Fig 1: Schematic depicting the effect of EZH2 S-nitrosylation on its stability, translocation and catalytic activity of Polycomb Repressive Complex-2.PRC2 consists mainly of EZH2, SUZ12, and EED, residing primarily in the nucleus. The major subunit of PRC2 i.e., histone methyltransferase EZH2 localizes primarily in the nucleus and is responsible for deposition and maintenance of the levels of H3K27me3. These cells also have a limited cell migration capacity. On exposure of these cells to SNP/GSNO/bradykinin/VEGF, S-Nitrosylation of specific cysteine residues on EZH2 occur leading to a decline in their stability which is marked by reduction in EZH2 protein levels (by 2 h) along with early dissociation of SUZ12 (by 0.5 h) from EZH2-bound PRC2 complex. Upon disassembly, S-Nitrosylated EZH2 translocate from the nucleus to the cytosol (by 0.5 to 1 h) where it undergoes ubiquitination followed by its degradation primarily through the autophagosome-lysosome pathway. Additionally, there is a significant effect on the catalytic activity of EZH2, which is shown by a reduction in the deposition of H3K27me3 marks on histone H3 (by 1 h). Such reduction in H3K27me3 level upon catalytic inactivation of EZH2 through S-Nitrosylation was contributed by demethylase UTX and JMJD3, both of which remained unchanged upon SNP/GSNO challenge to EC. Such reduction in H3K27me3 through EZH2 S-nitrosylation was responsible for NO dependent induction of endothelial gene expression and migration. Moreover, abrogating EZH2-H3K27me3 axis using GSNO in hyperglycemia exposed EC, reversed endothelial inflammation and monocyte adhesion. The schematic diagram was prepared using BioRender (Agreement number: HD280FT6PP). Created in BioRender. Chowdhury, R. (2025) https://BioRender.com/t71o935.
Fig 2: SNP or GSNO challenge to endothelial cells caused degradation of EZH2 primarily through the autophagosome-lysosome pathway while blocking endogenous NO production machinery in bradykinin induced endothelial cells reversed EZH2 and H3K27me3 level.A EA.hy926 cells exposed to SNP (500 μM followed by co-immunoprecipitation with EZH2 and immunoblotted with ubiquitin antibody. (n = 3, biological replicate) B Immunoblotting analysis of EZH2 in EA.hy926 cells treated with MG132 (MG) (1 μM) and SNP (500 μM). (n = 4, biological replicate) C Immunoblotting for EZH2 in EA.hy926 cells treated with Bafilomycin A1 (Baf) (100 nM) and SNP (500 μM). (n = 4, biological replicate) D, E Immunoblotting EZH2 (D) and H3K27me3 (E) in cultured EA.hy926 cells treated with both MG132 (1 μM) and bafilomycinA1 (100 nM) followed by exposing to SNP (500 mM). (n = 4 for EZH2, and n = 3 for H3K27me3, biological replicate) F, G Immunoblotting EZH2 (F) and H3K27me3 (G) in cultured EA.hy926 cells pretreated with both MG132 (1 μM) and bafilomycinA1 (100 nM) followed by exposing to GSNO (100 mM). (n = 3, biological replicate) (H) Immunoblotting for p62 in EA.hy926 cells treated with bafilomycin A1 (100 nM). I Immunoblotting for eNOS in HUVEC transfected with eNOS siRNA. (n = 3, biological replicate) J, K Immunoblotting for EZH2 (J) and H3K27me3 (K) in HUVEC transfected with eNOS siRNA and induced with VEGF (10 ng/mL). (n = 3, biological replicate) (L) Immunoblotting for EZH2 in EA.hy926 cells treated with L-NAME (1 mM) and bradykinin (10 mM). (n = 3, biological replicate) M, N Rat aortic rings were treated with L-NAME (1 μM) and bradykinin (10 μM) followed by immunoblotting to analyze the EZH2 (M) and H2K27me3 (N). (n = 3, biological replicate) (O) EZH2 (green) along with F‐actin (Red) staining of EA.hy926 cells after treating with L-NAME (1 μM) and bradykinin (10 μM). DAPI shown in blue. (Scale bar: 10 μm) (n = 3, biological replicate). All data are presented as mean values ± SD. All statistical analyses are either performed by One-way ANOVA with a post-hoc Tukey test for multiple groups or by two-tailed unpaired t-test for two groups.
Fig 3: External nitric oxide supplementation, endogenous induction of nitric oxide producing machinery, or treatment with GSNO caused S-nitrosylation of EZH2 and dissociation of SUZ12 and histone H3.A Dot blot of protein lysates collected from EA.hy926 cells exposed to SNP (500 μM) for 0.5 h and processed through biotin-switch assay followed by immunoprecipitation with EZH2 antibody. Blots were incubated with Streptavidin-HRP followed by developing with chemiluminescence substrate for visualization. (n = 3, biological replicate) B Same samples were run through SDS-PAGE followed by transfer to nitrocellulose membrane followed by incubation with Streptavidin-HRP and developing the blot with chemiluminescence substrate for visualization. (n = 3) C, D EA.hy926 cells were exposed to either SNP (500 μM, C) or bradykinin (10 μM, D) for 0.5 h followed by immunoprecipitation with EZH2 antibody and further immunoblotting to show the presence of S-nitrosylation of EZH2 using Nitro-Cysteine antibody. (n = 3, biological replicate) E Plasmid containing HA-tagged EZH2 were transfected in HEK-293 cells followed by exposing to SNP (500 μM) for 0.5 h. Cell lysates were then immunoprecipitated using HA antibody followed by immunoblotting with respective antibodies. (n = 3, biological replicate) F, G Dot blot (F) and SDS-PAGE followed by immunoblot (G) of protein lysates collected from EA.hy926 cells exposed to GSNO (100 μM) for 0.5 h followed by immunoprecipitation with EZH2 antibody and processing through iodoTMT protocol. Blots were incubated with anti-IodoTMT antibody. Blots were developed with chemiluminescence substrate for visualization. (n = 3, biological replicate) H, I Histone methyltransferase activity (H) and inhibition (I) analysis of EZH2 in EA.hy926 cells exposed to GSNO (100 μM) or SNP (500 μM) for 2 h. (n = 5, biological replicate). All data are presented as mean values ± SD. All statistical analyses are either performed by One-way ANOVA with a post-hoc Tukey test for multiple groups or by two-tailed unpaired t-test for two groups.
Fig 4: GSNO exposure caused reduction in EZH2 and H3K27me3 level along with altering the interacting partners of EZH2.A Immunoblotting for EZH2 and H3K27me3 in cultured EA.hy926 cells exposed to GSNO (100 μM). (n = 4, biological replicate) B, C Immunoblot analysis of EZH2 (B) and H3K27me3 (C) in rat aortic explants exposed to GSNO (100 μM). (n = 3, biological replicate) D Immunoblotting analysis of EZH2 in nuclear and cytosolic fractions obtained from EA.hy926 cells treated with GSNO (100 μM). (n = 3, biological replicate) E Immunofluorescence followed by confocal imaging of EA.hy926 cells exposed to 1 h of GSNO (100 μM) and labeled for EZH2 (green) and F-actin (red). DAPI staining is shown in blue. White and yellow arrow head indicates EZH2 not bound and bound to F-actin respectively. (Scale bar: 10 μm), n = 3, biological replicate. F EA.hy926 cells treated with GSNO (100 μM) for 0.5 h were subjected to co-immunoprecipitation using EZH2 antibody followed by immunoblotting for SUZ12. (n = 3, biological replicate) G, H En face preparations were double stained for VE-cadherin (green) and EZH2 (red, G) or H3K27me3 (red, H). Images were captured from the luminal surface of the aorta (Scale bar: 10 μm). The levels of nuclear EZH2 and H3K27me3 were analyzed using Image J. Both EZH2 (G) and H3K27me3 (H) analysis, control (n = 112) and GSNO (n = 113) from three independent biological replicate. Middle: Box whisker plots show nuclear EZH2, with minimum, first quartile (lower bound), median, third quartile (upper bound), maximum values, whiskers down to the minimum and up to the maximum value. I–L Venn-diagram (I) representing the percentage of common/overlapping proteins in control and 0.5 h GSNO (100 μM) exposed HUVEC. Data (J) showing the number of total and unique interacting proteins. Heat map-based visualization of all the uniquely interacting proteins (K) or proteins with a PEP score ≥ 5. (n = 2, biological replicate). All data are presented as mean values ± SD. All statistical analyses are either performed by One-way ANOVA with a post-hoc Tukey test for multiple groups or by two-tailed unpaired t-test for two groups.
Fig 5: Performing Molecular Dynamic (MD) simulation studies through GROMACS 5.1.5 version using WT and S-nitrosylated EZH2 at C324 and C695.A–C Structural illustration of the initial structural alignment of the wildtype and S-nitrosylated EZH2 protein of the EZH2-SUZ12 complex (A). The magenta and green color represents the EZH2 chain of both WT and S-nitrosylated version, while the cyan and red color indicates the SUZ12 chain. It also provides a close-up view of S-nitrosylation at position C324 of EZH2 in the S-nitrosylated protein (B) and zooms in on the S-nitrosylation at position C695 of EZH2 in the S-nitrosylated protein (C). D, E Backbone RMSD of whole EZH2 WT and EZH2 S-nitrosylated (D). Backbone RMSD of EZH2 WT and EZH2 S-nitrosylated specifically within the residue ranges between 112 and 121 of SAL (E). Backbone RMSD of residues in SUZ12 ranging between 584 and 588 (F). G, H Structural illustration of EZH2-SUZ12 complex near the SAL (112–121) domain of EZH2 in both EZH2 WT and EZH2 S-nitrosylated forms at 100 ns and 800 ns of simulation. Structural data reflects the distance between unique residues of EZH2 (LEU116 and GLN117) and SUZ12 (ASP585 and LYS587) to reflect potential for hydrogen bonds which were lost at 800 ns only in EZH2-SUZ12 complex having S-nitrosylated form of EZH2 at C324 and C695.
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