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 1/10 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.