Figure 5(A) Simulation analysis of the potential KLF4 PARylation site (PBZ domain) with or without DNA binding. The triple amino acids Y430, Y451, R452 on KLF4 zinc finger 2 (Znf2) is identified as the potential YYR motif involved in mediating PARP1 modification. (B) The effect of YYR motif mutations on KLF4 PARylation. Constructs of KLF4-Zinc 1-YKH/AAA mutation, KLF4-Zinc 2-YYR/AAA mutation and KLF4-Zinc-2-Y451A mutation were co-transfected with Myc-PARP1 into 293T cells, respectively, and then pulled-down using M2 agarose followed by measuring KLF4 PARylation with anti-PAR antibody. (C) KLF4-Zinc 2-YYR/AAA mutation has only effect on KLF4 PARylation but not on other types posttranslational modifications such as protein methylation, ubiquitylation, acetylation and sumoylation in 293T cells. The FLAG-tagged wild-type or mutant KLF4 were immunoprecipitated with FLAG and detected by anti-SYM10 (methylation), anti-SUMO (sumoylation), Anti-ac-lysine (acetylation), anti-Ub (ubiquitination) and anti-PAR (PARylation). Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol, soluble nucleus and chromatin in MDA-MB-231 cells. While the activation of PARP1 in response to genotoxic stress enhances chromatin recruitment of KLF4 ORC2 were the loading control for chromatid fraction. Ponceau S were the control of nuclear soluble fraction. E) Effect of PARP1 on the distribution of KLF4 subcellular localization among cytosol, soluble nucleus and chromatin in MDA-MB-231 cells. , inhibition of PARP1 by Olaparib attenuates the KLF4 accumulation in chromatin fraction H3 were the loading control for chromatid fraction. PARP1 were the control of nuclear soluble fraction. (F) KLF4-Zinc 2-YYR/AAA mutation remarkably reduces DNA damage-induced chromatin recruitment of ectopic expressed KLF4 in U2OS cells. ORC2 is the loading control for chromatid fraction. (G). Confocal analysis of PAR and FLAG-tagged KLF4 showing that KLF4-Zinc 2-YYR/AAA mutation significantly reduces KLF4 PARylation in U2OS cells. Scale bars, 10 μm. (H) Two interaction sites are observed in PARP1-KLF4 interaction pose. One of them is at znf1 and the other is at znf2 (at the same site where RFA binds). H909 and T824 on PARP1 are the potential core amino acids mediating the interaction pose. (I) The effect of PARP1 mutations (H909A or T824A) on KLF4 PARylation, confirming the critical role of H909 in mediating KLF4 PARylation in 293T cells. Myc-PARP1 wildtype or mutations (H909A or T824A) and FLAG-KLF4 were co-transfected into 293T cells and then the FLAG-KLF4 was pulldown by anti-FLAG antibody, the PARylation modification of KLF4 was blotted with anti-PAR antibody, and the binding PARP1 was blotted with anti-myc antibody. (J). Replacement of this histidine by alanine on PARP1 or replacement of the YYR motif of KLF4 by triple alanines leads to the attenuation of KLF4 PARylation. In PARP1-/- MEFs, wild-type or mutant PARP1 (H909A) were co-transfected with FLAG-tagged wild-type or KLF4-Zinc 2-YYR/AAA mutation followed by immunoprecipitation using M2-agarose and Western blotting by anti-PAR antibody. (K) KLF4 PARylation enhances its transcriptional function demonstrated by p21 promoter luciferase assay. 293T cells were co-transfected with p21 luciferase reporter plasmid with wild-type or mutant KLF4 (YYR/AAA) and wild-type or mutant PARP1 (H909A) and then submitted to luciferase assay. n=3, p=4.74x10-6 (PARP1WT vs PARP1H909A), Data are mean ± SEM; one-way ANOVA was used for the statistical analysis.