Fig 2: Dicer acts as a transcriptional repressor of p53.(A) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. (B) Western blot analysis of the effect of Dicer knockout in p53+/+ or p53−/− HCT116 cells. (C) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. (D) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. (E) Quantitative polymerase chain reaction (qPCR) assay of the p21, PUMA, and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. (F and G) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. (H and I) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.

Fig 3: Regulation of the Dicer-p53 interaction by acetylation.(A) Schematic diagram of the p53 DBD. (B) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. (C) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. (D) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. (E) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. (F) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. (G) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. (H and I) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. (J) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.

Fig 4: Mechanistic insights into Dicer-mediated regulation of p53 functions.(A) ChIP analysis of the Dicer overexpression mediated effect on p53 at the p21 or PUMA promoter in HCT116 cells. (B) ChIP analysis of the p53 effect on Dicer at the p21 or PUMA promoter in H1299 cells by transfected Dicer with or without p53. (C) ChIP analysis of the Dicer knockout mediated effect on H3K9me3 at the p21 promoter in HCT116 cells. Two negative control clones and four Dicer knockout clones were examined. Significance was assessed in indicated comparisons by one-way analysis of variance (ANOVA). (D) Western blot analysis of endogenous interaction between Dicer and SUV39H1 in HCT116 cells. (E) Western blot analysis of the interaction among overexpressed p53, Dicer, and SUV39H1 in H1299 cells. (F) ChIP analysis of the overexpressed Dicer effect on H3K9me3 at the p21 promoter in H1299 by transfected p53 plus Vector, Dicer, or Dicer-∆. (G) ChIP analysis of the H3K9me3 at the p21 promoter in MEF p53+/+ (left) or MEF p533KR/3KR (right) cells with TSA/NAM or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment. (H) ChIP analysis of the Dicer effect on H3K9me3 at the p21 promoter in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells. (I) Work model of Dicer-mediated transcriptional regulation on p53. Error bars indicate means ± SD. n = 3 for technical replicates. All data are shown as representative of three experiments.