Fig 1: K362 methylation enhances ERG transcriptional and oncogenic activity.a Luciferase activity of the ETS responsive reporter in LNCaP cells expressing WT, K362A, K362A/E412A ERG, or empty vector (EV). Bottom, protein expression verified by immunoblotting. b Luciferase activity of the ETS responsive reporter in RWPE1 cells expressing WT, K362A, E412A, K362A/E412A ERG, or empty vector (EV). Bottom, protein expression verified by immunoblotting. c Total modulated genes in LNCaP cells expressing WT or K362A ERG. d Heat map showing the intensity of the transcripts significantly modulated in LNCaP cells expressing WT, K362A ERG, or empty vector (EV). Scale bar shows LogRatio range. Red, upregulation; green, downregulation (n = 2/group). e Scatter plot of the LogRatios of genes deregulated in LNCaP-ERG vs control (logFC ERG WILD) and LNCaP-ERG-K362A vs control (logFC ERG MUT). Regression line was calculated by means of the lm function (regression line coefficient = 0.44). f Venn diagram showing the convergence of genes modulated by WT-ERG and ERG-K362A. g Convergence between genes modulated by WT-ERG or ERG-K362A and ERG genomic occupancy in VCaP cells. h Soft agar, clonogenic, and sphere forming assays in LNCaP cells expressing WT, K362A ERG or an empty vector (EV). i Growth of xenografts of LNCaP cells expressing WT-ERG, ERG-K362A, or an empty vector (EV) injected in NSG mice (n = 3/group). Right, histological and Ki67 immunostaining scores in tumor xenografts. Scale bars represent 200 µm. j Growth of xenografts of RWPE1 cells expressing WT, ERG-K362A, K362A/E412A (DM) ERG, or an empty vector (EV) injected in NSG mice (n = 4, biological independent samples). Right, histological and Ki67 immunostaining scores in tumor xenografts. Scale bars represent 200 µm. All error bars, mean ± s.d. P-values were determined by one-way ANOVA test. Source data are provided as a Source Data File.
Fig 2: K362 methylation affects protein conformation.a ERG domain structure and organization of the ETS DNA binding domain and auto-inhibitory modules. b Solvent accessible surface area for non-methylated (K362) and methylated (Me-K362) form of the ERGi domain. c Representative structures from molecular dynamic simulation of non-methylated (left) and methylated (right) ERGi domain. d Overlay of ERG domain structure of active DNA-bound ERG derived from the X-ray model (blue ribbon) and structures of ERGi, methylated ERG, and indicated ERG mutants derived from molecular dynamic simulations. e Diagram of single (E412A) and double mutant (K362A/E412A) ERG constructs. f Inter-molecular distance in MD simulated ERG domain structures. The distance between residue Leu320 in a1 helix and Ala413 in a4 helix was calculated in all systems over the simulation time to estimate the degree of divergence relative to the active DNA-bound ERG (4iri) structure defined by X-ray crystallography. The same parameter measured in the X-ray structure identified by the pdb code 4iri is depicted as red square. ERGi (Violet), mERG (green), K362A/E412A (light blue) K362A (orange), K362M (yellow), and K362R (blue).
Fig 3: ERG and EZH2 co-occupy and co-regulate a specific network of genes.a Venn diagram showing the number of ERG, EZH2, and ERG/EZH2 co-localized peaks (=1-kb) in VCaP cells extracted by ChIP-seq. b Distance of ERG and EZH2 peaks at ERG/EZH2 co-occupied sites. c Distribution of total ERG and EZH2 (top panel) and ERG–EZH2 co-occupied (lower panel) sites in intergenic, promoter, enhancer, intron, and exon regions. d Enrichment of ERG binding motif at ERG/EZH2 co-occupied sites by de-novo motif analysis. e Venn diagram showing the convergence of ERG and mERG genomic occupancy determined by ChIP-seq in VCaP cells. f Distribution of active and repressive histone marks among ERG, EZH2, and ERG/EZH2 targets. g Pie chart showing percentage of active and inactive genes among ERG/EZH2 targets in VCaP cells. h ChIP–reChIP analysis to evaluate co-occupancy by ERG and EZH2 at the indicated gene promoters in VCaP cells. P-values were determined by unpaired, two-tailed Student’s t-test. i Expression of ERG/EZH2 co-occupied genes after EZH2 (upper) or ERG (lower) knockdown in VCaP cells evaluated by qRT-PCR. All error bars, mean ± s.d. (n = 3, technical replicates). P-values were determined by one-way ANOVA test. Source data are provided as a Source Data File.
Fig 4: ERG and EZH2 co-occupy gene promoters forming activatory and repressory complexes.a ChIP analysis of ERG and EZH2 occupancy on the IL-6 promoter in VCaP cells. Data are presented as fold enrichment relative to input. b ChIP–reChIP analysis of ERG and EZH2 to evaluate co-occupancy at the IL-6 promoter in VCaP cells. Data are presented as fold enrichment relative to input. c qRT-PCR analysis of IL-6 mRNA and immunoblotting analysis of the indicated proteins (right) upon ERG and EZH2 knockdown in VCaP cells. d qRT-PCR analysis of IL-6 mRNA (left) and IL-6 promoter activity by luciferase reporter assay (middle) in LNCaP cells with ERG overexpression and EZH2 knockdown. Expression of the indicated proteins was verified by immunoblotting (right panel). e ChIP analysis of ERG and EZH2 occupancy in the IL-6 promoter at the ETS binding site (EBS) in LNCaP and LNCaP-ERG stable cell lines. f IL-6 mRNA determined by qRT-PCR (left) and IL-6 promoter activity by luciferase reporter assay (middle) in LNCaP transfected with the indicated plasmids. Expression of the indicated proteins was verified by immunoblotting (right). g ChIP–reChIP analysis of EZH2, ERG, and SUZ12 to evaluate co-occupancy at the IL-6 and Nkx3.1 promoters in VCaP cells. Data are presented as fold enrichment relative to IgG of the Re-ChIP. All error bars, mean ± s.d. (n = 3, technical replicates). P-values were determined by one-way ANOVA test. Source data are provided as a Source Data File.
Fig 5: EZH2 methylates ERG at lysine K362.a Sequence alignment of ERG domain containing the EZH2 recognized R-K-S motif from diverse species. b Detection of methylated ERG in VCaP cells by immunoblotting with anti-mERG antibody and competition with methylated (M) and non-methylated (C) peptides (n = 2). c Detection of mERG in VCaP cells by immunofluorescence microscopy with anti-mERG antibody pre-incubated with the specific competitor and control peptides (n = 2). Scale bar = 20 µm. d Detection of ERG, EZH2, and mERG in VCaP cells by immunofluorescence microscopy (n = 2). Scale bar = 20 µm. e Detection of ERG and mERG by IB in control and HA-tagged K362A ERG transfected VCaP cells (n = 2). f In vitro methylation assay with recombinant ERG and EZH2 followed by immunoblots with indicated antibodies (left) and in the presence of the EZH2 inhibitor GSK343 (right) (n = 2). g Detection of mERG, ERG, and EZH2 by IB in VCaP cells upon EZH2 knockdown by two siRNA (siEZH2 and siEZH2 3'UTR) (n = 2). h Detection of mERG, ERG, and EZH2 by IB in VCaP cells upon treatment with 10 µM DZNep (H) at indicated time points (n = 2). i Immunoblots of mERG, ERG, and EZH2 in RWPE1 cells transiently transfected with the indicated ERG and EZH2 expression vectors (n = 2). j Binding of recombinant ERG and EZH2 determined by microscale thermophoresis (MST). Insert, MST tracing. k Co-IP of ERG and EZH2 in PC3 cells transiently transfected with Ha-tagged ERG expression vector (n = 2). l Co-IP of ERG and EZH2 in VCaP cells with ERG and EZH2 specific antibodies and control IgG (n = 2). m Diagram of truncated ERG constructs. n Co-IP and His-pulldown in PC3 cells transiently transfected with the His-?N-ERG constructs and immunoblotting with anti-His and anti-EZH2 antibodies (n = 2). o Diagram of truncated EZH2 constructs. p Binding of Myc-EZH2-?SET to Ha-ERG assessed by co-immunoprecipitation in PC3 cells transiently transfected with the truncated EZH2 constructs along with ERG plasmid (n = 2). Molecular weights are indicated in kilodaltons (kDa). Source data are provided as a Source Data File.
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