rabbit monoclonal anti-ezh2  (Cell Signaling Technology Inc)

Journal: Nature communications

Article Title: EZH2 mutations in follicular lymphoma distort H3K27me3 profiles and alter transcriptional responses to PRC2 inhibition.

doi: 10.1038/s41467-024-47701-x

Figure Lengend Snippet: Fig. 2 | Ezh2Y641F is sufficient to drive aberrant H3K27me3 patterns and strengthened H3K27ac peaks. a, b Western blot analysis in isogenic iMEFs. Identical samples were run in parallel on different blots. Source data are provided as a Source Data file. c In vitro histone methyltransferase assay using 3H-labeled S- adenosyl-methionine comparing the activity of reconstituted PRC2 variants on recombinant nucleosomes in the presence of JARID2, AEBP2 or H3K27me3 peptide. Source data are provided as a Source Data file. d H3K27me3 and H3K27me2 CUT&RUN tracks for WT and Ezh2Y641F/WT iMEFs with corresponding genomic annotations shown below (note that the H3K27me3 signal for WT cells exceeds the range displayed). e Heatmap of changes in enrichment for histone modifications across 10 emission states defined by ChromHMM (see Supplementary Fig. 1j) comparing Ezh2Y641F/WT iMEFs to WT. f H3K27ac CUT&RUN tracks for WT and Ezh2Y641F/WT iMEFs with corresponding genomic annotations shown below. g Violin

Article Snippet: The resulting Table 1 | List of antibodies used in the study Antibody Host Application Source Clone/identifier EZH2 rabbit polyclonal WB homemade33 N/A H3K27me3 rabbit monoclonal WB, C&R,ChIP CST C36B11 H3K27me2 mouse monoclonal WB, C&R Active Motif 324 H3K27me1 mouse monoclonal WB Active Motif 321 EED rabbit polyclonal WB homemade33 N/A H3.3K27M rabbit monoclonal WB Millipore RM192 H2Aub rabbit monoclonal C&R CST 8240S H3K27ac rabbit polyclonal C&R Abcam Ab4729 H3K4me3 rabbit monoclonal C&R CST C42D8 H3K36me3 rabbit polyclonal C&R Abcam Ab9050 H4 rabbit polyclonal WB Active Motif AB_2636967 SUZ12 rabbit monoclonal C&R CST D39F6 Rabbit IgG goat polyclonal WB secondary BioRad 12004161 Mouse IgG goat polyclonal WB secondary BioRad STAR117D800GA WB Western blot, C&R CUT&RUN, ChIP chromatin immunoprecipitation, CST Cell Signaling Technology.

Techniques: Western Blot, In Vitro, HMT Assay, Labeling, Activity Assay, Recombinant

Journal: Molecular Therapy. Nucleic Acids

Article Title: Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression

doi: 10.1016/j.omtn.2024.102173

Figure Lengend Snippet: EZH2-FLASH identifies direct endothelial RNA targets (A) Experimental approach used in the study with plan and listed experiments. (B) Schematic representation of steps in FLASH (formaldehyde and UV crosslinking, ligation, and sequencing of hybrids) with EZH2 immunoprecipitation using lysates from UV crosslinked HUVECs. Dynamic EZH2:RNA complex formation occurs as represented. Following RNA ligation and hybrid formation between interacting RNAs, sequencing is performed. Further analysis of single and hybrid reads bound by EZH2, reveals interacting RNA molecules. (C) Distribution of annotated reads over genome, with gene classification (biotype), from statistically filtered EZH2-FLASH data with two biological replicates in HUVECs and MEG3-lncRNA (0.04%, red line) as the lncRNA candidate (14.6%, red wedge). (D) ( i and ii) Enriched motifs with sequences in MEG3 mRNA of EZH2-FLASH that uniquely overlap exons; the logos were drawn using the top 4–8 nt K-mers for each experimental replicate ( top and middle ) and Z - score for each. Motif analysis was performed using the MEME suite. ( iii ) Enriched motif within the fragments of MEG3:MEG3 hybrids. (E) Total RNA-RNA interactions associated with MEG3 at chr14:100,829,033-100,836,300 (Hg38), (MEG3 id = NR_002766.2) and distribution of all MEG3 interactions among various classes of RNAs as captured by EZH2-FLASH. (F) Intermolecular MEG3-RNA interactions captured by EZH2-FLASH. Hybrid counts were mapped for all annotated hybrids' genomic features, and those of MEG3 were plotted in the Circos plot, aligning with their position along the MEG3 genomic sequence. The main MEG3 hybrid detected is MEG3, that is represented by the number of interactions in red. Red circle shows the position within the MEG3 gene in kilobases with ∗50–55 kb falling within exon 3. The blue circle is a visual representation of MEG3 exons. Regions overlapping exons are represented in solid blue. The purple broad circle shows the nucleotides at each position: (A) dark blue, (B) light blue, (T) light red, (G) dark red. The inner part of the white circle shows MEG3:MEG3 hybrids; arcs connecting the center of each hybrid fragment are shown in red, and the regions spanned by the hybrid fragments are shown in light green.

Article Snippet: To precipitate EZH2, we used 20 μL of anti-EZH2 rabbit monoclonal antibody (D2C9 clone, CST, no. 5246S) and crosslinked cell lysates of HUVECs.

Techniques: Ligation, Sequencing, Immunoprecipitation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression

doi: 10.1016/j.omtn.2024.102173

Figure Lengend Snippet: MEG3 directly targets angiogenic genes (A) ChIRP-seq analysis showing percent of peak elements in the genomic features upon MEG3-ChIRP pull-down using biotinylated probes against the MEG3 gene to isolate associated DNA for sequencing and RNA for qPCR validation of probes ( Figure S3 C). The ChIRP pull-down with LacZ oligos was used as negative control. n=2 biological replicates with odd and even probes against MEG3 was followed by bioinformatics analysis to merge the individual replicates and boost the signals. (B) ChIRP-seq as in (A) with a display of percent peaks over named regulatory elements. (C) Enriched motifs with sequences in MEG3 mRNA of ChIRP-seq peaks that uniquely overlap promoter, enhancer, TF, open chromatin, and CTCF binding region were assessed (see ); logos were drawn using the top 50 nt K-mers for each experimental replicate and a Z score was calculated for each. Top enriched motifs by E value (statistical significance as calculated by MEME) within the (i) promoter sequence and (ii) enhancer binding, that uniquely overlapped regions associated with MEG3 probes. Motif analysis was performed using the MEME suite and all motifs are listed in the . (D) Volcano plot of gene enrichment analysis for all MEG3 ChIRP peak-associated genes (left) with top-rated GO biological pathway annotations (right). EnrichR analysis with Panther Pathway resource was used to associate most represented genes with pathways and p value was calculated by the Binomial statistic with a cutoff of 0.05 used as a start point. (E) MEG3-associated pathways with p values, classified using Panther Pathway analysis for MEG3-ChIRP-seq mRNAs targets. (F) Overlap between MEG3 mRNA targets obtained from ChIRP-seq experiments and RNA-seq of MEG3 KD performed in HUVECs (shown in Figures 3 E and ; Table S7 ). (G) Overview of the critical steps to obtain MEG3-bound genomic loci and intersections with EZH2 and H3K27me3 ChIP signals (GEO databases for HUVECs). The intersection between GEO EZH2 ChIP, GEO H3K27me3 ChIP, and statistically filtered MEG3-ChIRP data from two biological replicates was performed. Overlapping features were mapped and enhancer regions exposed. The number of genes and degree of overlap is obtained between MEG3- and PRC2-dependent genes. The p values are a result of hypergeometric test.

Article Snippet: To precipitate EZH2, we used 20 μL of anti-EZH2 rabbit monoclonal antibody (D2C9 clone, CST, no. 5246S) and crosslinked cell lysates of HUVECs.

Techniques: Sequencing, Biomarker Discovery, Negative Control, Binding Assay, RNA Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression

doi: 10.1016/j.omtn.2024.102173

Figure Lengend Snippet: Overlapping targets between MEG3 and repressive chromatin (EZH2 and H3K27me3) in ECs (A) Convergence of MEG3-ChIRP peaks overlapping EZH2-ChIP peaks or H3K27me3 peaks at genomic loci with the named gene regions. (B) Distribution of MEG3-ChIRP peaks overlapping EZH2-ChIP peaks or H3K27me3 peaks with intersecting reads in relation to gene type. (C) Maximum peak score of ChIP signal for EZH2 and H3K27me3 intersecting the top enriched MEG3 peaks associated with nearest genes. Highest EZH2 peak score is over ITGA4, with H3K27me3 activity also detected in ITGA4, ITGA7, ITGA8, and ITGA9, members of the ITGA family. (D) Heatmap showing distribution of reads from EZH2 ChIP-seq experiment. Signal densities at all unique RefSeq genes within TSSs ±3 kb are sorted by EZH2 occupancy, in control vs. MEG3-deficient (10 nM) HUVECs. (i) Percent total changed representable peaks in MEG3 KD (mRNA, antisense, and lncRNA genes) from ChIP-seq. (ii) Depletion of MEG3 gene in HUVECs (10 nM LNA GapmeRs) was achieved with MEG3 relative expression showing ∼70% reduction compared with control (Ctr). (E) Volcano plot of differentially expressed genes with log fold change (logFC) on the x -axis from MEG3 KD in HUVECs and the in-house ChIRP-seq (MEG3, human, HUVECs) –log10p adj on the y -axis. Representative co-detected genes (red) between two datasets are statistically significant and represent bona fide MEG3 targets. Among the overlapping targets we highlighted in green the genes commonly seen between the ChIP-seq experiment ( Figure 3 D) and MEG3 KD. Finally, in blue are genes from the ChIP-seq experiment ( Figure 3 D) commonly found between all three datasets MEG3 KD, ChIP-seq, and ChIRP-seq. We highlighted the ITGA4 gene that belongs to the angiogenesis pathway and is mutually regulated by MEG3:EZH2. Similar overlap with murine dataset is represented in D and S6E. (F) Maximum peak scores of the overlapping signal over the ITGA4 promoter obtained by intersection of the EZH2 ChIP signal (D) with the MEG3-ChIRP signal (A) at this promoter region (chr2:181,457,035–181,458,302). Upon depletion of MEG3 the EZH2 signal is significantly reduced and there is no overlap with the MEG3 ChIRP signal.

Article Snippet: To precipitate EZH2, we used 20 μL of anti-EZH2 rabbit monoclonal antibody (D2C9 clone, CST, no. 5246S) and crosslinked cell lysates of HUVECs.

Techniques: Activity Assay, ChIP-sequencing, Control, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression

doi: 10.1016/j.omtn.2024.102173

Figure Lengend Snippet: Functional profiling of overlapping genes with maximum peak scores between ChIP signal for EZH2 and H3K27me3 vs. MEG3-ChIRP peak s

Article Snippet: To precipitate EZH2, we used 20 μL of anti-EZH2 rabbit monoclonal antibody (D2C9 clone, CST, no. 5246S) and crosslinked cell lysates of HUVECs.

Techniques: Functional Assay, Clinical Proteomics, Membrane

Journal: Molecular Therapy. Nucleic Acids

Article Title: Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression

doi: 10.1016/j.omtn.2024.102173

Figure Lengend Snippet: Functional profiling of differentially expressed genes in EZH2 ChIP-seq from control and MEG3-depleted ECs

Article Snippet: To precipitate EZH2, we used 20 μL of anti-EZH2 rabbit monoclonal antibody (D2C9 clone, CST, no. 5246S) and crosslinked cell lysates of HUVECs.

Techniques: Functional Assay, Control, Binding Assay, Activity Assay, Clinical Proteomics, Membrane

Journal: Molecular Therapy. Nucleic Acids

Article Title: Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression

doi: 10.1016/j.omtn.2024.102173

Figure Lengend Snippet: MEG3:EZH2-regulated targets in ECs (A) Using a Venn diagram, logical relationships are displyed between genes by plotting the EZH2-FLASH RNA gene interactome (pink) against RNA-seq data for genes that are differentially expressed following EZH2 knockdown in HUVECs (blue) (these are de novo analyzed GEO RNA-seq data ( GSE71164 ) of Scr vs. EZH2). Next, MEG3 ChIRP-seq peaks (green) are displayed against EZH2 ChIP-seq intensities over loci (yellow) obtained following MEG3 KD in HUVECs. We focused on targets in Group 1, that are regulated by EZH2 at the DNA level, in a MEG3-dependent manner. (B) Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red, expressed ITGA4 gene; green , ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164 ). (C) Genomic tracks showing ChIRP-seq signal (MEG3 and LacZ) in HUVECs over the ITGA4 gene region. The updated tracks for ChIRP-seq show called peaks using MACS2. The two ChiRP tracks are from biological replicates of even and odd probes, which have been combined in all MEG3 tracks from two experiments. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2 signals, and it overlaps with the EZH2 signal in the promoter region of the gene. The EZH2 ChIP-seq tracks over ITGA4 regions are also presented in duplicates for Scr control and MEG3-depleted HUVECs (MEG3 KD, 10 nM). Within the ITGA4 promoter region where the named signals converge there is a CpG island of 1,268 bp covering chr2:181,457,035–181,458,302 (green). This same pattern of EZH2 and other PRC2 components occupancy is observed on UCSC representation of ITGA4 regulatory region (see Figure S8 A). (D) MEG3-ChIRP validation by qPCR, n=3 independent experiment in duplicates. Analysis of MEG3 binding was done using primers against ITGA4 probe areas 1, 2, and 3 as marked in corresponding signal under (C). qPCR was performed in triplicate. The signal was related to LacZ control ChIRP to calculate the enrichment and background level was <1. (E) ITGA4 expression in HUVECs depleted of MEG3 (10 nM, 48 h) vs. LNA GapmerR control from n=6 independent experiments compared using a t test. (F) ChIP-qPCR enrichment for EZH2 and H3K27me3 over the ITGA4 promoter region using primer set ITGA4 -2 in HUVECs depleted of MEG3 (10 nM) vs. LNA GapmeR control (control). qPCR was performed in triplicates from n=4 independent experiments. The signal is expressed as percent IgG control. Both EZH2 and H3K27me3 signals are reduced in MEG3 KD samples. A representative graph is from n=3 independent qPCR experiments with data showing mean ± SEM.

Article Snippet: To precipitate EZH2, we used 20 μL of anti-EZH2 rabbit monoclonal antibody (D2C9 clone, CST, no. 5246S) and crosslinked cell lysates of HUVECs.

Techniques: RNA Sequencing, Knockdown, ChIP-sequencing, Gene Expression, Binding Assay, Control, Biomarker Discovery, Expressing, ChIP-qPCR

Journal: Molecular Therapy. Nucleic Acids

Article Title: Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression

doi: 10.1016/j.omtn.2024.102173

Figure Lengend Snippet: Inhibition of EZH2 de-represses ITGA4 and improves EC function (A) ChIP signal enrichment vs. 1% input for EZH2 and H3K27me3 mark over the ITGA4 promoter (regions 1 and 2) in HUVECs treated with A-395 (5 μM, 24 h) vs. control (DMSO). The expression was measured using two sets of primers against the same promoter region of ITGA4 . Representative graphs are average of 3 independent qPCR experiments and data are mean ± SEM. (B) ITGA4 expression in the presence of A-395 vs. DMSO control, n=6 independent experiments compared using a t test. (C) (i) Immunostaining for ITGA4 protein levels in ECs treated with A-395 (5 μM) vs. DMSO, or upon MEG3 depletion (MEG3 KD, using LNA GapmeRs, 10 nM 48 h). Scale bars, 400 μm (magnification ×200). (ii) Western blot staining for ITGA4 protein as in (i). Staining was related to β-tubulin control and quantification with densitometry measurement and a full blot in Figures S8 A and . (D) Intracellular localization of MEG3 (chromatin-associated lncRNA) between different cellular compartments in HUVECs treated with A-395 vs. DMSO. Using A-395 (5 μM, 24 h) chemical probe, the distribution of MEG3 has shifted from the nucleus (where it was highly chromatin bound) into the cytoplasm. Representative bars were compared by t test and one-way ANOVA. (E) MEG3-ChIRP followed by qPCR, n=3, analysis of MEG3 binding over the ITGA4 promoter region in HUVECs treated with A-395 (5 μM, 24 h) vs. DMSO. MEG3-ChIRP lysates from HUVECs treated with A-395 resulted in reduced engagement of MEG3 with the ITGA4 site compared with either DMSO control or ChIRP with non-biotinylated probes. The non-biotin probes served as a negative control, and we detected the background level <1. (F) Measure of cell migratory capacity using ECIS functional analysis in ECs depleted of ITGA4 (50 nM) and treated with control or A-395 (5 μM, 24 h). The data showing ECIS trace (left-hand side) are mean ± SD as calculated by the ECIS machine. The graph on the right is mean ± SEM with n=4 replicates, and each value obtained as mean of three technical replicates. p values were further obtained by Student’s t test comparisons of individual groups, as shown. (G) Adhesion assay was assessed using ECIS functional analysis. Fibronectin, FN (20 μg/mL) was used to coat the culture plates and assess adhesion of ECs following cell pre-treatment with A-395 (5 μM, 24 h). The cell index was determined as a measure of cell attachment to FN that leads to increase of resistance and adhesion. The difference in resistance change was calculated over 3 h. Experiments were performed in triplicate (technical replicates). The data showing ECIS trace (left-hand side) are mean ± SD as calculated by the ECIS machine, and the graph on the right is mean ± SEM with n=6 replicates.

Article Snippet: To precipitate EZH2, we used 20 μL of anti-EZH2 rabbit monoclonal antibody (D2C9 clone, CST, no. 5246S) and crosslinked cell lysates of HUVECs.

Techniques: Inhibition, Control, Expressing, Immunostaining, Western Blot, Staining, Binding Assay, Negative Control, Functional Assay, Cell Adhesion Assay, Cell Attachment Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression

doi: 10.1016/j.omtn.2024.102173

Figure Lengend Snippet: Hindlimb ischemia was performed in mice (n=13) that were injected with vehicle (water) or A-395 (i.p. 10 mg/kg twice/week) to inhibit EZH2 enzymatic activity for 3 weeks. Muscle tissue was collected at day 21 and processed for histology. (A) Staining was done for H3K27me3 and isolectin B4 (Iso-B4), displaying nuclear positivity with strong intensity in vehicle control. A-395 treatment decreased total H3K27me3 staining, as compared by t test (p < 0.0001), while seemingly increasing isolectin B4. Scale bars, 400 μm (magnification ×200). (B) Staining for capillaries (Iso-B4) and arterioles (α-SMA) was also performed to assess arteriogenesis in mice. The data show increased area of staining for Iso-B4 dye and α-SMA in A-395 vs . vehicle control treated mice with limb ischemia, p < 0.05. Scale bars, 400 μm (magnification ×200). (C) The ratios of ischemic to contralateral foot blood flow represented at 0, 3, 7, 14 and 21 days post hindlimb ischemia in mice (n=13) remained unchanged between A-395 treatment and control; data is mean ± SEM, compared using 2-way ANOVA. (D) i In the muscle sections A-395 has increased the total percentage (%) of vessels positive for ITGA4 (red, left) within/in the vicinity of the Iso-B4-positive cells (green, right), when compared with the vehicle control Scale bars, 100 μm (magnification ×400); ii Percentage (%) arterioles stained with α-SMA and positive for ITGA4, has increased in sections of A-395 treated mice comapred with control. Representative points were compared using t -test, p < 0.01.

Article Snippet: To precipitate EZH2, we used 20 μL of anti-EZH2 rabbit monoclonal antibody (D2C9 clone, CST, no. 5246S) and crosslinked cell lysates of HUVECs.

Techniques: Injection, Activity Assay, Staining, Control