pgex 4p 1 expression  (Addgene inc)

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 1 Epigenetic regulation of skeletal development of fetuses from HFD (high fat diet) rat dams. A, Representative images from Alizarin red/Alcian blue staining of E18.5 embryos from dams fed either control diet or HFD. Cartilage and calcification stained dark blue; arrows indicate differences of skeletal ossification in the head. B, Western blots for H3K27me3, Ezh2, p-Ezh2 and CDK1 in proteins from EOCCs (embryonic rat osteogenic calvarial cells) either from six HFD obese dams or six control diet dams. M, male; F, female. C, Real-time PCR for Ezh2 mRNA expression in total RNA from EOCCs either from six HFD obese dams or six control diet dams. *P < .05 by t-test. D, and E, representing top genes and significantly H3K27me3 enriched or decreased gene body and gene promoter using Heat Map analysis of ChIP-seq data from EOCCs either from six HFD obese dams or six control diet dams (pooled)

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Staining, Control, Western Blot, Real-time Polymerase Chain Reaction, Expressing, ChIP-sequencing

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 2 Identification of epigenetic target genes affected by maternal obesity during fetal skeletal development. A, SATB2 was significantly enriched for H3K27me3 within the gene body, especially in the promoter region based on the detection of a peak (black and red arrow head) of enrichment by Illumina DNA sequencing (male and female are mixed, and samples were pooled to three per group). B, Real-time PCR for SATB2 and ALP mRNA expression in total RNA from EOCCs either from six HFD obese dams or six control diet dams. *P < .05 by t-test. C, ChIP of rat SATB2 enhancer elements by specific anti H3K27me3 and Ezh2 antibodies. D, ChIP of enrichment of SATB2 after IP with H3K27me3 and Ezh2 antibodies. Fold enrichment relative to IgG. *P < .05 by t-test EOCCs from control vs EOCCs from HFD obese dams. E, Control EOCCs were treated with vehicle (control), NEFA mixture 400 µM with Palmitic and Oleic acid 2:1 mixture, Sigma-Aldrich), Palmitic acid (270 µM) or Oleic acid (130 µM) for 48 hours: Western blots for H3K27me3, Ezh2, p-Ezh2, SATB2 and Col 1 (collagen 1). n = 3/treatment

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: DNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 4 Increased Ezh2/H3K27me3 but decreased SATB2 expression in human UC MSCs from obese mothers. A, UC MSCs from either lean or obese mothers were cultured, passage 2 cells were immune-stained with anti-Ezh2 antibody (red, white arrows) or anti-SATB2 antibody (green, yellow arrows). B, and C, Real-time PCR (Box & Whiskers graphs) and Western blots (under Box & Whiskers graphs) of Ezh2 and SATB2 mRNA and protein expression in UC MSCs either from lean or obese mothers. *P < .05 vs lean, t-test. D, ChIP of human Ezh2, SATB2 and GAPDH enhancer elements by specific anti H3K27me3 antibody, and E, ChIP of enrichment of human Ezh2, SATB2 and GAPDH after IP with H3K27me3 antibody, fold enrichment relative to IgG. *P < .01 by t-test vs lean

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Expressing, Cell Culture, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 6 Increased trabecular bone mineral density in Ezh2 osteoblastic cell specific deletion male mice. A, Sagittal views of total, trabecular and cortical bone mineral density, and Strength Strain Index (SSI) from a represented mouse from cko and their respective control mice. B, Total bone mineral content in all female mice. B, Tibia pQCT parameters, TOT-BMC (total bone mineral content), P = .0499 by one-way ANOVA; TOT-BMD (total bone mineral density), P = .0365 by one-way ANOVA; TRAB-BMD (trabecular bone mineral density), P = .0188 by one-way ANOVA; CRT-BMD (cortical bone mineral density), P = .0620 by one-way ANOVA in male cko mice compared to their respective genotypic control mice, followed by Tukey's post hoc test comparing cko with its respective control genotype group, *means P < .05 significantly different, n = 4

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Control

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) Immunoprecipitation of EZH2 with PARP1 under physiological conditions and after induction of DNA damage. His-PARP1 was expressed in HeLa cells by transfection. Cells were treated with 100 uM of the alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) for 10 minutes to induce DNA damage and activate PARP1. Input corresponds to 1/20 th of protein extracts from transfected cells used for the tag-construct pulldown. (B) Proteins interacting with EZH2 were analyzed by His pulldown or immunoprecipitated with non-immunogenic IgG (control) followed by western blot analysis with anti-EZH2 (top), anti-PAR (middle) and anti-PARP1 (bottom) antibodies. (C) Immunoprecipitation of EZH2 with PAR-affinity resin after induction of DNA damage. LCLs and HeLa cells were treated with or without 100 uM MNNG for 10 minutes. Cellular protein extracts were immunoprecipitated with a PAR affinity resin or PAR negative control resin and analyzed by western blot with anti-EZH2 and anti-PARP1 antibodies. Input corresponds to 1/10 th the amount of cell extracts used for immunoprecipitation.

Article Snippet: The plasmid encoding GST-fused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [ ].

Techniques: Immunoprecipitation, Transfection, Construct, Control, Western Blot, Negative Control

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) PARylation of EZH2 by PARP1 in vitro . Human EZH2/PRC2 complex (EZH2, EED, SUZ12, RbAP48 and AEBP2) was incubated alone (lane 1) or with the agents indicated at the top (250 nM of olaparib (PARP inhibitor) was used and NAD+ is necessary for PARP1 activity). After 1 hour, PARylation was blocked by adding olaparib to all samples and PARylated proteins were pulled-down by PAR-affinity resin and analyzed by western blot with anti-EZH2 and anti-PAR antibodies. PARylation appears as a smear due to the different sizes of the various PAR polymers. Input corresponds to 1/10 th the amount of protein used for immunoprecipitation. Input was immunoblotted with anti-PARP1 and anti-EZH2 antibodies. (B) Schematic of the experimental strategy for C) and D). Briefly, the EZH2/PRC2 complex was incubated with PARP1 in the presence or absence of NAD+ as in A). After 1 hour, PARylation was stopped with olaparib and PARP1 was removed by immunoprecipitation with an anti-PARP1 antibody. The EZH2/PRC2 complex was incubated with EZH2 substrates histone H3 and S-adenosyl methionine (SAM) to allow histone methylation to occur. After 30 minutes, histone methytransferase activity was determined by assessing H3K27me3 levels. (C) In vitro histone methyltransferase assay. As indicated in B), purified histone H3 and SAM were incubated with the agents indicated at the top. After 30 minutes, proteins were analyzed by western blot using anti-Histone H3, anti-H3K27me3 and anti-PAR antibodies. Input corresponds to 1/20 th the amount of the protein used for immunoblotting. Input was probed with an anti-EZH2 antibody. (D) Levels of EZH2 activity with (black) and without (grey) PARP1 activity. Extracts from the EZH2/PRC2 complex incubated with histone H3 and SAM as in lanes 2 and 4 from C) were assessed for H3K27me3 levels by ELISA. N=3 ± SD.

Article Snippet: The plasmid encoding GST-fused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [ ].

Techniques: In Vitro, Incubation, Activity Assay, Western Blot, Immunoprecipitation, Methylation, HMT Assay, Purification, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) Time course of in vitro PARylation assay. EZH2/PRC2 complex was incubated with PARP1, NAD+ and DNA fragments to allow in vitro PARylation. The reaction was blocked at different time points by adding the PARP inhibitor olaparib. After removing PARP1 from the reaction, PARylated proteins were pulled down with a PAR-affinity resin and analyzed by western blot with an anti-EZH2 antibody. Input corresponds to 1/10 th the amount of protein used for PAR pulldown. Input was probed with an anti-EZH2 antibody. (B) In vitro histone methylation assay. EZH2/PRC2 complex treated as in A) was incubated with histone H3 and SAM to allow methylation of lysine 27 of histone H3. After 30 minutes, histone H3 was extracted and H3K27me3 levels were measured by ELISA. EZH2 activity was calculated by setting H3K27me3 levels at time 0 as 100% EZH2 activity. N=3 mean ± SD. (C) In vitro histone methyltransferase activity assay. EZH2/PRC2 complex was incubated with PARP1 in the presence (PARylated) or absence (unmodified) of NAD+. After 1 hour, the reaction was blocked as in A) and EZH2/PRC2 complex was incubated with SAM and different concentrations of histone H3 to allow histone H3-K27 methylation to occur. After 30 minutes, the reaction was blocked and the amount of methylated histone H3-K27 generated by EZH2 activity was measured using an H3K27me3 ELISA kit. N=3, mean ± SD. (D) Time course of in vitro histone methyltransferase (HMT) activity. EZH2/PRC2 complex was treated as in C) and incubated with SAM and histone H3 to allow methylation of H3-K27. The reaction was blocked at different time points and the amount of methylated histone H3-K27 generated by EZH2 activity was measured by an H3K27me3 ELISA. N=3, mean ± SD.

Article Snippet: The plasmid encoding GST-fused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [ ].

Techniques: In Vitro, Incubation, Western Blot, Methylation, Enzyme-linked Immunosorbent Assay, Activity Assay, Generated

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) In vitro PARG assay. EZH2/PRC2 complex and PARP1 were incubated with or without PARG as indicated (Note: NAD+ is required for PARylation). After 1 hour, the reaction was blocked by addition of the PARP inhibitor olaparib and the EZH2/PRC2 complex was incubated with (lanes 2 and 4) or without (lanes 1 and 3) PARG to allow degradation of PAR polymers. After 1 hour, the reaction was stopped by adding Laemmli buffer and the proteins were analyzed by western blot using anti-EZH2 and anti-PAR antibodies. The upper band in the top panel represents PARylated EZH2. PARG activity was confirmed by reduction of PAR smear. (B) In vitro histone methyltransferase (HMT) activity assay. EZH2/PRC2 complex treated as in A) was subsequently assayed for histone methyltransferase activity using an HMT assay kit. The activity of EZH2 under the indicated conditions was calculated based on the amount of H3-K27 converted in the assay. As a control, the activity of EZH2/PRC2 complex alone was also determined. N=3, mean ± SD.

Article Snippet: The plasmid encoding GST-fused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [ ].

Techniques: In Vitro, PARG Assay, Incubation, Western Blot, Activity Assay, HMT Assay, Control

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) Schematic of experimental approach that couples histone PARylation and methylation in vitro . First, histone H3 was incubated with PARP1 in the presence or absence of NAD+ and olaparib to allow for PARylation. After 60 minutes, the reaction was blocked by addition of olaparib, PARP1 was removed by immunoprecipitation and the remaining histone H3 was either assessed for PARylation by PAR-resin pulldown or incubated with EZH2/PRC2 and SAM to allow H3-K27 methylation in vitro . After 30 minutes, the histone methyltransferase reaction was blocked and H3K27me3 levels were determined by different approaches. (B) Histone H3 PARylation decreases subsequent H3-K27 methylation. Histone H3 proteins treated as in A) were analyzed by western blot using an anti-H3K27me3 antibody and an anti-histone H3 antibody as a control. The signal intensity of H3K27me3 relative to H3 was measured using ImageJ software and normalized to the signal from unmodified histone H3 (H3 incubated with PARP1 in the absence of NAD+, lane 1). PARP1 activity was confirmed by western blot using an anti-PAR antibody. The western blot is representative of three independent experiments. (C) PARylation reduces histone methylation in vitro . Histone H3 samples treated as in A) were used to determine EZH2 activity toward unmodified and PARylated histone H3 by measuring H3K27me3 levels using an ELISA kit. H3K27me3 levels from unmodified histone H3 were set as 100% EZH2 activity. N=3, mean ± SD. (D) In vitro PARylation of histone H3. Histone H3 proteins treated as in A) were immunoprecipitated using the PAR-affinity resin and PARylation of H3 was confirmed by western blot using an anti-H3 antibody. PAR-resin specificity and PARylation levels were determined by western blot analysis of purified proteins with an anti-PAR antibody. The smear observed in lane 2 indicated PARylation. H. (E) PARP1 Immunoprecipitation. PARP1 removal after PARylation of histone H3 treated as above was confirmed by western blot analysis of proteins immunoprecipitated with an anti-PARP1 antibody.

Article Snippet: The plasmid encoding GST-fused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [ ].

Techniques: Methylation, In Vitro, Incubation, Immunoprecipitation, Western Blot, Control, Software, Activity Assay, Enzyme-linked Immunosorbent Assay, Purification

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) Schematic of histone peptide pull-down after PARylation. (B) Histone peptide pull-down for EZH2. Synthesized histone H3 peptide, corresponding to residues 21-44 of human histone H3, was conjugated with biotin and incubated with PARP1 in the presence or absence of NAD+ to allow for PARylation. After 1 hour, the reaction was blocked with 250 nM olaparib and the H3 peptide was immunopurified with streptavidin-magnetic beads and subsequently incubated with EZH2/PRC2 complex. After 4 hours, the peptide-coated, streptavidin-conjugated beads were washed to remove unbound proteins and bound proteins were analyzed by western blot using an anti-EZH2 antibody. PARylation of the peptide was confirmed by western blot using an anti-PAR antibody. Top panel shows short film exposure; lower panel longer film exposure. (C) Schematic of histone peptide pull-down after in vitro histone methyltransferase assay. (D) Histone peptide pull-down assay for PARP1. Synthesized histone H3 peptide containing tri-methylated lysine 27 was conjugated with streptavidin magnetic beads followed by incubation with purified PARP1. After 4 hours, the peptide-coated, streptavidin-conjugated beads were washed to remove unbound proteins and bound proteins were analyzed by western blot using an anti-PARP1 antibody.

Article Snippet: The plasmid encoding GST-fused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [ ].

Techniques: Synthesized, Incubation, Magnetic Beads, Western Blot, In Vitro, HMT Assay, Pull Down Assay, Methylation, Purification

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A and B) EZH2 association with chromatin after DNA damage. HEK293 cells and HeLa cells were treated with or without 100 uM MNNG to induce DNA damage. After 10 minutes, proteins were extracted and fractionated to obtain nuclear soluble and chromatin-bound protein extracts. The fractionated proteins were analyzed for EZH2 expression by western blot using an anti-EZH2 antibody. The effectiveness of separation of nuclear soluble and chromatin-bound proteins was determined by western blot with an anti-histone H3 antibody. All western blots are representative of at least three independent experiments. (C) Global levels of nuclear soluble proteins and chromatin-bound proteins are unaffected by PARP1 activation. Nuclear soluble and chromatin-bound protein extracts from HEK293 cells and HeLa cells treated as in B) were separated by gel electrophoresis on a 4-20% polyacrylamide gel and stained with coomassie brilliant blue to confirm both correct fraction separation and equal protein quantity. The image is representative of at least three independent experiments.

Article Snippet: The plasmid encoding GST-fused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [ ].

Techniques: Expressing, Western Blot, Activation Assay, Nucleic Acid Electrophoresis, Staining

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) PARylation of EZH2 after DNA damage by UV radiation. HeLa cells were irradiated with UVA and UVB for 2 minutes and then recovered in media. PARylated proteins were pulled down at the indicated recovery times using PAR-resin and analyzed by western blot using anti-PARP1, anti-EZH2 and anti-PAR antibodies. The shift of EZH2 to a higher molecular weight isoform indicates its PARylation. EZH2 PARylation increases and reaches a maximum at 3h after the initial treatment with UV. Input corresponds to 1/20th the amount of protein extract. (B and C) HeLa cells were treated as above and proteins were pulled down using either an anti mono-ADP-ribose antibody or an anti mono/poly-ADP-ribose antibody. Immunoprecipitated proteins were then analyzed by western blot with anti-EZH2 and anti-PARP1 antibodies. The western blot is representative of at least three independent experiments. Untreated cells served as control. (D) Histones were extracted from HeLa cells treated as described above. H3K27me3 levels were analyzed by western blot with an anti-H3K27me3 antibody or anti-histone H3 as control. (E) Quantification of H3K27me3 levels after UV irradiation-induced DNA damage. HeLa cells were exposed to UV as described in A) and recovered in media. After 3 hours, histones were purified and assessed for H3K27me3 levels by ELISA. The level of K27 methylation under the indicated conditions were calculated based on the amount of H3-K27 converted in the assay, divided by the amount of total histones loaded. Data were normalized to the untreated and expressed as % of H3K27me3. Data were N=3, mean ± SD. Statistically significant differences between experimental conditions and control samples were determined by Student's t test.

Article Snippet: The plasmid encoding GST-fused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [ ].

Techniques: Irradiation, Western Blot, Molecular Weight, Immunoprecipitation, Control, Purification, Enzyme-linked Immunosorbent Assay, Methylation

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: (A) BRCA1 -mutated and BRCA1 -reconstituted MDA-MB-436 human breast carcinoma cells were treated with or without the PARP1 inhibitor olaparib in the presence or absence of the EZH2 inhibitor UNC1999 at the indicated concentrations. After 4 days, cell count/viability was determined by Trypan blue exclusion using a Bio Rad TC20 Automated Cell Counter. Results show mean ± SD of living cells. N=3. * p<0.05 compared to individual treatment using Student t test. (B) Lin-CD34 + AML primary cells from two patients and from healthy bone marrow donor (NBMCs) were treated with 2μM UNC1999, 5 μM olaparib, or were left untreated. After 4 days, live cell number was determined by Trypan blue exclusion using a Bio Rad TC20 Automated Cell Counter. Results show mean × SD number of live cells. N=3. * p<0.05 compared to individual treatment using Student t test. (C) Lin-CD34 + AML primary cells (patient #1) were treated with olaparib and UNC1999 as indicated. After 48 hours, DNA damage was evaluated by measuring the percentage of γ-H2AX-positive Lin-CD34 + AML#1 cells by flow cytometry. Data are mean × SD. N=3. * p<0.001 in comparison to Control; ** p<0.05 in comparison to UNC1999 or olaparib treatment. (D) Lin-CD34 + AML #2 cells and NBMCs were isolated as in B) and treated with UNC1999, 0.5 μg/μl DNR, 1 μM olaparib, the indicated drug combinations, or were left untreated. After 4 days living cells were determined by Trypan blue exclusion assay. Data are mean × SD. N=3; * p<0.01 compared to individual/dual treatment using Student t test. (E) Proteins were extracted from Lin-CD34 + AML cells and NBMCs after 2 days of treatment with the indicated drug combination and analyzed by western blot with antibodies specific for H3K27me3, histone H3, RAD51 and PARP1 proteins. Blots are representative of three independent experiments.

Article Snippet: The plasmid encoding GST-fused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [ ].

Techniques: Cell Counting, Flow Cytometry, Comparison, Control, Isolation, Trypan Blue Exclusion Assay, Western Blot

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: Figure 1: EZH2 interacts with PARP1 and is PARylated after DNA damage induction. (A) Immunoprecipitation of EZH2 with PARP1 under physiological conditions and after induction of DNA damage. His-PARP1 was expressed in HeLa cells by transfection. Cells were treated with 100 uM of the alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) for 10 minutes to induce DNA damage and activate PARP1. Input corresponds to 1/20th of protein extracts from transfected cells used for the tag-construct pulldown. (B) Proteins interacting with EZH2 were analyzed by His pulldown or immunoprecipitated with non-immunogenic IgG (control) followed by western blot analysis with anti-EZH2 (top), anti-PAR (middle) and anti-PARP1 (bottom) antibodies. (C) Immunoprecipitation of EZH2 with PAR-affinity resin after induction of DNA damage. LCLs and HeLa cells were treated with or without 100 uM MNNG for 10 minutes. Cellular protein extracts were immunoprecipitated with a PAR affinity resin or PAR negative control resin and analyzed by western blot with anti-EZH2 and anti-PARP1 antibodies. Input corresponds to 1/10th the amount of cell extracts used for immunoprecipitation.

Article Snippet: The plasmid encoding GSTfused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [55].

Techniques: Immunoprecipitation, Transfection, Construct, Control, Western Blot, Negative Control

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: Figure 2: PARP1 PARylates EZH2 and inhibits EZH2 activity in vitro. (A) PARylation of EZH2 by PARP1 in vitro. Human EZH2/PRC2 complex (EZH2, EED, SUZ12, RbAP48 and AEBP2) was incubated alone (lane 1) or with the agents indicated at the top (250 nM of olaparib (PARP inhibitor) was used and NAD+ is necessary for PARP1 activity). After 1 hour, PARylation was blocked by adding olaparib to all samples and PARylated proteins were pulled-down by PAR-affinity resin and analyzed by western blot with anti-EZH2 and anti-PAR antibodies. PARylation appears as a smear due to the different sizes of the various PAR polymers. Input corresponds to 1/10th the amount of protein used for immunoprecipitation. Input was immunoblotted with anti-PARP1 and anti-EZH2 antibodies. (B) Schematic of the experimental strategy for C) and D). Briefly, the EZH2/PRC2 complex was incubated with PARP1 in the presence or absence of NAD+ as in A). After 1 hour, PARylation was stopped with olaparib and PARP1 was removed by immunoprecipitation with an anti-PARP1 antibody. The EZH2/PRC2 complex was incubated with EZH2 substrates histone H3 and S-adenosyl methionine (SAM) to allow histone methylation to occur. After 30 minutes, histone methytransferase activity was determined by assessing H3K27me3 levels. (C) In vitro histone methyltransferase assay. As indicated in B), purified histone H3 and SAM were incubated with the agents indicated at the top. After 30 minutes, proteins were analyzed by western blot using anti-Histone H3, anti-H3K27me3 and anti-PAR antibodies. Input corresponds to 1/20th the amount of the protein used for immunoblotting. Input was probed with an anti-EZH2 antibody. (D) Levels of EZH2 activity with (black) and without (grey) PARP1 activity. Extracts from the EZH2/PRC2 complex incubated with histone H3 and SAM as in lanes 2 and 4 from C) were assessed for H3K27me3 levels by ELISA. N=3 ± SD.

Article Snippet: The plasmid encoding GSTfused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [55].

Techniques: Activity Assay, In Vitro, Incubation, Western Blot, Immunoprecipitation, Methylation, HMT Assay, Purification, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: Figure 3: PARylation of EZH2 stably inhibits EZH2 enzymatic activity. (A) Time course of in vitro PARylation assay. EZH2/ PRC2 complex was incubated with PARP1, NAD+ and DNA fragments to allow in vitro PARylation. The reaction was blocked at different time points by adding the PARP inhibitor olaparib. After removing PARP1 from the reaction, PARylated proteins were pulled down with a PAR-affinity resin and analyzed by western blot with an anti-EZH2 antibody. Input corresponds to 1/10th the amount of protein used for PAR pulldown. Input was probed with an anti-EZH2 antibody. (B) In vitro histone methylation assay. EZH2/PRC2 complex treated as in A) was incubated with histone H3 and SAM to allow methylation of lysine 27 of histone H3. After 30 minutes, histone H3 was extracted and H3K27me3 levels were measured by ELISA. EZH2 activity was calculated by setting H3K27me3 levels at time 0 as 100% EZH2 activity. N=3 mean ± SD. (C) In vitro histone methyltransferase activity assay. EZH2/PRC2 complex was incubated with PARP1 in the presence (PARylated) or absence (unmodified) of NAD+. After 1 hour, the reaction was blocked as in A) and EZH2/PRC2 complex was incubated with SAM and different concentrations of histone H3 to allow histone H3-K27 methylation to occur. After 30 minutes, the reaction was blocked and the amount of methylated histone H3-K27 generated by EZH2 activity was measured using an H3K27me3 ELISA kit. N=3, mean ± SD. (D) Time course of in vitro histone methyltransferase (HMT) activity. EZH2/PRC2 complex was treated as in C) and incubated with SAM and histone H3 to allow methylation of H3-K27. The reaction was blocked at different time points and the amount of methylated histone H3-K27 generated by EZH2 activity was measured by an H3K27me3 ELISA. N=3, mean ± SD.

Article Snippet: The plasmid encoding GSTfused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [55].

Techniques: Stable Transfection, Activity Assay, In Vitro, Incubation, Western Blot, Methylation, Enzyme-linked Immunosorbent Assay, Generated

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: Figure 4: PARG reverses EZH2 PARylation and restores EZH2 enzymatic activity. (A) In vitro PARG assay. EZH2/PRC2 complex and PARP1 were incubated with or without PARG as indicated (Note: NAD+ is required for PARylation). After 1 hour, the reaction was blocked by addition of the PARP inhibitor olaparib and the EZH2/PRC2 complex was incubated with (lanes 2 and 4) or without (lanes 1 and 3) PARG to allow degradation of PAR polymers. After 1 hour, the reaction was stopped by adding Laemmli buffer and the proteins were analyzed by western blot using anti-EZH2 and anti-PAR antibodies. The upper band in the top panel represents PARylated EZH2. PARG activity was confirmed by reduction of PAR smear. (B) In vitro histone methyltransferase (HMT) activity assay. EZH2/PRC2 complex treated as in A) was subsequently assayed for histone methyltransferase activity using an HMT assay kit. The activity of EZH2 under the indicated conditions was calculated based on the amount of H3-K27 converted in the assay. As a control, the activity of EZH2/ PRC2 complex alone was also determined. N=3, mean ± SD.

Article Snippet: The plasmid encoding GSTfused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [55].

Techniques: Activity Assay, In Vitro, PARG Assay, Incubation, Western Blot, HMT Assay, Control

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: Figure 5: PARylation of Histone H3 decreases EZH2-mediated histone methylation. (A) Schematic of experimental approach that couples histone PARylation and methylation in vitro. First, histone H3 was incubated with PARP1 in the presence or absence of NAD+ and olaparib to allow for PARylation. After 60 minutes, the reaction was blocked by addition of olaparib, PARP1 was removed by immunoprecipitation and the remaining histone H3 was either assessed for PARylation by PAR-resin pulldown or incubated with EZH2/PRC2 and SAM to allow H3-K27 methylation in vitro. After 30 minutes, the histone methyltransferase reaction was blocked and H3K27me3 levels were determined by different approaches. (B) Histone H3 PARylation decreases subsequent H3-K27 methylation. Histone H3 proteins treated as in A) were analyzed by western blot using an anti-H3K27me3 antibody and an anti-histone H3 antibody as a control. The signal intensity of H3K27me3 relative to H3 was measured using ImageJ software and normalized to the signal from unmodified histone H3 (H3 incubated with PARP1 in the absence of NAD+, lane 1). PARP1 activity was confirmed by western blot using an anti-PAR antibody. The western blot is representative of three independent experiments. (C) PARylation reduces histone methylation in vitro. Histone H3 samples treated as in A) were used to determine EZH2 activity toward unmodified and PARylated histone H3 by measuring H3K27me3 levels using an ELISA kit. H3K27me3 levels from unmodified histone H3 were set as 100% EZH2 activity. N=3, mean ± SD. (D) In vitro PARylation of histone H3. Histone H3 proteins treated as in A) were immunoprecipitated using the PAR-affinity resin and PARylation of H3 was confirmed by western blot using an anti-H3 antibody. PAR-resin specificity and PARylation levels were determined by western blot analysis of purified proteins with an anti-PAR antibody. The smear observed in lane 2 indicated PARylation. H. (E) PARP1 Immunoprecipitation. PARP1 removal after PARylation of histone H3 treated as above was confirmed by western blot analysis of proteins immunoprecipitated with an anti-PARP1 antibody.

Article Snippet: The plasmid encoding GSTfused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [55].

Techniques: Methylation, In Vitro, Incubation, Immunoprecipitation, Western Blot, Control, Software, Activity Assay, Enzyme-linked Immunosorbent Assay, Purification

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: Figure 6: PARylation of histone H3 decreases EZH2 affinity for H3, while methylation of histone H3 has no effect on the ability of PARP1 to interact with histone H3. (A) Schematic of histone peptide pull-down after PARylation. (B) Histone peptide pull-down for EZH2. Synthesized histone H3 peptide, corresponding to residues 21-44 of human histone H3, was conjugated with biotin and incubated with PARP1 in the presence or absence of NAD+ to allow for PARylation. After 1 hour, the reaction was blocked with 250 nM olaparib and the H3 peptide was immunopurified with streptavidin-magnetic beads and subsequently incubated with EZH2/PRC2 complex. After 4 hours, the peptide-coated, streptavidin-conjugated beads were washed to remove unbound proteins and bound proteins were analyzed by western blot using an anti-EZH2 antibody. PARylation of the peptide was confirmed by western blot using an anti-PAR antibody. Top panel shows short film exposure; lower panel longer film exposure. (C) Schematic of histone peptide pull-down after in vitro histone methyltransferase assay. (D) Histone peptide pull-down assay for PARP1. Synthesized histone H3 peptide containing tri-methylated lysine 27 was conjugated with streptavidin magnetic beads followed by incubation with purified PARP1. After 4 hours, the peptide-coated, streptavidin-conjugated beads were washed to remove unbound proteins and bound proteins were analyzed by western blot using an anti- PARP1 antibody.

Article Snippet: The plasmid encoding GSTfused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [55].

Techniques: Methylation, Synthesized, Incubation, Magnetic Beads, Western Blot, In Vitro, HMT Assay, Pull Down Assay, Purification

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: Figure 7: EZH2 interaction with chromatin decreases after DNA damage. (A and B) EZH2 association with chromatin after DNA damage. HEK293 cells and HeLa cells were treated with or without 100 uM MNNG to induce DNA damage. After 10 minutes, proteins were extracted and fractionated to obtain nuclear soluble and chromatin-bound protein extracts. The fractionated proteins were analyzed for EZH2 expression by western blot using an anti-EZH2 antibody. The effectiveness of separation of nuclear soluble and chromatin-bound proteins was determined by western blot with an anti-histone H3 antibody. All western blots are representative of at least three independent experiments. (C) Global levels of nuclear soluble proteins and chromatin-bound proteins are unaffected by PARP1 activation. Nuclear soluble and chromatin-bound protein extracts from HEK293 cells and HeLa cells treated as in B) were separated by gel electrophoresis on a 4-20% polyacrylamide gel and stained with coomassie brilliant blue to confirm both correct fraction separation and equal protein quantity. The image is representative of at least three independent experiments.

Article Snippet: The plasmid encoding GSTfused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [55].

Techniques: Expressing, Western Blot, Activation Assay, Nucleic Acid Electrophoresis, Staining

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: Figure 8: EZH2 PARylation decreases global H3K27me3 levels after DNA damage. (A) PARylation of EZH2 after DNA damage by UV radiation. HeLa cells were irradiated with UVA and UVB for 2 minutes and then recovered in media. PARylated proteins were pulled down at the indicated recovery times using PAR-resin and analyzed by western blot using anti-PARP1, anti-EZH2 and anti- PAR antibodies. The shift of EZH2 to a higher molecular weight isoform indicates its PARylation. EZH2 PARylation increases and reaches a maximum at 3h after the initial treatment with UV. Input corresponds to 1/20th the amount of protein extract. (B and C) HeLa cells were treated as above and proteins were pulled down using either an anti mono-ADP-ribose antibody or an anti mono/poly-ADP-ribose antibody. Immunoprecipitated proteins were then analyzed by western blot with anti-EZH2 and anti-PARP1 antibodies. The western blot is representative of at least three independent experiments. Untreated cells served as control. (D) Histones were extracted from HeLa cells treated as described above. H3K27me3 levels were analyzed by western blot with an anti-H3K27me3 antibody or anti-histone H3 as control. (E) Quantification of H3K27me3 levels after UV irradiation-induced DNA damage. HeLa cells were exposed to UV as described in A) and recovered in media. After 3 hours, histones were purified and assessed for H3K27me3 levels by ELISA. The level of K27 methylation under the indicated conditions were calculated based on the amount of H3-K27 converted in the assay, divided by the amount of total histones loaded. Data were normalized to the untreated and expressed as % of H3K27me3. Data were N=3, mean ± SD. Statistically significant differences between experimental conditions and control samples were determined by Student’s t test.

Article Snippet: The plasmid encoding GSTfused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [55].

Techniques: Irradiation, Western Blot, Molecular Weight, Immunoprecipitation, Control, Purification, Enzyme-linked Immunosorbent Assay, Methylation

Journal: Oncotarget

Article Title: Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

doi: 10.18632/oncotarget.24291

Figure Lengend Snippet: Figure 9: EZH2 inhibitor UNC1999 enhanced PARP1 inhibitor olaparib-mediated synthetic lethality in BRCA- deficient cell lines and acute myeloid leukemia (AML) primary cells. (A) BRCA1-mutated and BRCA1-reconstituted MDA- MB-436 human breast carcinoma cells were treated with or without the PARP1 inhibitor olaparib in the presence or absence of the EZH2 inhibitor UNC1999 at the indicated concentrations. After 4 days, cell count/viability was determined by Trypan blue exclusion using a Bio Rad TC20 Automated Cell Counter. Results show mean ± SD of living cells. N=3. *p<0.05 compared to individual treatment using Student t test. (B) Lin-CD34+ AML primary cells from two patients and from healthy bone marrow donor (NBMCs) were treated with 2μM UNC1999, 5 μM olaparib, or were left untreated. After 4 days, live cell number was determined by Trypan blue exclusion using a Bio Rad TC20 Automated Cell Counter. Results show mean × SD number of live cells. N=3. *p<0.05 compared to individual treatment using Student t test. (C) Lin-CD34+ AML primary cells (patient #1) were treated with olaparib and UNC1999 as indicated. After 48 hours, DNA damage was evaluated by measuring the percentage of γ-H2AX-positive Lin-CD34+ AML#1 cells by flow cytometry. Data are mean × SD. N=3. *p<0.001 in comparison to Control; **p<0.05 in comparison to UNC1999 or olaparib treatment. (D) Lin-CD34+ AML #2 cells and NBMCs were isolated as in B) and treated with UNC1999, 0.5 μg/µl DNR, 1 μM olaparib, the indicated drug combinations, or were left untreated. After 4 days living cells were determined by Trypan blue exclusion assay. Data are mean × SD. N=3; *p<0.01 compared to individual/ dual treatment using Student t test. (E) Proteins were extracted from Lin-CD34+ AML cells and NBMCs after 2 days of treatment with the indicated drug combination and analyzed by western blot with antibodies specific for H3K27me3, histone H3, RAD51 and PARP1 proteins. Blots are representative of three independent experiments.

Article Snippet: The plasmid encoding GSTfused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [55].

Techniques: Cell Counting, Flow Cytometry, Comparison, Control, Isolation, Trypan Blue Exclusion Assay, Western Blot