1. Volume 69, Issue 2, Pages 279-291.e5 (January 2018)
Phosphorylation of EZH2 by AMPK Suppresses PRC2 Methyltransferase Activity and Oncogenic Function 
Lixin Wan, Kexin Xu, Yongkun Wei, Jinfang Zhang, Tao Han, Christopher Fry, Zhao Zhang, Yao Vickie Wang, Liyu Huang, Min Yuan, Weiya Xia, Wei-Chao Chang, Wen-Chien Huang, Chien-Liang Liu, Yuan-Ching Chang, Jinsong Liu, Yun Wu, Victor X. Jin, Xiangpeng Dai, Jianfeng Guo, Jia Liu, Shulong Jiang, Jin Li, John M. Asara, Myles Brown, Mien-Chie Hung, Wenyi Wei 
Molecular Cell 
Volume 69, Issue 2, Pages e5 (January 2018)
DOI: /j.molcel
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2. Molecular Cell 2018 69, 279-291.e5DOI: (10.1016/j.molcel.2017.12.024)
Copyright © 2018 Elsevier Inc. Terms and Conditions

3. Figure 1 AMPK Suppresses EZH2-Mediated Histone H3K27 Trimethylation
(A) Immunoblot (IB) analysis of whole-cell lysates (WCLs) derived from Ampkα WT and Ampkα1−/−; Ampkα2−/− double knockout (DKO) MEFs.
(B) Ampkα1−/−; Ampkα2−/− DKO MEFs were infected with the retroviral construct expressing HA-AMPKα1. Infected cells were selected with 1 μg/mL puromycin for 72 hr to eliminate the non-infected cells before harvesting.
(C) Ampkα WT and DKO MEFs were treated with 100 μM A for the indicated period of time before harvesting.
(D) T98G cells were treated with 2 mM metformin for 2 days before harvesting.
(E) Ampkα WT and DKO MEFs were infected with shGFP control or shEzh2 lentiviral shRNA. The infected cells were selected with 1 μg/mL puromycin for 72 hr to eliminate the non-infected cells before harvesting.
(F) Quantification 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. ∗p < 0.05, Student’s t test.
(G) shGFP- (as a negative control) and shEZH2-MDA-MB-231 cells were treated with or without 10 μM AMPK inhibitor Compound C for 12 hr before harvesting for IB analysis.
See also Figure S1.
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4. Figure 2
AMPK Abrogates EZH2-Mediated Epigenetic Silencing of PRC2 Target Genes
(A) Heatmap depicting the H3K27me3 ChIP-seq signal ± 10 kb around its peak summit in both Ampkα WT and DKO MEFs.
(B) Scatter density plot comparing H3K27me3 signals in Ampkα WT (x axis) and DKO (y axis) MEF cells. Read counts within a 5 kb window around the center of each H3K27me3 peak, which was pooled from both cell conditions, were normalized by 10 million total reads. The color scale indicates the density of peaks.
(C) Ampkα WT and DKO MEFs were subjected to quantitative real-time RT-PCR analyses for relative RNA levels of the indicated genes. Expression levels of these genes were normalized and compared to those of Ampkα WT. Data are means ± SD (n = 3). ∗p < 0.05, Student’s t test.
(D) Ampkα WT and DKO MEFs were treated with 100 μM A as indicated for 48 hr. Total RNAs were extracted for quantitative real-time RT-PCR analyses for relative RNA levels of the indicated genes. Expression levels of these genes were normalized and compared to those of Ampkα WT without treatment. Data are means ± SD (n = 3).
(E) Control and 2 mM metformin-treated OVCAR8 cells were subjected to quantitative real-time RT-PCR analyses for relative RNA levels of the indicated genes. Expression levels of these genes were normalized and compared to those of the untreated control cells. Data are means ± SD (n = 3). ∗p < 0.05; ns, not significant, Student’s t test.
See also Figure S2.
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5. Figure 3
Identification of EZH2 as a Downstream Phosphorylation Substrate of AMPK
(A) Sequence alignments of the T311- and the S366/T367-containing regions between EZH2 among various species as well as human EZH1. A schematic representation of EZH2 protein domain structure was shown beneath to illustrate the position of these candidate sites.
(B) Immunoblot (IB) analysis of whole-cell lysates (WCLs) and anti-EZH2 immunoprecipitates derived from MDA-MB-231 cells.
(C) IB analysis of WCLs and anti-HA immunoprecipitates derived from 293T cells transfected with Flag-EZH2 and the indicated HA-AMPKα constructs.
(D) In vitro kinase assays showing that bacterially purified recombinant WT EZH2 and, to a much lesser extent, S366A/T367A-EZH2, but not the T311A-EZH2 mutant, could be phosphorylated by the AMPK kinase in vitro.
(E) AMPK primarily phosphorylated EZH2 at T311. IB analysis of WCLs and anti-Flag immunoprecipitates derived from 293T cells transfected with the indicated constructs.
(F) IB analysis of WCLs and anti-HA immunoprecipitates derived from 293T cells transfected with the indicated HA-EZH2, HA-SUZ12, and HA-EED constructs.
See also Figure S3.
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6. Figure 4 Activation of AMPK Promotes EZH2 T311 Phosphorylation
(A) Immunoblot (IB) analysis of whole-cell lysates (WCLs) derived from Ampkα WT and DKO MEFs treated with 100 μM A for the indicated periods of time before harvesting.
(B and C) IB analysis of OVCAR5 (B) and OVCAR8 (C) cells treated with 100 μM A for the indicated time periods before harvesting.
See also Figure S4.
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7. Figure 5
Phosphorylation of EZH2 at T311 by AMPK Suppresses PRC2 Methyltransferase Activity
(A) T98G cells stably expressing empty vector (EV; as a negative control) or the indicated HA-EZH2 constructs were infected with shGFP or shEZH2 lentiviral constructs. Infected cells were selected with 1 μg/mL puromycin for 72 hr to eliminate the non-infected cells before harvesting for immunoblot (IB) analysis.
(B) shEZH2-OVCAR5 cells stably expressing WT or T311A-EZH2 were treated with 100 μM A for 48 hr before harvesting.
(C) shEZH2-OVCAR5 cells stably expressing WT or T311E-EZH2 were treated with 10 μM Compound C for 48 hr before harvesting.
(D) Ampkα WT and DKO MEFs stably expressing WT or T311A-EZH2 were further depleted with lentiviral shEzh2 construct before harvest for IB analysis.
(E) In vitro methyltransferase assays using immuno-purified PRC2 containing the indicated EZH2 constructs and oligonucleosomes as the substrate.
(F) IB analysis of whole-cell lysates (WCLs) and anti-Flag immunoprecipitates derived from 293T cells transfected with HA-SUZ12 and the indicated Flag-EZH2 constructs.
(G) Autoradiography of 35S-labeled SUZ12 bound to bacterially purified His-EZH2 recombinant proteins.
(H) IB analysis of WCLs and anti-EZH2 immunoprecipitates derived from OVCAR5 cells infected with EV or pLenti-CMV-HA-AMPKα (1–312) as indicated.
(I) IB analysis of WCLs and anti-EZH2 immunoprecipitates derived from OVCAR5 cells treated with 10 mM 2DG for 1 hr as indicated.
(J) Gel filtration experiments to illustrate that EZH2-containing protein complex migrated differently in T311E-EZH2-expressing OVCAR8 cells than in OVCAR8 cells expressing WT or T311A-EZH2. OVCAR8 cells stably expressing GFP (as a negative control) or the indicated HA-EZH2 constructs were infected with shGFP or shEZH2 lentiviral constructs. The infected cells were selected with 1 μg/mL puromycin for 72 hr to eliminate the non-infected cells before harvesting for gel filtration analysis using Superose 6 gel filtration chromatography. Prior to running cell lysates, the molecular weight resolution of the column was first estimated by running native molecular weight markers (∼669 KD thyroglobulin, ∼440 KD ferritin, ∼158 KD aldolase, ∼75 KD conalbumin, and ∼43 KD ovalbumin) and determining their retention times on Coomassie-stained SDS-PAGE protein gels.
(K) A schematic illustration of the proposed model in which AMPK-mediated phosphorylation of EZH2 disrupts the binding between EZH2 and SUZ12.
See also Figure S5.
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8. Figure 6
AMPK-Mediated Phosphorylation of EZH2 at T311 Attenuates PRC2 Oncogenic Function
(A) EZH2-depleted OVCAR5 cells stably reintroducing control, WT, T311A, R308L, T311E, and S21D EZH2 were quantified for mRNA level expression of EZH2 target genes. mRNA levels of HOXA11, OLIG2, SOX17, and GATA6 were measured by qRT-PCR, and are normalized to GAPDH expression. Data are means ± SEM (n = 3).
(B and C) EZH2-depleted OVCAR5 cells stably expressing empty vector (EV; as a negative control) or the indicated HA-EZH2 constructs were infected with shGFP or shEZH2 lentiviral constructs. Infected cells were selected with 1 μg/mL puromycin for 72 hr to eliminate the non-infected cells. These cells were subjected to clonogenic survival assays for 10 days. Crystal violet was used to stain the formed colonies (B). The colony numbers were calculated as mean ± SD (n = 3). ∗p < 0.05, Student’s t test (C).
(D and E) OVCAR5 cells generated in (B) were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS and layered onto 0.8% agarose in DMEM with 10% FBS. The plates were cultured for 21 days whereupon the colonies >50 μm were counted under a light microscope (D). The colony numbers were plotted as mean ± SD (n = 3). ∗p < 0.05, Student’s t test (E).
(F and G) The tumor size (F) and growth curves (G) for the xenograft experiments using shEZH2-OVCAR5 cells expressing GFP (as a negative control) or the indicated EZH2 construct. In each flank of six nude mice, 1 × 106 cells were injected subcutaneously. The visible tumors were measured at the indicated days. Error bars represent ± SEM, n = 6.
(H and I) EZH2-depleted OVCAR5 cells stably expressing WT or R308L-EZH2 constructs were subjected to clonogenic survival assays for 14 days. A total of 100 μM A was added immediately after plating cells as indicated. Crystal violet was used to stain the formed colonies (H). The colony numbers were calculated as mean ± SD (n = 3) (I).
(J and K) EZH2-depleted OVCAR5 cells stably expressing WT or R308L-EZH2 constructs were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS and layered onto 0.8% agarose in DMEM with 10% FBS. The plates were cultured for 21 days whereupon the colonies >50 μm were counted under a light microscope. A total of 100 μM A was added immediately after plating cells as indicated (J). The colony numbers were calculated as mean ± SD (n = 3) (K).
See also Figure S6.
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9. Figure 7
EZH2 Phosphorylation at T311 Correlates with Better Survival in Cancer Patients
(A and B) Immunohistochemical staining of p-T172-AMPKα and p-T311-EZH2 in representative ovarian cancer (A) or breast cancer (B) specimens. Brown staining indicates positive immunoreactivity.
(C and D) A positive correlation between p-T172-AMPKα and p-T311-EZH2 levels in human ovarian cancer (C) or breast cancer (D) clinical samples. Statistical significance was determined by a χ2 test.
(E and F) Kaplan-Meier curves showing the overall survival of patients with high or low expression of p-T311-EZH2 in their ovarian cancer (E) or breast cancer (F). Statistical significance was determined by a log-rank test.
See also Figure S7.
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