In this study, we present evidence demonstrating that Ezh2 is subject to phosphorylation at two highly conserved residues, Thr-345 and Thr-487, which are bona fide CDK1 substrates. Consistent with the cell cycle-regulated activity of CDK1, Ezh2 phosphorylation is enriched during mitosis. Phospho-Ezh2 exhibits reduced stability due to its ubiquitination and degradation by the proteasome. In addition, cells expressing Ezh2 where both threonines are mutated manifest a proliferative disadvantage in at least two cell types. Thus, our studies establish a role for Thr-345 and Thr-487 in regulating Ezh2 stability, a novel consequence of CDK1-mediated phosphorylation.
The significance of CDK1-mediated regulation of Ezh2 stems from the necessity for a cell to transmit its epigenetic marks to daughter cells during replication. The mechanisms underlying cellular memory are poorly understood, although it is evident that Ezh2 (as well as other Polycomb group proteins) plays a significant role in this process. Interestingly, the PRC2 complex can bind to H3K27me3 and was observed to co-localize with this mark during G1
as well as sites of ongoing DNA replication (26
), which led to the proposal that PRC2 binds to established H3K27me3 during DNA replication and transmits the mark to the newly synthesized histones incorporated into the daughter strand. Given the amount of epigenetic information that must be transmitted to the daughter strands, one might expect to observe up-regulation of Ezh2 prior to DNA replication. Consistent with this notion, the pRB-E2F pathway up-regulates Ezh2
at both mRNA and protein levels at the G1
to S transition (12
After the cell has successfully replicated its DNA and transmitted its epigenetic marks, excess Ezh2 accumulated during the G1
to S transition might need to be purged to restore the normal level. Thus, mitotic phosphorylation of Ezh2 and subsequent degradation by the ubiquitination pathway might serve such a purpose (). Because Ezh2 binds and represses the CDKN2A tumor suppressor locus (16
), degradation of excess Ezh2 after mitosis may be important to keep cell proliferation in check.
FIGURE 6. A model depicting the role of Ezh2 phosphorylation during mitosis. During mitosis when CDK1 kinase activity is high, Ezh2 is phosphorylated (circled P) at Thr-345 and Thr-487 (Box 1). Phosphorylation of Thr-345 facilitates Ezh2 interaction with lncRNAs (more ...)
At the time this work was nearing completion, three studies concerning CDK1-mediated phosphorylation at Thr-345 and Thr-487 were published (27
). Chen et al.
) provided evidence demonstrating that phosphorylation of Thr-350 (Thr-345 in mouse Ezh2) is important for the recruitment of Ezh2 and maintenance of H3K27 trimethylation at PRC2 target loci including HOXA9
. This could be explained by the observation that the phospho-mimetic mutant T345E exhibits enhanced binding to the lncRNAs HOTAIR and XIST, which are known to recruit Ezh2 to target loci (28
). On the other hand, Wei et al.
) reported that phosphorylation at Thr-492 (Thr-487 in mouse Ezh2) disrupts Ezh2 binding with other PRC2 components, Suz12 and Eed, thereby attenuating Ezh2 methyltransferase activity.
Collectively, these studies show that Thr-345 and Thr-487 phosphorylation can function distinctively. Expression of human T350A, but not T492A, resulted in slower cell proliferation (27
), suggesting that the proliferative disadvantage we observed in cells expressing mouse T345A/T487A was due to loss of phosphorylation of Thr-345, but not Thr-487. Additionally, cancer cell migration and invasion required Thr-350 (27
), but expression of the phospho-deficient mutant T492A enhanced these two processes (29
). Thus, Thr(P)-350 and Thr(P)-492 appear to play opposing roles in cancer cell migration and invasion. It will be interesting to determine which of these modifications dominates given that these two phosphorylation sites can exist simultaneously within mouse Ezh2 (C
A role for Thr-350 phosphorylation in regulating Ezh2 protein stability was also investigated by Chen et al.
), but the authors concluded that it had no effect. However, the basis of their conclusion stemmed from an experiment where the half-lives of wild-type and T350A Ezh2 were compared and found to be similar. We also initially compared the half-lives of wild-type, Mut-A, and Mut-E Ezh2 and came to the same conclusion (supplemental Fig. S3B
). However, phosphorylation of Ezh2 occurs during mitosis (E
), and it is estimated that in asynchronous cells, only 1% of Ezh2 is phosphorylated at Thr-345 (28
). For this reason, we compared the half-life of phospho-Ezh2 with total Ezh2 and concluded that phospho-Ezh2 has a shorter half-life when compared with the total Ezh2 (A
). We further substantiated our conclusion by demonstrating that Mut-A is degraded less rapidly after mitosis (B
) and that ubiquitination of Ezh2 is impaired in the mutants Mut-A and Mut-E (, C
At first glance, it may seem contradictory that phosphorylation of Thr-345 leads to Ezh2 instability given that this modification is also important for lncRNA binding to facilitate recruitment of Ezh2 to target promoters (28
). However, it should be noted that these two consequences are not necessarily mutually exclusive. Because Thr(P)-345 constitutes a small fraction of total Ezh2, Kaneko et al.
) suggest that this form of Ezh2 may be important for the initial establishment of the H3K27me3 mark. This mark can later be propagated by other PRC2 complexes regardless of their Ezh2 phosphorylation status especially because PRC2 can bind to H3K27me3 (26
). Thus, Ezh2 Thr(P)-345 may be degraded after the recruitment of PRC2 to their targets by lncRNAs and the initial establishment of H3K27me3 marks (). This model is particularly attractive because initial recruitment of Ezh2 may be more specific due to the use of lncRNAs. Given that we presently do not know the kinetics of Ezh2 recruitment, we cannot confirm this possibility.
In summary, the findings described in this study coupled with the studies by other groups demonstrate an important role for CDK1-mediated phosphorylation of Ezh2. Like Ezh2, CDK1 activity is positively associated with cell proliferation and cancer (30
) and has emerged as a central player in controlling ES cell self-renewal and lineage specification (31
). Thus, this mechanism of Ezh2 regulation may provide valuable insight into the development of therapeutic drugs as well as methods in regenerative medicine.