designed the experiments and supervised the study. deficiency in maintaining pluripotency (Margueron and Reinberg, 2011), suggesting that the integrity of the PRC2 core complex is essential to its enzymatic activity. mutations or amplifications have been found in a broad spectrum of human cancers including B-cell lymphoma, ovarian cancer, breast cancer, melanoma, bladder cancer, gastric cancer and other cancers (Kim and Roberts, 2016). Given the evidence of EZH2 as a cancer driver, NECA numerous efforts have been made that led to the development of EZH2 inhibitory compounds including EPZ-6438 (Knutson et al., 2013) and GSK126 (McCabe et al., 2012), both of which are currently used in clinical trials primarily against EZH2-mutated B-cell lymphoma and advanced solid tumors (Kim and Roberts, 2016). However, mixed responses of anti-EZH2 single agent therapies have been reported in both clinical and pre-clinical studies, particularly in the settings of solid tumors, advocating novel combination therapies for EZH2 hyperactive solid tumor patients (Kim and Roberts, IL5R 2016). Here we found that AMPK directly phosphorylates EZH2 at Thr311 to disrupt its interaction with SUZ12 and to inhibit PRC2 enzymatic activity, which is supported by the increased expression of PRC2-repressed genes. Furthermore, the T311E-EZH2 mutant that mimics AMPK-mediated phosphorylation status suppresses tumor cell growth both and and double knockout (thereafter termed DKO) MEFs (Tsou et al., 2011), we observed an upregulation of methylated histone H3K27 and to a lesser extent, elevation in H3K4me3, but not other histone methylation markers we examined (Figure 1A). Re-introducing AMPK1 largely suppressed deletion-induced of H3K27me3 (Figure 1B), and H3K27me3 levels were downregulated after ectopic expression of constitutively active AMPK1 in breast cancer cells (Figure S1A). These results indicate a direct connection between genetic status and the H3K27 methylation levels. Furthermore, activating AMPK NECA by a specific AMPK agonist, A769662 (Cool et al., 2006), attenuated H3K27me3 in WT, but not DKO MEFs (Figure 1C). Furthermore, A769662 treatment also led to a decrease of H3K27me3 in various ovarian cancer cell lines (Figure S1B). These findings suggest that the kinase activity of AMPK is required to suppress H3K27me3 in cells. Open in a separate window Figure 1 AMPK Suppresses EZH2-mediated Histone H3K27 Trimethylation(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from WT and double knock out (DKO) MEFs. (B) DKO MEFs were infected with the retroviral construct expressing HA-AMPK1. Infected cells were selected with 1 g/ml puromycin for 72 hours to eliminate the non-infected cells before harvesting. (C) WT and DKO MEFs were treated with 100 M A769662 for the indicated period of time before harvesting. (D) T98G cells were treated with 2 mM metformin for 2 days before harvesting. (E) WT and DKO MEFs were infected with shGFP control or shlentiviral shRNA. The infected cells were selected with 1 g/ml puromycin for 72 hours to eliminate the non-infected cells before harvesting. (F) Quantification NECA of the relative H3K27me3 band intensities from three independent experiments. H3K27me3 bands were normalized to TUBULIN, and then normalized to the first lane. Data are represented as mean SD, n=3. * 0.05, Students test. (G) shGFP- (as a negative control) and shin DKO MEFs decreased H3K27me3 levels (Figures 1ECF). Moreover, compared to control cells, inhibiting AMPK by Compound C failed to induce H3K27me3 in (Figure S1G). However, phosphorylated oligonucleosomes could still be efficiently methylated by the PRC2 complex in methyltransferase experiments (Figure S1G), indicating that phosphorylation of histones by AMPK does not interfere with PRC2-mediated H3K27 trimethylation failed to eliminate H3S10p (Figure 1A). On the other hand, in ovarian cancer cell line OVCAR5, but not OVCAR8, treatment with the specific AMPK agonist A769662 led to a moderate increase of H3S10p (Figure S1B), while H3K27me3 downregulation was observed in both cell lines treated with A769662. These results suggest that although AMPK controls both histone phosphorylation and EZH2-mediated H3K27me3, we failed to demonstrate an obvious correlation between H3S10p and H3K27me3. Numerous reports showed that S10 of H3 is subjected to phosphorylation by a handful of other kinases such as Aurora A (Crosio et al., 2002), Aurora B (Wilkins et al., 2014), and CDK8 (Meyer et al., 2008), thus it is plausible that AMPK-mediated H3 S10 phosphorylation occurs in more specific settings, likely to be in a cellular and/or tissue context-dependent manner. In skeletal muscle cells, AMPK promotes HDAC5 phosphorylation and abrogates HDAC5-mediated suppression of GLUT4 transcription (McGee et al., 2008)..