Fig. 3: PARG inhibition facilitates BRD4–MZ1–CRL2^VHL ternary complex formation and K29/K48-branched ubiquitylation.

a PDD promotes ubiquitylation of BRD4 and BRD2. HeLa cells were treated with the indicated concentration of MZ1 for 1 h. MG132 was added 5 min prior to the treatment with MZ1. The ubiquitylated proteins were purified from the cell lysates using TUBE-conjugated agarose. Input or TUBE-pulldown samples were subjected to western blotting, as indicated. b TUBE pulldown using either K29-, K48-, or K63-specific TUBEs. c BRD4 was modified with K48- and K29-linked ubiquitin chains. Endogenous BRD4 was immunopurified from HCT116 cells treated with PDD (3 μM, 6 h) and MZ1 (100 nM, 1 h) together with MG132 (20 μM, 1 h) and subsequently subjected to PRM-based ubiquitin linkage quantification (n = 2, biological replicates). d HCT116 cells were treated with PDD (3 μM, 6 h) and/or MZ1 (100 nM, 1 h), and BRD4-modified ubiquitin chains were analyzed using PRM. The data show abundance (normalized to the vehicle) of signature peptides for K29 ubiquitin linkages and total ubiquitin (n = 2, biological replicates). e HT1080 cells transfected with the indicated siRNAs were treated as in (a, b) to pulldown K29-linked ubiquitin chains. f Knockdown of TRIP12 partially canceled PDD-dependent promotion of BRD4 degradation. HeLa cells were transfected with the indicated siRNAs and treated with PDD (3 μM, 6 h) and/or MZ1 (100 nM, 1 or 2 h). g BRD4–MZ1–CRL2^VHL ternary complex assembly. 293T cells were transfected with FLAG-BRD4 (lanes 2–5) and/or HA-VHL (lanes 1–5), and cell lysates were subjected to immunoprecipitation using anti-FLAG antibody. h The samples in (g) were subjected to LC-MS and label-free quantification (n = 3, biological replicates, ANOVA). i Heatmaps of ChIP-seq signals of BRD4 in the cells treated either with control, PDD17273, or MZ1. BRD4-binding regions in control cells (n = 3562) were subtracted for plotting, and peaks were divided into three clusters. j Percentage of BRD4-binding regions consisting of the three clusters. k Metaplots of ChIP-seq signals of BRD4 over the center of peaks. l Metaplots depicting H3K27ac ChIP-seq signals in HCT116 cells over BRD4-binding regions. m HCT116 cells were treated with PDD (5 μM, 4 h), and cell fractionation was performed. PARylation of chromatin fractions was analyzed.