysine (K48) linked polyubiquitination on EZH2 using co-immunoprecipitation and western blotting techniques. Further we characterized the impact of the identified degron in EZH2 stability using cycloheximide chase mediated protein turnover analysis of wild type and EZH2 degron deletion and serine (S601A/S605A) substitution mutants. Moreover, we investigated the impact of increased stability of EZH2 on its H3K27 trimethylation activity using wild type and degron deletion and serine substitution mutants by western blot analysis. We also investigated the impact of increased stability of EZH2 via degron modification by analyzing its downstream target p21.

Results: Extensive interaction between substrates and R474 and Y488 residues in the 7^th WD repeat domain of βTrCP is critical for stable binding and this is mediated via the conserved degron motif. In co-immunoprecipitation experiments, the mutation of R474A or deletion of 7^th WD repeat domain in βTrCP abrogated the EZH2-βTrCP interaction. Further, the deletion of the identified degron DSKNVS in EZH2 or substitution of critical serine residues residing within this degron abrogated EZH2-βTrCP interaction and consequent lysine K48-linked polyubiquitination and proteasomal degradation. Half-life measurements by cycloheximide chase experiments demonstrated that the degron deletion or substitution mutations exhibit increased protein stability as compared to wild type EZH2. Motif analysis revealed that the novel EZH2 degron harbors the GSK3β recognition and phosphorylation motif (DpSKNVpS) involved in the phosphorylation of several known βTrCP substrates. Therefore, we examined the involvement of GSK3β in EZH2-βTrCP interaction. Pharmacologic inhibition of GSK3β compromised EZH2- βTrCP interaction in a dose dependent manner. Further, deletion of the degron or alanine substitution mutation of the critical serine residues (S601A and S605A) within the degron increased H3K27 trimethylation activity of EZH2 and consequently increased its repression on its downstream target p21.

Conclusion: In the present study, we identify the presence of a novel non-canonical degron and GSK3β-mediated phosphorylation of the site that regulates EZH2 stability and activity. Our studies demonstrate that βTrCP/GSK3β axis plays an important role in controlling H3K27 trimethylation activity by targeting EZH2 for degradation. We propose that this newly identified mechanism might help in designing novel therapeutic approaches for clinical management of hematologic malignancies driven by aberrant activity of EZH2.