Journal: Nature Communications

Article Title: EZH2-induced lysine K362 methylation enhances TMPRSS2-ERG oncogenic activity in prostate cancer

doi: 10.1038/s41467-021-24380-6

Figure Lengend Snippet: 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-ΔN-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.

Article Snippet: To perform immunofluorescence for anti-pS21 EZH2 (Bethyl Laboratories) cells were grown in μ-slide (chambered coverslip) with 8 wells (ibidi).

Techniques: Sequencing, Methylation, Western Blot, Immunofluorescence, Microscopy, Incubation, Control, Transfection, In Vitro, Recombinant, Knockdown, Expressing, Binding Assay, Microscale Thermophoresis, Co-Immunoprecipitation Assay, Plasmid Preparation, Construct, Immunoprecipitation

Journal: Nature Communications

Article Title: EZH2-induced lysine K362 methylation enhances TMPRSS2-ERG oncogenic activity in prostate cancer

doi: 10.1038/s41467-021-24380-6

Figure Lengend Snippet: 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.

Article Snippet: To perform immunofluorescence for anti-pS21 EZH2 (Bethyl Laboratories) cells were grown in μ-slide (chambered coverslip) with 8 wells (ibidi).

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Activity Assay, Luciferase, Reporter Assay, Over Expression, Expressing, Binding Assay, Stable Transfection, Transfection

Journal: Nature Communications

Article Title: EZH2-induced lysine K362 methylation enhances TMPRSS2-ERG oncogenic activity in prostate cancer

doi: 10.1038/s41467-021-24380-6

Figure Lengend Snippet: 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.

Article Snippet: To perform immunofluorescence for anti-pS21 EZH2 (Bethyl Laboratories) cells were grown in μ-slide (chambered coverslip) with 8 wells (ibidi).

Techniques: ChIP-sequencing, Binding Assay, Two Tailed Test, Expressing, Knockdown, Quantitative RT-PCR

Journal: Nature Communications

Article Title: EZH2-induced lysine K362 methylation enhances TMPRSS2-ERG oncogenic activity in prostate cancer

doi: 10.1038/s41467-021-24380-6

Figure Lengend Snippet: a Box-plots of prostate weights evaluated at 16-week from wild-type (ERG flox/flox) ERG (PbCre; ERG flox/flox) and ERG/PTEN (PbCre; ERG flox/flox PTEN flox/flox) mice ( n = 4/group). Minima in WT group is 13, maxima is 17, median is 15, percentile (0,25 th ) is 13.25; minima in R26 ERG group is 39.2, maxima is 56.1, median is 49.05, percentile (0,25 th ) is 41.57; minima in ERG/PTEN group is 165.2, maxima is 183.6, median is 172.2, percentile (0,25 th ) is 161.3. b Histological and IHC evaluation from 16-week old prostate from wild-type, ERG, and ERG/PTEN mice. FFPE representative prostate sections were examined by IHC to assess ERG and EZH2 ( n = 3). Scale bars represent 200 µm. c Prostates of 16-week-old wild-type, ERG, and ERG/PTEN mice ( n = 3) were examined by immunoblots to assess mERG, ERG, and EZH2. Lysate were collected at 16 weeks from ventral prostate lobes and analyzed with indicated antibodies. d Densitometric analysis of pAKT relative total AKT (left panel), mERG relative to ERG (middle panel) mERG and EZH2 relative to GAPDH and (right panel). e Cytoplasmic and nuclear localization of ERG, EZH2, and mERG in prostate tissues from WT and ERG/PTEN mice ( n = 2). f Expression of selected ERG/EZH2 target genes evaluated by qRT-PCR in prostatic tissue from 16-week-old ERG (PbCre; ERG flox/flox) and ERG/PTEN (PbCre; ERG flox/flox PTEN flox/flox) mice. ( n = 3/ biological independent samples). g ChIP-qPCR analysis of ERG and EZH2 occupancy at the indicated promoters in prostatic tissue from 16-week-old ERG (PbCre; ERG flox/flox) and ERG/PTEN (PbCre; ERG flox/flox PTEN flox/flox) mice. ( n = 3/biological independent samples). h Experimental plan. 25-week-old ERG/PTEN mice ( n = 5/group) were treated with vehicle or 15 mg/kg GSK343 by intraperitoneal injection three times/week for 2 weeks. i Immunoblotting analysis of the indicated proteins in prostatic tissue from control and GSK343-treated ERG/PTEN mice ( n = 2). j Expression of ERG/EZH2 co-regulated genes in control and GSK343-treated ERG/PTEN mice evaluated by qRT-PCR. k ChIP-qPCR analysis of mERG and EZH2 occupancy at the indicated promoters in prostatic tissue from control and GSK343-treated ERG/PTEN mice. l Prostate tumor development in ERG/PTEN mice treated with vehicle (control) or GSK343. 25-week-old ERG/PTEN mice ( n = 5/group) were treated with vehicle or 15 mg/kg GSK343 by intraperitoneal injection three times/week for 2 weeks. Left, representative images of prostates. Right, prostate weights at the end of the experiment. Minima in CTRL group is 0.362, maxima 0.6031, median is 0.467, percentile (0,25 th ) is 0.385; minima in GSK-343 group is 0.145, maxima 0.39, median is 0.263, percentile (0,25 th ) is 0.16. m H&E staining and IHC evaluation of ERG, EZH2, and Ki67 in ERG/PTEN mice following treatment with vehicle or GSK343 as described above ( n = 2). Scale bars represent 200 µm. n Quantitative IHC scores of Ki67 immunostaining in control and GSK343-treated ERG/PTEN mice. P -values were determined by one-way ANOVA test. Minima in CTRL group is 45, maxima 75, median is 63, percentile (0,25 th ) is 52.5; minima in GSK-343 group is 10, maxima 35, median is 20, percentile (0,25 th ) is 12.5. All error bars, mean ± s.d. ( n = 3, technical replicates). Source data are provided as a Source Data File.

Article Snippet: To perform immunofluorescence for anti-pS21 EZH2 (Bethyl Laboratories) cells were grown in μ-slide (chambered coverslip) with 8 wells (ibidi).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, ChIP-qPCR, Injection, Control, Staining, Immunostaining

Journal: Nature Communications

Article Title: EZH2-induced lysine K362 methylation enhances TMPRSS2-ERG oncogenic activity in prostate cancer

doi: 10.1038/s41467-021-24380-6

Figure Lengend Snippet: a Immunoblots of mERG and the indicated proteins in control (EV) and stable PTEN knockdown (shPTEN) VCaP cells ( n = 3). b Expression of ERG/EZH2 co-occupied genes in VCaP-shPTEN and control (EV) VCaP cells evaluated by qRT-PCR. c ERG, mERG, and EZH2 occupancy at the indicated gene promoters in VCaP-shPTEN and control (EV) VCaP cells by ChIP-qPCR. d EZH2 phosphorylation (Ps21) evaluated by IF and quantitative assessment (right) in VCaP-shPTEN and control (EV) following treatment with MK-2206 (5 µM) for 24 h. Scale bar 10 µm ( n = 2). e Immunoblots of mERG and indicated proteins in VCaP-shPTEN (sh-PTEN) and control (EV) cells treated with DMSO and MK-2206 (5 µM) for 5 and 24 h ( n = 2). f Diagram of WT, S21A, and S21D EZH2 mutated constructs. g Immunoblots of mERG and indicated proteins in RWPE1 cells transfected with indicated expression plasmids. Right, densitometric assessment of mERG level relative to total ERG ( n = 2). h IHC evaluation of pS21 EZH2 in prostate tissue from ERG (PbCre; ERG flox/flox) and ERG/PTEN (PbCre; ERG flox/flox PTEN flox/flox) mice. Right, quantitative IHC scores of pS21EZH2 and EZH2. All error bars, mean ± s.d. ( n = 3, technical replicates) ( b , c ). P -values were determined by one-way ANOVA test. Scale bars represent 200 µm. Source data are provided as a Source Data File.

Article Snippet: To perform immunofluorescence for anti-pS21 EZH2 (Bethyl Laboratories) cells were grown in μ-slide (chambered coverslip) with 8 wells (ibidi).

Techniques: Western Blot, Control, Knockdown, Expressing, Quantitative RT-PCR, ChIP-qPCR, Phospho-proteomics, Construct, Transfection

Journal: Nature Communications

Article Title: EZH2-induced lysine K362 methylation enhances TMPRSS2-ERG oncogenic activity in prostate cancer

doi: 10.1038/s41467-021-24380-6

Figure Lengend Snippet: a Pathways enriched in the list of genes co-occupied by ERG/EZH2 (GS_ERG/EZH2, n = 1656 genes) using enrich tool. b Functional analysis of the GS_ERG/EZH2 set using enrichDGN tool. c , d Heat map ( c ) and principal component analysis ( d ) using the GS_ERG/EZH2 set applied to primary prostate tumors (PRIMARY) and castration-resistant prostate cancers (CRPC) with known ERG and EZH2 status in the Michigan dataset. Color bars indicate ERG and EZH2 expression status. e GSEA using the GS_ERG/EZH2 set comparing ERG fusion positive and negative prostate tumors in the Sboner dataset. f Heat map with the top deregulated ERG/EZH2 gene signature (GS_ERG/EZH2_50) applied to the TCGA dataset. Color bars indicate ERG fusion and PTEN status.

Article Snippet: To perform immunofluorescence for anti-pS21 EZH2 (Bethyl Laboratories) cells were grown in μ-slide (chambered coverslip) with 8 wells (ibidi).

Techniques: Functional Assay, Expressing