Figure EV2. PIAS4‐dependent DPC SUMOylation (related to Fig 2).

1. Sperm chromatin was left untreated or treated with 2,000 J/m² of UV‐C and added to non‐replicating egg extracts in the presence or absence of 10 µM of Talazoparib (PARPi). At the indicated time points, chromatin was recovered via chromatin spin‐down and samples were immunoblotted with the indicated antibodies. Note that UV‐C triggers PARylation at 2 min (lane 7), which is abolished in the presence of PARPi.
2. Sperm chromatin was treated with 2,000 J/m² of UV‐C and added to non‐replicating egg extracts in the presence of PARPi. SUMO E1 inhibitor was added where indicated. Chromatin was recovered via chromatin spin‐down and samples were immunoblotted with the indicated antibodies.
3. NPE was either mock‐ or PIAS4‐depleted, added to p4xDPC and analysed as in Fig 2A.
4. Schematic representation of PIAS4 functional domains and mutations introduced into the SIM motifs (Kaur et al, 2017).
5. NPE was either mock‐ or PIAS4‐depleted and supplemented with WT PIAS4 or a mutant containing inactivating substitutions in the SIM1 and SIM2 domains (SIM1/2*) (D). Protein samples were immunoblotted with the indicated antibodies.
6. Samples from (E) were added to p4xDPC^SUMO and analysed as in Fig 2A.
7. Sperm chromatin was left untreated or exposed to 2,000 J/m² of UV‐C and added to non‐replicating egg extracts that were either mock‐ or PIAS4‐depleted and supplemented with recombinant PIAS4 WT or SIM1/2*. PARPi was added where indicated. Chromatin was recovered via chromatin spin‐down, and samples were immunoblotted with the indicated antibodies.
8. Samples from Fig 2F were added to untreated sperm chromatin. At the indicated time points, chromatin was recovered via chromatin spin‐down and samples were immunoblotted with the indicated antibodies.
9. Samples from Fig 2F were added to UV‐C‐treated sperm chromatin in the presence of PARPi. At the indicated time points, chromatin was recovered via chromatin spin‐down and samples were immunoblotted with the indicated antibodies.
10. Immunoblot analysis of whole cell lysate of HeLa cells transfected or not with Myc‐PIAS4 expression construct. Lysate from Myc‐PIAS4‐expressing cells was distributed equally between the three individual IP conditions in Fig 2J (800 µg per sample).
11. HeLa/GFP‐DNMT1 cells transfected with non‐targeting control (CTRL) or PIAS4 siRNAs were treated with 5‐azadC for the indicated times, collected and subjected to GFP immunoprecipitation under denaturing conditions, and immunoblotted with indicated antibodies.
12. Immunoblot analysis of HeLa/GFP‐DNMT1 cells transfected with non‐targeting control (CTRL) or PIAS4 siRNAs.
13. U2OS cells were transfected with control (CTRL) or PIAS4 siRNA targeting the 3′UTR and subsequently transfected with plasmids encoding WT or catalytically inactive (CI) Myc‐PIAS4 expression plasmid. Cells were then exposed to 5‐azadC, fixed 1 h later and co‐immunostained with Myc and DNMT1 antibodies. Representative images are shown. Note that Myc‐PIAS4 CI is recruited to DNMT1 DPCs even in the absence of endogenous PIAS4 activity. Scale bar, 5 µm.
14. Immunoblot analysis of U2OS cells transfected with control (CTRL) or PIAS4 siRNA targeting the 3′UTR.