Journal: Nature Communications

Article Title: Lack of SMARCB1 expression characterizes a subset of human and murine peripheral T-cell lymphomas

doi: 10.1038/s41467-024-52826-0

Figure Lengend Snippet: A UMAP plot showing 24 clusters of the integrated scRNA-seq dataset from two control spleen samples (WT) and two PTCL-NOS Smarcb1− tumor samples. B Relative abundance of different cell types in murine WT spleens (left), PTCL spleens (middle), and human tumors (right; NB: in order to ensure comparability, the stromal cells were removed before quantification). The pie charts in the lower part show the ratio between B-cells and myeloid cells. C Multiplex immunofluorescence (IF) images of FFPE sections of murine PTCL-NOS Smarcb1− and control spleen samples (WT: upper panels; tumor: lower panels). For better visualization, the white boxed areas ( a to f ) are enlarged (2.5x; scale bar = 100 µm). DAPI (gray) provides a nuclear counterstain, Ezh2 (yellow) defines malignant cells (Ezh2 hi ), B220 (blue) is used as a pan B-cell marker (B220 + ), and Ly6g (pink) as a marker for neutrophils (Ly6g + ). D Quantitative analysis of IF images from ( C ). Four representative regions of interest (ROIs; size: 1500 × 1500 µm) were selected and analyzed for mouse WT and Tumor samples. A Wilcoxon-Mann-Whitney test was calculated to determine if there are differences between WT and Tumor samples for all comparisons (* p = 0.0286). Boxplot settings: middle, median; lower hinge, 25% quantile; upper hinge, 75% quantile; upper/lower whisker, largest/smallest observation less/greater than or equal to upper/lower hinge ±1.5 * IQR. E The heatmap shows the overlap between cluster-specific DEG lists and the cancer hallmark metaprograms. F Signature plots of the programs Cycling, MYC, EMT and Stress in cells from WT (left) and tumor (right) samples. G A split violin plot (left/gray half: WT; right/black half: tumor) illustrates the increase in T-cell exhaustion features (Exhaust.) with a simultaneous decrease in NK cytotoxicity (Cytotox.) markers (e.g., Ncr1/NKp46) as well as infiltration of immunosuppressive myeloid cells in tumor versus WT samples. Source data of B and D are provided as a Source Data file. B Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: For multiplexed immunofluorescence analysis, slices of PTCL-NOS Smarcb1− and corresponding murine control spleens were stained in the MACSima imaging system using antibodies against B220 (RA3-6B2, Miltenyi Biotec, APC, 1:50), Ly6G (1A8, Miltenyi Biotec, PE, 1:50) and EZH2 (REA907, Miltenyi Biotec, APC, 1:50).

Techniques: Control, Multiplex Assay, Immunofluorescence, Marker, MANN-WHITNEY, Whisker Assay

Journal: Journal of Nanobiotechnology

Article Title: MiR26a reverses enzalutamide resistance in a bone-tumor targeted system with an enhanced effect on bone metastatic CRPC

doi: 10.1186/s12951-024-02438-z

Figure Lengend Snippet: The mechanism of regulatory relationships among EZH2, SFRP1, and WNT5A. (A) The miR26a level in C4-2B and C4-2B EnzR cells. n = 3, mean ± SD, *** p < 0.001, one-way ANOVA. (B) Statistical analysis of each group. n = 5, mean ± SD, * p < 0.05, **** p < 0.0001, one-way ANOVA. (C) In vitro cell migration and invasion assays. Microscope images of anti-migration and anti-invasion effects in each group (visualized with 0.1% crystal violet, scale bar: 100 μm). C4-2B EnzR cells were incubated with miR26a mimic and miR26a inhibitor, respectively (miR26a mimic/inhibitor: 1 µg·mL − 1 ). (D) Dual luciferase reporter assays were used to verify the direct targeting of miR26a on EZH2 and WNT5A, h-EZH2-3’UTR/h-WNT5A-3’UTR: 0.16 µg, hsa-miR-26a-5p/NC: 5 pmol. n = 3, mean ± SD, **** p < 0.0001, one-way ANOVA. (E) Protein expressions of EZH2, WNT5A, H3K27me3, and SFRP1 were detected via western blotting in siEZH2, siSFRP1, and their negative control groups (siNC1: EZH2 negative control, siNC2: SFRP1 negative control). (F) Statistical difference of the ratio of gray values. (G) Protein expressions of WNT5A were detected via western blotting in siEZH2 + siSFRP1 and siEZH2 + siNC1 groups. (H) Statistical difference of the ratio of gray values. n = 3, mean ± SD, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA

Article Snippet: Keratinocyte medium was purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. EZH2 Ab (rabbit, Cat.#AF5150, Lot.#00095479, Affinity), H2K27me3 Ab (rabbit, Cat.#DF6941, Lot.#46k3813, Affinity), SFRP1 Ab (rabbit, Cat.#DF01172, Lot.#35u4433, Affinity), WNT5A Ab (rabbit, Cat.#BM5137, Lot.#BST17695137, Boster), AR Ab (rabbit, Cat.#AF6137, Lot.#84f0092, Affinity) and GAPDH Ab (mouse, Cat.#ab8245, Abcam) were purchased for Western blotting study.

Techniques: In Vitro, Migration, Microscopy, Incubation, Luciferase, Western Blot, Negative Control

Journal: Genes & Development

Article Title: Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis

doi: 10.1101/gad.269522.115

Figure Lengend Snippet: Ezh2 is dispensable in genetically engineered mouse models of prostate and breast cancers. ( A ) Western blot on whole-tissue lysates from dorso–lateral (DL), anterior (A), and ventral (V) lobes of a normal prostate and a Hi-Myc invasive prostate cancer. The specific antibodies used are indicated at the left . ( B ) Hematoxylin and eosin (HE) and IHC staining of different proteins, as indicated, in a normal mouse prostate (wild type) and Hi-Myc and Hi-Myc;PB-Cre;Ezh2 fl/fl invasive prostate cancer. ( C ) Impact of Ezh2 knockdown in Myc-CaP cells (derived from Hi-Myc mouse prostates) on Ezh2, H3K27me3, HDAC, and H3 (Western blot; left panel) as well as cell proliferation ( right panel). Mean ± SD. n = 3. ( D , left panel) RT-qPCR analysis of p16 , p19 , and p21 expression in Myc-CaP cells. RT-qPCR values indicate relative expression in sh-Ezh2 compared with sh-scramble cells after normalization to TBP. ( Right panel) Enrichment of H3K27me3 by chromatin immunoprecipitation (ChIP) and qPCR (ChIP-qPCR) at the corresponding loci. actin and foxf1a were used as negative and positive controls, respectively. ChIP-qPCR values indicate relative enrichment compared with histone H3. Mean ± SD. n = 3. ( E , left panel) Western blot showing loss of Ezh2 protein in MMTV-Cre;N1IC;Ezh2 fl/fl FACS-sorted luminal cells. MMTV-Cre;N1IC;Ezh2 wt/wt cells were used as a control. ( Right panel) Representative HE staining on mammary glands of wild-type, MMTV-Cre;N1IC;Ezh2 wt/fl , and MMTV-Cre;N1IC;Ezh2 fl/fl mice showing the presence of tumors in the presence or absence of Ezh2.

Article Snippet: Antibodies against Ezh1, Ezh2, Eed, Suz12, and H3K27me2/3 (Western blot and ChIP [chromatin immunoprecipitation]/ChIP-seq [ChIP combined with deep sequencing]) were previously described ( ); total H3 (39163) and H3K27me3 (39155) for ChIP-seq were purchased from Active Motif; Lamin B1 (ab16048) was purchased from Abcam; Ezh2 (NCL-L-EZH2) for IHC on PDXs was purchased from Novocastra; H3K27me3 (C36B11) for IHC on PDXs was purchased from Cell Signaling; Flag M2 was purchased from Sigma (F1804); Nkx3.1 antibody was a generous gift from Dr C. Abate-Shen; SMA antibody was purchased from Dako; PCNA, AR, and Sirt1 antibodies were purchased from Santa Cruz Biotechnology; and tubulin antibody was purchased from Sigma.

Techniques: Western Blot, Immunohistochemistry, Knockdown, Derivative Assay, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation, ChIP-qPCR, Control, Staining

Journal: Genes & Development

Article Title: Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis

doi: 10.1101/gad.269522.115

Figure Lengend Snippet: Coupling of EZH2 expression to proliferation is required for H3K27me3 homeostasis but perturbed in breast cancers. ( A , left panel) Heat map of hierarchical clustering of the most significantly varying transcripts in primary and metastatic (Met.) prostate cancers (PCa). Data are from . Samples are arranged horizontally, and transcripts are arranged vertically. The cluster containing the EZH2 transcript is shown in greater detail at the right . ( Right panel) Gene ontology (DAVID, http://david.abcc.ncifcrf.gov ) of the EZH2 cluster showing the 20 most significantly enriched categories, their fold enrichment, and corresponding P -values. ( B ) Western blot probed with antibodies recognizing Ezh2, H3K27me3, Lamin B1, or histone H3 in sh-scramble ( left panel) and sh-Ezh2 ( right panel) Myc-CaP cells. In order to modulate proliferation in vitro, cells were cultured in the presence of 0.5%, 2%, or 10% fetal calf serum (FCS) or 10% FCS medium plus a cocktail of growth factors (bovine pituitary extract, insulin, and epidermal growth factor, indicated as +GFs in the last lane). Corresponding dot plots show signal quantification of Ezh2 (blue) and H3K27me3 (red) abundance (arbitrary units) as a function of the growth rate (number of divisions per cell per hour), as assessed by proliferation assays carried out in parallel for each culture condition. ( C ) EZH2 and H3K27me3 IHC staining quantifications across two patient-derived xenografts (PDXs) treated with various combinations of drugs. Correlation plots of EZH2 versus Ki67 and H3K27me3 versus Ki67 signal intensities are shown. Intensity values were normalized to the control (untreated) condition (green triangles). The everolimus + fulvestrant-treated PDXs show strongly reduced proliferation (red triangles). Each dot corresponds to the mean of six measurements (two stainings on three biological replicates). The corresponding coefficient of determination ( R 2 ) and P -value of the linear regression are shown. Representative IHC staining for EZH2 and H3K27me3 in untreated and everolimus + fulvestrant treated PDXs are shown. Nuclei are counterstained in blue/purple.

Article Snippet: Antibodies against Ezh1, Ezh2, Eed, Suz12, and H3K27me2/3 (Western blot and ChIP [chromatin immunoprecipitation]/ChIP-seq [ChIP combined with deep sequencing]) were previously described ( ); total H3 (39163) and H3K27me3 (39155) for ChIP-seq were purchased from Active Motif; Lamin B1 (ab16048) was purchased from Abcam; Ezh2 (NCL-L-EZH2) for IHC on PDXs was purchased from Novocastra; H3K27me3 (C36B11) for IHC on PDXs was purchased from Cell Signaling; Flag M2 was purchased from Sigma (F1804); Nkx3.1 antibody was a generous gift from Dr C. Abate-Shen; SMA antibody was purchased from Dako; PCNA, AR, and Sirt1 antibodies were purchased from Santa Cruz Biotechnology; and tubulin antibody was purchased from Sigma.

Techniques: Expressing, Western Blot, In Vitro, Cell Culture, Immunohistochemistry, Derivative Assay, Control

Journal: Genes & Development

Article Title: Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis

doi: 10.1101/gad.269522.115

Figure Lengend Snippet: Genetic loss of EZH2 is linked to poor prognosis in breast cancer. ( A , top left panel) Correlation plot of EZH2 transcript and a proliferation metagene. Residual (adjusted) values of EZH2 transcripts to the proliferation metagene are shown in the bottom left panel. EZH2 copy number variations are color-coded, with normal copy number in gray, hemizygous loss in blue, and gain in red. The same procedure was applied to adjust ORC6 transcript values (data not shown). Kaplan-Meier plots of breast cancer-specific survival for patients with primary tumors with high (above median) or low (below median) EZH2 and ORC6 transcript levels are shown in the middle and right top panels. Kaplan-Meier plots of breast cancer-specific survival for patients with primary tumors with high versus low proliferation-adjusted levels of EZH2 and ORC6 transcripts are shown in the middle and right bottom panels. The hazard ratio (HR) between the highest and lowest survival groups and P -values are displayed on Kaplan-Meier plots. ( B ) Kaplan-Meier plot of breast cancer-specific survival for patients with primary tumors with normal EZH2 or hemizygous loss or gain of EZH2 . ( C ) Univariate analysis showing the association between genetic loss and death from breast cancer on all genes of chromosome 7. False discovery rate (FDR)-corrected P -values (log 10 scale) are plotted for all chromosome 7 genes, and significant values are highlighted in red (threshold of 0.15). A dashed green line indicates the position of the EZH2 locus. The analyses shown in A – C were performed on data from 2000 primary breast cancers of the METABRIC cohort. ( D , top ) Oncoprint generated on the cBioPortal OncoPrinter showing genomic alterations and mutations in genes encoding PRC2 core components in 58 breast cancer (BCa) metastases. Only altered cases are shown. ( Bottom ) Schematic representation of the EZH2 locus showing the position of a splice site mutation in position −1 of exon 11. ( E ) Oncoprint (cBioPortal) showing loss-of-function (LOF) mutations of core PRC2 genes in The Cancer Genome Atlas (TCGA) breast cancer data set. ( F ) Kaplan-Meier plot of overall survival associated with the corresponding tumors compared with the remaining (PRC2 wild-type) tumors.

Article Snippet: Antibodies against Ezh1, Ezh2, Eed, Suz12, and H3K27me2/3 (Western blot and ChIP [chromatin immunoprecipitation]/ChIP-seq [ChIP combined with deep sequencing]) were previously described ( ); total H3 (39163) and H3K27me3 (39155) for ChIP-seq were purchased from Active Motif; Lamin B1 (ab16048) was purchased from Abcam; Ezh2 (NCL-L-EZH2) for IHC on PDXs was purchased from Novocastra; H3K27me3 (C36B11) for IHC on PDXs was purchased from Cell Signaling; Flag M2 was purchased from Sigma (F1804); Nkx3.1 antibody was a generous gift from Dr C. Abate-Shen; SMA antibody was purchased from Dako; PCNA, AR, and Sirt1 antibodies were purchased from Santa Cruz Biotechnology; and tubulin antibody was purchased from Sigma.

Techniques: Generated, Mutagenesis

Journal: Genes & Development

Article Title: Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis

doi: 10.1101/gad.269522.115

Figure Lengend Snippet: Genetic disruption of EZH2 in a breast cancer cell line promotes tumorigenesis. ( A ) Western blot showing loss of the EZH2 protein and H3K27me3 mark in the EZH2 full knockout MDA-MB-231 cell line (indicated as EZH2-null) compared with the parental clone mutant for one allele out of three. ( B ) Proliferation curve of control and EZH2-null cells. ( C ) Multicellular spheroids of control or EZH2-null MDA-MB-231 cells were embedded in 3D acid-extracted type I collagen (T0) and further incubated for 2 d (T2). Images show representative phalloidin-labeled spheroids collected at T0 ( inset ) or T2. Bars, 200 μm. Data represent mean invasion area in type I collagen at T2 normalized to the mean invasion area at T0 ±SEM. n = 3; 15–20 spheroids were analyzed for each cell line, with a total of 52 and 45 measurements for control and EZH2-null cells, respectively. Red bars indicate mean ± SD. The P -value of the two-tailed unpaired t -test is indicated. ( D ) Representative pictures of orthotopic tumor xenografts developed from the control MDA-MB-231 clone and EZH2-null clone ( top panel) and a plot showing corresponding tumor volumes ( bottom panel). Red bars indicate mean ± SD. The P -value of the two-tailed unpaired t -test is indicated.

Article Snippet: Antibodies against Ezh1, Ezh2, Eed, Suz12, and H3K27me2/3 (Western blot and ChIP [chromatin immunoprecipitation]/ChIP-seq [ChIP combined with deep sequencing]) were previously described ( ); total H3 (39163) and H3K27me3 (39155) for ChIP-seq were purchased from Active Motif; Lamin B1 (ab16048) was purchased from Abcam; Ezh2 (NCL-L-EZH2) for IHC on PDXs was purchased from Novocastra; H3K27me3 (C36B11) for IHC on PDXs was purchased from Cell Signaling; Flag M2 was purchased from Sigma (F1804); Nkx3.1 antibody was a generous gift from Dr C. Abate-Shen; SMA antibody was purchased from Dako; PCNA, AR, and Sirt1 antibodies were purchased from Santa Cruz Biotechnology; and tubulin antibody was purchased from Sigma.

Techniques: Disruption, Western Blot, Knock-Out, Mutagenesis, Control, Incubation, Labeling, Two Tailed Test

Journal: Genes & Development

Article Title: Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis

doi: 10.1101/gad.269522.115

Figure Lengend Snippet: Impaired PRC2 function selectively affects H3K27me3-low genes. ( A ) Venn diagram showing the overlap between up-regulated transcripts upon Ezh2 deletion and Ezh2 targets defined by the presence of H3K27me3 in the promoter of wild-type versus Ezh2 Δ/Δ iMEFs. Up-regulated transcripts were identified with a minimum adjusted P -value of 15% and a minimum fold change of two. Targets that are H3K27me3-enriched and up-regulated upon loss of Ezh2 ( n = 217) are defined as “responsive targets” as opposed to H3K27me3-enriched “nonresponsive targets” ( n = 3974). Genes that are up-regulated but are H3K27me3-negative ( n = 226) correspond to indirect targets. ( B ) Time-course analysis by Western blot of Ezh2 and H3K27me3 before (time 0) and at different times after OHT-induced Ezh2 deletion. A two-point titration is provided for each condition. Tubulin and H3 were used as loading controls. ( C ) Time-course expression analysis of Ezh2-responsive direct targets. Red represents early responsive targets, and blue indicates late responsive genes. All values were normalized to initial (time 0) values. ( D ) H3K27me3 density plot around the transcription start site (TSS) of early responsive (in red) and late responsive targets (in blue). ( E ) Western blot of control, Ezh1-overexpressing, and Ezh2-overexpressing iMEFs untreated or treated with OHT to remove endogenous Ezh2 expression. Specific antibodies are indicated at the left . ( F ) Heat map representing the mean-centered expression of Ezh2-responsive targets in wild-type, Ezh2 Δ/Δ , and Ezh2 Δ/Δ iMEFs overexpressing either Ezh2 or Ezh1. ( G ) H3K27me3 density plot around the TSSs of genes for which the absence of endogenous Ezh2 is rescued by Ezh1 (blue line) or not (red line). Analyses presented in C , D , F , and G were performed using measurements on two biological replicates.

Article Snippet: Antibodies against Ezh1, Ezh2, Eed, Suz12, and H3K27me2/3 (Western blot and ChIP [chromatin immunoprecipitation]/ChIP-seq [ChIP combined with deep sequencing]) were previously described ( ); total H3 (39163) and H3K27me3 (39155) for ChIP-seq were purchased from Active Motif; Lamin B1 (ab16048) was purchased from Abcam; Ezh2 (NCL-L-EZH2) for IHC on PDXs was purchased from Novocastra; H3K27me3 (C36B11) for IHC on PDXs was purchased from Cell Signaling; Flag M2 was purchased from Sigma (F1804); Nkx3.1 antibody was a generous gift from Dr C. Abate-Shen; SMA antibody was purchased from Dako; PCNA, AR, and Sirt1 antibodies were purchased from Santa Cruz Biotechnology; and tubulin antibody was purchased from Sigma.

Techniques: Western Blot, Titration, Expressing, Control

Journal: Genes & Development

Article Title: Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis

doi: 10.1101/gad.269522.115

Figure Lengend Snippet: Impaired PRC2 function leads to transcriptional instability. ( A ) Nascent RNA FISH analysis of two responsive Ezh2 targets in Ezh2 wild-type and Ezh2 mutant iMEFs. The Cpa6 gene is autosomal, while the Tspan7 gene is localized on the X chromosome and thus only presents in one copy in this male cell line. The top panel shows representative examples of RNA FISH signals, and the bottom graph shows relative proportions of nuclei with no signal, one pinpoint (monoallelic), and two pinpoints (biallelic) over a minimum of 50 nuclei. ( B , top ) Experimental scheme for the single-cell analysis of PRC2 target genes. ( Bottom ) Single-cell analysis of the Ezh2 transcript and selected responsive (res.) and nonresponsive genes. Forty-nine Ezh2 wild-type and 37 Ezh2 mutant cells were analyzed by RT-qPCR on a Biomark-HD system. Ezh2 mRNA level in individual cells is plotted at the top ; red diamonds represent Ezh2 wild-type cells (DiI-positive), and green diamonds indicate Ezh2 Δ/Δ cells (DiO-positive). A heat map representing the mean-centered, log 2 transformed expression of selected target genes is displayed at the bottom . ( C ) Western blot of Ezh2, H3K27me3, and Lamin B1 as a loading control in different conditions as indicated at the top of each lane. ( D ) Heat map showing hierarchical clustering of transcripts in Ezh2 wild-type, Ezh2 Δ/Δ , and pre- and post-deletion rescue conditions.

Article Snippet: Antibodies against Ezh1, Ezh2, Eed, Suz12, and H3K27me2/3 (Western blot and ChIP [chromatin immunoprecipitation]/ChIP-seq [ChIP combined with deep sequencing]) were previously described ( ); total H3 (39163) and H3K27me3 (39155) for ChIP-seq were purchased from Active Motif; Lamin B1 (ab16048) was purchased from Abcam; Ezh2 (NCL-L-EZH2) for IHC on PDXs was purchased from Novocastra; H3K27me3 (C36B11) for IHC on PDXs was purchased from Cell Signaling; Flag M2 was purchased from Sigma (F1804); Nkx3.1 antibody was a generous gift from Dr C. Abate-Shen; SMA antibody was purchased from Dako; PCNA, AR, and Sirt1 antibodies were purchased from Santa Cruz Biotechnology; and tubulin antibody was purchased from Sigma.

Techniques: Mutagenesis, Single-cell Analysis, Quantitative RT-PCR, Transformation Assay, Expressing, Western Blot, Control

Journal: Journal of cellular physiology

Article Title: Mll-COMPASS complexes mediate H3K4me3 enrichment and transcription of the osteoblast master gene Runx2/p57 in osteoblasts

doi: 10.1002/jcp.27355

Figure Lengend Snippet: (A) MC3T3 cells were differentiated to mineralized osteoblasts for 9 days of culture (DIV) using Ascorbic Acid (AA) 50ug/mL. Nuclear extracts were collected at indicated the days and analyzed by western blot using specific antibodies against the indicated epigenetic regulators. TFIIB or RNA-polymerase II (RNAPII) was used to control for equal protein loading. (B-C and E-F) Binding of chromatin regulators to the Runx2 P1 promoter at the indicated differentiation days were analyzed by ChIP using antibodies against: (B) Wdr5, (C) Utx, (E) Ezh2, (F) Prmt5, and (G) Jarid1b. (D) Re-ChIP assay performed in chromatin samples obtained from differentiated cells (5 DIV) using first an antibody against Utx and subsequently an antibody against Wdr5. Results and statistical analyses are shown as described in figure legend 2. ***p<0.001, *p<0.05, ns = non-statistically significant differences.

Article Snippet: The following antibodies were used in ChIP assays: Wdr5 (ab56919, Abcam), Jarid1b/Kdm4b (ab50958, Abcam), Ezh2 (07–689, Merck Millipore, Danvers, MA, USA), Utx/Kdm6a (ab91231, Abcam), Prmt5/Jbp1 (611539, BD Biosciences), H3K27me3 (07–449, Merck Millipore), H3K4me1 (ab8895, Abcam), H3K4me3 (ab8580, Abcam), H4R3me2S (ab5823, Abcam), H3Ac (06–599, Merck Millipore), Cgbp (SC-25391, Santa Cruz Biotechnology), Menin (A300–105A, Bethyl Laboratories, Montgomery, TX, USA), Set1A (A300–290A, Bethyl Laboratories), Set1B (SC-248563, Santa Cruz Biotechnology), Mll1 (39829, Active Motif), Mll2 (SC-292359, Santa Cruz Biotechnology), Mll3 (ab71200, Abcam), Mll4 (ab60053, Abcam).

Techniques: Western Blot, Binding Assay

Journal: Journal of cellular physiology

Article Title: Mll-COMPASS complexes mediate H3K4me3 enrichment and transcription of the osteoblast master gene Runx2/p57 in osteoblasts

doi: 10.1002/jcp.27355

Figure Lengend Snippet: (A-B and D-E) Differentiating MC3T3 cells (3DIV) were infected with lentiviral particles containing shRNAs against the chromatin modifiers: (A) Wdr5, (B) Utx, (D) Ezh2 and (E) Prmt5. Knockdown efficiencies were confirmed by RT-qPCR and western blot (A-D, left panels) analyses, 48 h post-infection (5 DIV). TFIIB or RNAPII proteins were used to control for equal protein loading. (C) Effect of knocking down Wdr5 (left) and Utx (right) expression on Bglap gene transcription. mRNA expression values were normalized against Gapdh mRNA levels. Statistical analyses were assessed with respect to the mRNA levels obtained in cells infected with an empty vector (EV). *p<0.05, ***p<0.001, ns = non-statistically significant differences.

Article Snippet: The following antibodies were used in ChIP assays: Wdr5 (ab56919, Abcam), Jarid1b/Kdm4b (ab50958, Abcam), Ezh2 (07–689, Merck Millipore, Danvers, MA, USA), Utx/Kdm6a (ab91231, Abcam), Prmt5/Jbp1 (611539, BD Biosciences), H3K27me3 (07–449, Merck Millipore), H3K4me1 (ab8895, Abcam), H3K4me3 (ab8580, Abcam), H4R3me2S (ab5823, Abcam), H3Ac (06–599, Merck Millipore), Cgbp (SC-25391, Santa Cruz Biotechnology), Menin (A300–105A, Bethyl Laboratories, Montgomery, TX, USA), Set1A (A300–290A, Bethyl Laboratories), Set1B (SC-248563, Santa Cruz Biotechnology), Mll1 (39829, Active Motif), Mll2 (SC-292359, Santa Cruz Biotechnology), Mll3 (ab71200, Abcam), Mll4 (ab60053, Abcam).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing, Plasmid Preparation

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: EZH2 and HDAC1/2 expression in PTCL.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: Representative immunohistochemical features of HDAC1 (left), HDAC2 (middle), and EZH2 (right) in PTCL-NOS, ALCL, NK/T and AITL. All images were captured at ×200 and ×400 magnifications. HDAC, histone deacetylase; EZH2, enhancer of zeste homolog 2; PTCL-NOS, peripheral T cell lymphoma not otherwise specified; ALCL, anaplastic large cell lymphoma; NK/T, natural killer/T-cell; AITL, angioimmunoblastic T-cell lymphoma.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Immunohistochemical staining, Histone Deacetylase Assay

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: Correlations between EZH2 and HDAC1/2 in four subtypes of PTCL.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques:

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: Correlations between EZH2/HDAC1/2 expression and the clinicopathological characteristics in PTCL.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: Correlations between the EZH2/HDAC1/2 expression and the clinicopathological characteristics in PTCL-NOS.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: Correlations between the EZH2/HDAC1/2 expression and the clinicopathological characteristics in NK/TCL.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: OS rates based upon the expression levels of EZH2 and HDAC1/2 in PTCL. (A) Significant trend towards poorer OS rates for patients with high EZH2 expression (P=0.012). (C) High expression of HDAC2 was correlated with a poorer OS rate compared with low expression of HDAC2 (P<0.001), which was not observed in the HDAC1 group [(B); P=0.353]. OS, overall survival; HDAC, histone deacetylase; EZH2, enhancer of zeste homolog 2.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing, Histone Deacetylase Assay

Journal: Oncology Letters

Article Title: Clinical significance of enhancer of zeste homolog 2 and histone deacetylases 1 and 2 expression in peripheral T-cell lymphoma

doi: 10.3892/ol.2019.10410

Figure Lengend Snippet: OS based upon the levels of EZH2 and HDAC1/2 in PTCL-NOS. (A) Significant trend towards poorer OS rates for patients with high EZH2 (P=0.002). (C) High expression of HDAC2 was correlated with a poorer OS rate, compared with low expression of HDAC2 (P<0.001), which was not observed in the HDAC1 group [(B); P=0.339]. OS, overall survival; HDAC, histone deacetylase; EZH2, enhancer of zeste homolog 2; PTCL-NOS, peripheral T cell lymphoma not otherwise specified.

Article Snippet: Following rinsing with phosphate-buffered saline, the slides were incubated with polyclonal rabbit anti-HDAC1 (catalog no. BS6485; 1:100 dilution; Bioworld Technology, Inc., St. Louis Park, MN, USA), polyclonal rabbit anti-HDAC2 (catalog no. 12922-3-AP; 1:200 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and polyclonal rabbit anti-EZH2 (catalog no. BS90776; 1:50 dilution; Bioworld Technology, Inc.) primary antibodies overnight at 4°C.

Techniques: Expressing, Histone Deacetylase Assay

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: CDYL enhances PRC2 activity in vitro. A, Coomassie Blue staining of PRC2 complexes (containing EZH2, SUZ12, and EED) purified from Sf9 cells. B, MNase digestion of reconstituted oligonucleosomes resolved by 2% agarose gel. Left panel: lane 1 shows the pure pG5E4 plasmid DNA, and lane 2 shows band shift of pG5E4 DNA assembly into oligonucleosomes. Right panel: equimolar amounts of reconstituted oligonucleosomes were digested with increasing amounts of MNase (Sigma). The DNA was isolated and subjected to electrophoresis on a 2% agarose gel in the presence of ethidium bromide. Partial MNase digestion (oligonucleosome: MNase = 2 μg: 0.5 μl) generated a nucleosomal DNA ladder with visible mono-, di-, and trinucleosomal fragments, which are indicated by corresponding numbers of asterisks. Mononucleosomal DNA runs as a 147 bp fragment. C, CDYL stimulates PRC2 activity in vitro. Reconstituted recombinant oligonucleosomes were incubated with EZH2/SUZ12/EED complexes (PRC2-core) in the absence or presence of increasing amounts of baculovirus generated CDYL and histone methyltransferase activity was determined by standard HMT assays. The reaction products were examined by Western blotting with the antibodies indicated on the right. Ponceau staining of histones is shown in the bottom panel to show equal amounts of substrates used in each reaction. D, CDYL only stimulates PRC2 methyltransferase activity toward oligonucleosome, but not mononucleosome substrates. Reconstituted Xenopus oligonucleosomes were digested with MNase (oligonucleosome: MNase = 1 μg: 1 μl) at room temperature for 5 min. This treatment yielded mainly mononucleosomes (see Fig. 4B). Equal amounts of mononucleosomes were used as substrates for the HMT assay in the top panel, whereas equal amounts of undigested oligonucleosomes were used as substrates in the bottom panel. Commercially available EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (PRC2-full) were used to provide methyltransferase activity as indicated. The mild increase of PRC2 activity seen upon CDYL addition in the top panel was mainly due to incomplete digestion of oligonucleosomes (see Fig. 4B). E, binding affinity between CDYL and H3K27me3 is much stronger than the affinity between EED and H3K27me3. In the top panel, histone peptide binding assays show that CDYL, but not EED, binds to H3K27me2 when the same amounts of FLAG-tagged proteins (0.5 μg) were used in the assay. To compare the binding affinity for H3K27me3, 0.2, 0.4, or 1 μg of baculovirus-expressed FLAG-CDYL and 0.6, 1.2, or 3 μg of FLAG-EED proteins were used in the peptide binding assay. Ten percent of total proteins were used as loading controls (middle panel). Peptide-protein complexes were pulled down by streptavidin beads and bound proteins were examined by Western blotting using anti-FLAG antibodies (bottom panel). Ponceau staining of baculovirus-expressed FLAG-CDYL and FLAG-EED is shown in the right panel.

Article Snippet: Anti-EZH2 and anti-Myc (MBL) antibodies were used to detect the respective translated proteins.

Techniques: Activity Assay, In Vitro, Staining, Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Isolation, Electrophoresis, Generated, Recombinant, Incubation, Western Blot, HMT Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: CDYL is physically associated with the PRC2 complex. A, in vivo immunoprecipitation. Top two panels: endogenous IP of MCF-7 cell lysates using antibodies against CDYL. Antibodies (EZH2 or SUZ12) used for Western blotting are indicated on the right. Bottom panel: MCF-7 cells were transfected with a FLAG-CDYL construct and subjected to co-IP assays after 48 h. Cell protein extracts were immunoprecipitated with polyclonal antibodies against EED, and blotted with monoclonal anti-FLAG antibodies. An immunoglobulin G (IgG) control is included in each experiment. B, GST pull-down assays. Purified GST or GST-CDYL proteins immobilized on glutathione Sepharose 4B beads were incubated with in vitro translated EZH2, SUZ12, or EED. Bound proteins were detected with monoclonal anti-EZH2 antibodies (top panel) or anti-MYC tag antibodies (lower two panels). C, Superose 6 gel filtration analysis of the MCF-7 nuclear extracts. Migration of molecular markers is indicated above the panels and the antibodies for Western blotting are indicated on the right. Equal volumes from each fraction were analyzed. D, similar FPLC experiments as in C using a Superdex 200 10/300 GL column. The chromatographic fractions were analyzed by Western blotting using the indicated antibodies. Bottom panel: HMT assays were performed using the indicated eluted fractions. Recombinant Xenopus H3 proteins were used as substrates, and the reaction products were analyzed by Western blotting with anti-H3K27me3 antibodies.

Article Snippet: Anti-EZH2 and anti-Myc (MBL) antibodies were used to detect the respective translated proteins.

Techniques: In Vivo, Immunoprecipitation, Western Blot, Transfection, Construct, Co-Immunoprecipitation Assay, Purification, Incubation, In Vitro, Filtration, Migration, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: Mapping the domains responsible for the interaction between CDYL and EZH2. A, schematic drawing of CDYL protein. CDYL deletion mutants including del1 (1–309 aa), del2 (1–60 aa, the chromodomain), del3 (61–545 aa), del4 (310–545 aa, the coAP domain), and del5 (61–309 aa) were fused to GST. B, GST pull-down experiments were performed with in vitro translated FLAG-EZH2 and purified GST or GST-CDYL deletion mutants. The precipitated complexes were examined by Western blotting using monoclonal anti-EZH2 antibodies (the upper panel). Only CDYL mutants containing the middle region from 61–309 aa (del1, del3, del5) efficiently pulled down EZH2. The lower panel shows the Ponceau staining of purified GST fusion proteins added to the reaction. The arrows indicate the positions of the respective GST fusion proteins as labeled on the top. C, schematic drawing of EZH2 protein. EZH2 deletion mutants (del1 to del5) were cloned into the pGBKT7 plasmid, which contains a c-Myc epitope tag and can be transcribed/translated in vitro. Del8 and Del9 were fused to GST. D, GST pull-down experiments were performed with in vitro translated Myc-EZH2 deletion mutants and purified GST or GST-CDYL in the left panels. Right panel: GST pull-down assays were performed with in vitro translated Myc-CDYL, which was incubated with purified GST, GST-del8, GST-del9, or GST-SET8 (negative control protein). Bound proteins were examined by Western blotting using monoclonal anti-Myc antibodies.

Article Snippet: Anti-EZH2 and anti-Myc (MBL) antibodies were used to detect the respective translated proteins.

Techniques: In Vitro, Purification, Western Blot, Staining, Labeling, Clone Assay, Plasmid Preparation, Incubation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: Validation of common target genes of CDYL and PRC2. A, quantitative ChIP assays were performed in MCF-7 cells with primer pairs specific to indicated gene promoters (see supplemental Table S1). Normal rabbit IgG, as well as polyclonal antibodies against CDYL, EZH2, and H3K27me3 were used to immunoprecipitate the protein-DNA complex. B, conventional semi-quantitative ChIP assays performed at the MYT1 and BASE promoters. C, CDYL and PRC2 exist in the same protein complex at the MYT1 and BASE promoters. ChIP and re-ChIP experiments were performed with the indicated antibodies and primer pairs. D, CDYL expression was efficiently knocked down by specific siRNAs. Non-silencing or CDYL specific siRNAs were transfected into MCF-7 cells. Total proteins were extracted and the expression of CDYL and EZH2 proteins were examined by Western blotting. Actin protein levels were measured to indicate equal loading of protein lysates. E, CDYL is required for PRC2 chromatin targeting at the MYT1 and BASE promoters. MCF-7 cells were transfected with control siRNA or CDYL-specific siRNA. 48 hours after the transfection, cell lysates were collected, and ChIP experiments were performed using the indicated antibodies. Real-time PCR assays were performed for the measurement. F, CDYL mainly represses the expression of target genes. MCF-7 cells were transfected with control or CDYL-specific siRNAs. Total RNAs were prepared and the mRNA levels of the indicated genes were examined by real-time RT-PCR. The data were normalized against the expression of GAPDH. Each bar represents the mean ± S.D. for triplicate measurements.

Article Snippet: Anti-EZH2 and anti-Myc (MBL) antibodies were used to detect the respective translated proteins.

Techniques: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: iScience

Article Title: TNIK drives castration-resistant prostate cancer via phosphorylating EGFR

doi: 10.1016/j.isci.2023.108713

Figure Lengend Snippet:

Article Snippet: Anti-EZH2 antibody , Affinity Biosciences , Cat# AF5150; RRID: AB_2837636.

Techniques: Virus, Bacteria, Recombinant, Lysis, Chromatin Immunoprecipitation, Control, Isolation, Labeling, Software