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: After PARylation, the human EZH2/EED/SUZ12/RbAp48/AEBP2 Complex (BPS Bioscience) was incubated in 30 mL HMT buffer containing 50 μM Tris, pH 8, 10 μM MgCl 2 , 10 μM DTT, and 3 μg purified Histone H3 (Active Motif, C110A) with or without 40 μM [S-(5’-Adenosyl)-L-methionine] (SAM) (BPS Bioscience).

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: After PARylation, the human EZH2/EED/SUZ12/RbAp48/AEBP2 Complex (BPS Bioscience) was incubated in 30 mL HMT buffer containing 50 μM Tris, pH 8, 10 μM MgCl 2 , 10 μM DTT, and 3 μg purified Histone H3 (Active Motif, C110A) with or without 40 μM [S-(5’-Adenosyl)-L-methionine] (SAM) (BPS Bioscience).

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: After PARylation, the human EZH2/EED/SUZ12/RbAp48/AEBP2 Complex (BPS Bioscience) was incubated in 30 mL HMT buffer containing 50 μM Tris, pH 8, 10 μM MgCl 2 , 10 μM DTT, and 3 μg purified Histone H3 (Active Motif, C110A) with or without 40 μM [S-(5’-Adenosyl)-L-methionine] (SAM) (BPS Bioscience).

Techniques: In Vitro, PARG Assay, Incubation, Western Blot, Activity Assay, HMT 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) 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: After PARylation, the human EZH2/EED/SUZ12/RbAp48/AEBP2 Complex (BPS Bioscience) was incubated in 30 mL HMT buffer containing 50 μM Tris, pH 8, 10 μM MgCl 2 , 10 μM DTT, and 3 μg purified Histone H3 (Active Motif, C110A) with or without 40 μM [S-(5’-Adenosyl)-L-methionine] (SAM) (BPS Bioscience).

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