Fig 1: Mechanism by which elevated H3K27me3 level upon intermittent high glucose exposure promotes inflammatory signaling in endothelial cells. In normal human endothelial cells, the histone methyltransferase EZH2 localizes primarily in cytosol and thus causes restricted tri-methylation of lysine residue 27 (K27) on histone H3 (H3K27me3). During such condition, the expression levels of essential signaling molecules such as KLF2, KLF4, and eNOS are maintained in endothelial cells to allow physiological function. In human endothelial cells exposed to intermittent hyperglycemia, this causes nuclear localization of EZH2 by de-phosphorylating Thr367 phosphorylation. Once localized in the nucleus, it assembles PRC2 and binds to histone H3 to cause an elevated quantity of H3K27me3. Such gain of H3K27me3 upon intermittent hyperglycemic exposure results in repression of KLF2 and KLF4 through promoter-level enrichment of H3K27me3. Repression of KLF2 and KLF4 initiates inflammatory signaling in endothelial cells through downregulation of eNOS and upregulation of ICAM1.
Fig 2: Genetic ablation or pharmacological inhibition of EZH2 resulted in FOSB gene expression in TNBC cells. a Heatmap demonstrated the common differentially expressed genes in MDA-MB-231, MDA-MB-436 and MDA-MB-453 cells either treated with 2 µM GSK343, or stably transduced with shEHZ2, compared to untreated controls. b The mRNAs of FOSB in MDA-MB-231 and MDA-MB-436 cells with or without 2 µM GSK343 treatment were determined by real-time PCR assay. c Immunoblot analysis of the indicated proteins in lysates from cells as in (a) with GAPDH as loading controls. d The mRNA levels of FOSB and EZH2 in MDA-MB-231 and MDA-MB-436 cells transfected with con-siRNA or siRNA against EZH2 were determined by real-time PCR assay. e The mRNAs of FOSB and SUZ12 in MDA-MB-231 cells transfected with con-siRNA or siRNA against SUZ12 were determined by real-time PCR assay
Fig 3: Intermittent high glucose induces nuclear localization of EZH2 through de-phosphorylation of threonine 367. (A,B) Immunoblotting and quantitation of differential hyperglycemia-treated HUVEC lysates for H3K27me3 ((A), n = 3) and EZH2 ((B), n = 4). (C) Subcellular fractionation, immunoblotting, and quantitation of nuclear and cytosolic level of EZH2 in intermittent high glucose-challenged HUVEC (n = 4). (D) Immunofluorescence and quantitation of HUVEC for EZH2 (green) after intermittent high glucose treatment. F-actin staining through phalloidin shown in red and DAPI staining shown in blue. Fluorescence intensity values taken for individual cells are each indicated by dots, and at least n > 100 cells were analyzed per group from three independent experiments. (E) Immunoblotting and quantitation of threonine 367 phosphorylated EZH2 in intermittent high glucose-exposed HUVEC (n = 4). (F,G) Tissue lysates of intermittent high glucose-treated rat aortic rings were immunoblotted and quantified for H3K27me3 (F) and EZH2 (G) (n = 3). Control treatment condition (cells constantly exposed to normal glucose (5.5 mM)) represented as “Ct” in the figure. All analyzed data were normalized to the control treatment condition. Values represent the mean ± SD. * p < 0.05 and *** p < 0.001 by unpaired t test.
Fig 4: Intermittent hyperglycemia causes EZH2 coupling with PRC2 proteins, leading to H3K27 trimethylation at promoter regions of the KLF2 and KLF4 genes. (A) Co-immunoprecipitation with EZH2 antibody, followed by immunoblotting for Suz12, EED, and histone H3 in both immuno-precipitated and total cell lysate (input) sample (n = 3). (B,C) ChIP assay using H3K27me3 antibody, followed by qPCR using primers specific to amplify promoter regions of KLF2 gene ((B), n = 5) and KLF4 gene ((C), n = 3) in HUVEC cells treated with intermittent high glucose. (D,E) Transcriptomic quantitation of KLF2 ((D), n = 3) and KLF4 ((E), n = 3) in HUVEC challenged with intermittent high glucose. (F,G) Immunoblotting and quantitation for KLF2 ((F), n = 4) and KLF4 ((G), n = 4) in intermittent high glucose-challenged HUVEC. (H,I). Tissue lysates of intermittent high glucose-treated rat aortic rings were immunoblotted for KLF2 ((H), n = 3) and KLF4 ((I), n = 3). Control treatment condition (cells constantly exposed to normal glucose (5.5 mM)) represented as “Ct” in the figure. All analyzed data were normalized to the control treatment condition. Values represent the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 by unpaired t test.
Fig 5: DDX11 suppresses p21 via enhance the protein stability of EZH2 in HCC cells. (A) GSEA indicated that EZH2 signaling was activated in patients with high expression of DDX11. (B) In TCGA cases, a positive correlation between DDX11 mRNA and EZH2 mRNA expression was found. (C) The protein expression of DDX11 was associated with EZH2 protein expression in 24 fresh HCC specimens in SYSUCC cohort. (D) HepG2 and PLC8024 cells were transfected with DDX11 shRNA or overexpression vectors. The mRNA expression of EZH2 was examined by qRT-PCR. (E) The expression of DDX11, EZH2 and p21 in stable cells with DDX11 knockdown or overexpression was examined by western blot. (F) DDX11-expressing cells were transfected with EZH2 siRNA for 36 h. The effect of EZH2 on DDX11-mediated p21 suppression was tested. (G) The protein binding of EZH2 and DDX11 was confirmed by co-IP experiments. (H) The EZH2 protein stability was measured by CHX treatment in cells with DDX11 knockdown. *P < 0.05, **P < 0.01. (I) Cells were cultured with 20 mg/L CHX for indicated time, with or without pretreatment of MG132 (20 µM). The protein degradation of EZH2 was determined by western blot. (J) HCC cells with or without DDX11 knockdown were transfected with Ub for 48 h. The ubiquitination of EZH2 protein was examined by co-IP mediated by Ub antibody. (K) The role of EZH2/p21 axis was assessed by rescue experiment, using colony formation. *P < 0.05.
Supplier Page from Cell Signaling Technology for SignalSilence ® Ezh2 siRNA I