Journal: Nucleic Acids Research

Article Title: A cryptic transactivation domain of EZH2 binds AR and AR’s splice variant, promoting oncogene activation and tumorous transformation

doi: 10.1093/nar/gkac861

Figure Lengend Snippet: EZH2 non-canonically binds genomic sites with gene-active markers, Pol II and AR or AR-V7, in prostate cancer cells. ( A , B ) Averaged intensities (A) and heatmaps (B) of EZH2 or H3K27me3 CUT&RUN and ATAC-seq signals ± 5 kb from the centers of canonical EZH2 + /H3K27me3 + /low-accessibility peaks (i.e. EZH2-ensemble, 8436 peaks; top panels) or non-canonical EZH2 + /H3K27me3 − /high-accessibility peaks (i.e. EZH2-solo, 15 987 peaks; bottom panels) in 22Rv1 cells. ( C ) Heatmap showing the signals of EZH2 (in duplicate), H3K27me3 (in duplicate), H3K27ac (in triplicate), H3K4me2, H3K4me3, BRD4 and RNA Pol II ± 5 kb from the centers of EZH2-solo (top) or EZH2:PRC2-ensemble peaks (bottom). Except H3K4me2, H3K4me3 and Pol II data which were from LNCaP-abl cells, all the others were generated in 22Rv1 cells. EZH2 and H3K27me3 datasets were from CUT&RUN experiments and all the others from ChIP-seq. ( D , E ) Heatmap showing the EZH2, H3K27me3, AR and AR-V7 signals ± 5 kb from the centers of either EZH2-solo (D) or EZH2:PRC2-ensemble (E) peaks identified in 22Rv1 cells. Mock, ligand-stripped condition; DHT, ligand-treated condition. ( F ) Venn diagram showing the overlapping of EZH2-solo peaks with either AR (left) or AR-V7 peaks (right) in 22Rv1 cells. ( G ) Venn diagram showing a significant overlap between EZH2-solo:AR- and EZH2-solo:AR-V7-co-occupied sites in 22Rv1 cells. ( H ) Pie chart showing genomic annotation of EZH2-solo:AR (left) or EZH2-solo:AR-V7 (right) peaks in 22Rv1 cells. ( I , J ) Averaged intensities (top) and heatmaps (bottom) of EZH2, H3K27me3 and AR ChIP-seq signals ± 5 kb from the centers of those EZH2-solo:AR-co-occupied sites identified from 22Rv1 cells, in LNCaP-abl (I) or VCaP (J) cells. ( K ) Heatmap showing the signals of AR (only 22Rv1 cells shown; left) or EZH2 binding, ± 5 kb from the centers of those EZH2-solo:AR-co-bound peaks identified in 22Rv1 cells (second column), in the AR-negative DU145 and PC3 cells (two right columns).

Article Snippet: Briefly, 0.5 × 10 6 cells were first collected, washed in the CUT&RUN wash buffer and then bound to activated concanavilin A (ConA) beads (Bangs Laboratories, #BP531).

Techniques: Generated, ChIP-sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: A cryptic transactivation domain of EZH2 binds AR and AR’s splice variant, promoting oncogene activation and tumorous transformation

doi: 10.1093/nar/gkac861

Figure Lengend Snippet: Interaction between EZH2 TAD and AR is required for establishment of EZH2-solo binding at AR sites and for the malignant growth of prostate cancer cells. ( A ) Anti-Flag immunoprecipitation (IP; top panel) for Flag-tagged AR and the indicated HA-tagged EZH2, either WT or with the indicated EZH2 TAD -dead mutation (FA or FK) or the SET deletion, in 293T cells. FA, F145A + F171A; FK, F145K + F171K. ( B ) GST pull-down using the indicated GST protein, either GST alone or fused with WT or mutant EZH2 TAD , and the total lysate of 22Rv1 cells, followed by anti-AR immunoblotting (top). AR-FL, full-length AR. ( C ) Scheme of the domain organization of AR-FL and AR-V7. NTD, N-terminal domain; DBD, DNA-binding domain; LBD, ligand-binding domain; (Q) n , poly(Q) region; (G) n , poly(G) region. ( D ) Pull-down using GST-tagged WT EZH2 TAD and total lysate of 293T cells transfected with the indicated Flag-tagged AR, followed by anti-Flag immunoblotting (top). ( E ) Averaged intensity (top) and heatmap (bottom) showing the EZH2 binding [exogenously expressed HA-tagged EZH2, either WT or TAD-mutated (FA), as assayed by anti-HA CUT&RUN], ± 5 kb from the center of called peaks in 22Rv1 cells. Cells with empty vector (EV) served as a CUT&RUN control. ( F ) Boxplot showing log 2 -transformed FPKM counts of EZH2, WT or TAD mutated, at EZH2-‘solo’ (right) or EZH2:PRC2-ensemble (left) target sites in 22Rv1 cells. The boundaries of box plots indicate the 25th and 75th percentiles, the center line indicates the median, and the whiskers (dashed) indicate 1.5× the interquartile range. ( G ) ChIP-qPCR of binding by HA-tagged EZH2, either WT or TAD mutant (FA), at the indicated EZH2 target genes, either those EZH2-‘solo’ ones co-targeted by EZH2:AR:AR-V7 (left) or the canonical EZH2:PRC2 targets (right). The y -axis shows signals after normalization to those of input ( n = 3; mean ± SD; unpaired two-tailed Student's t -test). EV-transduced cells served as a negative control. ( H– J ) Immunoblotting for EZH2 (H), RT–qPCR for EZH2-solo:AR:AR-V7-co-activated genes (I), and measurement for 22Rv1 cell growth (J) after mock treatment [scramble (Scr)] or depletion of endogenous EZH2 (shEZH2) in 22Rv1 cells, pre-rescued with exogenous shEZH2-resistant HA-EZH2, either WT or TAD mutant (FA or FK). For (I) and (J), n = 3, mean ± SD; unpaired two-tailed Student's t -test. ( K , L ) Averaged size (K) and weight (L) of xenografted tumors by using 22Rv1 cells, which were pre-rescued with exogenous shEZH2-resistant HA-EZH2, either WT or TAD mutant, and then subjected for mock treatment (Scr) or KD of endogenous EZH2. Tumor weight was measured at day 25 post-xenograft in NSG mice ( n = 10, mean ± SD; unpaired two-tailed Student's t -test).

Article Snippet: Briefly, 0.5 × 10 6 cells were first collected, washed in the CUT&RUN wash buffer and then bound to activated concanavilin A (ConA) beads (Bangs Laboratories, #BP531).

Techniques: Binding Assay, Immunoprecipitation, Mutagenesis, Western Blot, Ligand Binding Assay, Transfection, Plasmid Preparation, Control, Transformation Assay, Two Tailed Test, Negative Control, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: A cryptic transactivation domain of EZH2 binds AR and AR’s splice variant, promoting oncogene activation and tumorous transformation

doi: 10.1093/nar/gkac861

Figure Lengend Snippet: CUT&RUN-based profiling demonstrates effect by MS177 on decreasing genomic binding of both EZH2 and AR/AR-V7 in prostate cancer cells. ( A , E , G ) Average intensity (top) and heatmap (bottom) showing CUT&RUN signals of EZH2 (A), H3K27me3 (E) and AR (G) (normalized against spike-in control and sequencing depth) ± 5 kb around the peak centers of EZH2-solo (left) or EZH2:PRC2-ensemble target sites (right) identified in 22Rv1 cells, treated for 24 h with DMSO (black) or 2.5 μM MS177 (red). AR CUT&RUN (G) was conducted with a pan-AR antibody recognizing the N-terminal region of both AR-FL and AR-V7. ( B , F, H ) Log 2 -transformed RPKM counts for CUT&RUN signals of EZH2 (B), H3K27me3 (F) and AR (H) at EZH2-solo (left) or EZH2:PRC2-ensemble target sites (right) identified in 22Rv1 cells, treated for 24 h with DMSO or 2.5 μM MS177. ( C , D ) IGV views of EZH2 and H3K27me3 binding (spike-in control and depth normalized) at PRC1 (C) or CCND2 (D) post-treatment of 22Rv1 cells with DMSO or 2.5 μM MS177. ( I ) IGV views of AR binding (spike-in control and depth normalized) at KLK3 and KLK2 (left) and FKBP5 (right) post-treatment of 22Rv1 cells with DMSO or 2.5 μM MS177.

Article Snippet: Briefly, 0.5 × 10 6 cells were first collected, washed in the CUT&RUN wash buffer and then bound to activated concanavilin A (ConA) beads (Bangs Laboratories, #BP531).

Techniques: Binding Assay, Control, Sequencing, Transformation Assay