Fig 2: The homeodomain of Isl1 and ring domain of PIASy are responsible for coactivation of the insulin promoter.(a) A schematic illustration of mutated Isl1 constructs. (b) A schematic illustration of mutated PIASy constructs. The different vectors are named after the domains of Isl1 or PIASy shown above the scheme of the full length protein structure. (c) and (d) Isl1 or PIASy mutants (0.4 µg) were transfected into NIT cells as indicated, and then the relative luminescence activities were tested as described. (e) Mapping the full-length PIASy with various Isl1 domains for the coactivation of the insulin promoter. Isl1 and/or PIASy mutants (0.4 µg) were transfected into NIT cells as indicated. (f) Mapping the full length Isl1 with various PIASy domains for the coactivation of the insulin promoter. Isl1 and/or PIASy mutants (0.4 µg) were transfected into NIT cells as indicated. Statistical analysis was completed with the Student’s t-test. Each bar is the mean ± SD from three independent experiments. Significant differences are indicated by *(p < 0.05) and **(p < 0.01).

Fig 3: PIAS4-dependent DPC SUMOylation (related to Fig 2) 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.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.NPE was either mock- or PIAS4-depleted, added to p4xDPC and analysed as in Fig 2A.Schematic representation of PIAS4 functional domains and mutations introduced into the SIM motifs (Kaur et al, 2017).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.Samples from (E) were added to p4xDPCSUMO and analysed as in Fig 2A.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.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.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.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).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.Immunoblot analysis of HeLa/GFP-DNMT1 cells transfected with non-targeting control (CTRL) or PIAS4 siRNAs.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.Immunoblot analysis of U2OS cells transfected with control (CTRL) or PIAS4 siRNA targeting the 3'UTR.

Fig 4: The interaction between Isl1 and PIASy protein promotes insulin gene expression and secretion.(a) Relative mRNA expression level of insulin in NIT cells transfected with Isl1 or PIASy overexpression vectors for various times was measured with real-time PCR. (b) Relative mRNA expression level of insulin in NIT cells transfected with different amount of Isl1 or PIASy overexpression vectors for 24 hours was measured with real-time PCR. (c) and (d) Shown are the relative insulin mRNA expression level and insulin concentration in NIT and primary pancreatic islet cells transfected with Isl1 and/or PIASy overexpression vectors at an amount of 0.4 µg for 24 hours. (e) and (f) NIT and primary pancreatic islet cells were transfected with PIASy-siRNA, Isl1-siRNA or nc-siRNA at an amount of 0.6 µg for 24 hours, and the relative insulin mRNA expression level and insulin concentration were detected. (g) Shown is the relative mRNA expression levels of insulin, PIASy and Isl1 in db/db and db/+ mouse islets (n = 3). The Insulin mRNA levels was quantified and normalized with that of GAPDH. Statistical analysis was completed using the Student’s t-test. Each bar is the mean ± SD from three samples. Significant differences are indicated by *(p < 0.05) and **(p < 0.01).

Fig 5: Isl1 and PIASy activate the insulin promoter in a synergistic manner.(a) Overexpression plasmids for 0.2 µg PIASy and different doses of Isl1 were transfected into NIT cells with luciferase reporter plasmids. The relative luminescence intensities were determined as described in the Materials and Methods. (b) Overexpression plasmids for 0.2 µg Isl1 and different doses of PIASy were transfected into NIT cells with luciferase reporter plasmids. The empty vector pcDNA3.1 was used to normalize the transfection efficiency. Relative luminescence intensities were determined as described in the Materials and Methods. Statistical analysis was completed using the Student’s t-test. Each bar is the mean ± SD from three independent experiments. Significant differences are indicated by *(p < 0.05) and **(p < 0.01).