DNA damage induced p53 downregulates Cdc20 by direct binding to its promoter causing chromatin remodeling

Abstract

CDC20 is a critical molecule in the Spindle Assembly Checkpoint (SAC). It activates the Anaphase promoting complex and helps a dividing cell to proceed towards Anaphase. CDC20 is overexpressed in many tumor cells which cause chromosomal instability. There have been limited reports on the mechanism of SAC's response to genotoxic stress. We show that ectopically expressed p53 or DNA damage induced endogenous p53 can downregulate Cdc20 transcriptionally. We have identified a consensus p53-binding site on the Cdc20 promoter and have shown that it is being used by p53 to bind the promoter and bring about chromatin remodeling thereby repressing Cdc20. Additionally, p53 also downregulates Cdc20 promoter through CDE/CHR element, but in a p21 independent manner. This CDE/CHR element-mediated downregulation occurs only under p53 overexpressed condition but not in the context of DNA damage. The present results suggest that the two CCAAT elements in the Cdc20 promoter are not used by p53 to downregulate its activity, as reported earlier.

Figure 1.

(A) HCT116 p53^−/− cells were transiently transfected with 0, 1, 2, 3 and 4 µg of pCMVp53 expression plasmids. RT–PCR was done for p53 and Cdc20 and β-actin taken as internal control. It was seen that with increase in the level of p53 mRNA, the Cdc20 mRNA decreases. (B) HCT116 p53^−/− cells transiently transfected with 0, 250 and 500 ng of pCMVp53 vector and the total protein isolated followed by western blot analysis with anti-p53, anti-CDC20 and anti-β-actin antibodies. It was observed that, with increase in p53 protein, the level of CDC20 protein decreased. (C) HepG2 cells were treated with 0, 1 and 10 µg/ml 5-FU for 48 h followed by western blot analysis. With increasing doses of the DNA damaging drug within the cell, endogenous p53 protein level increased with subsequent decrease in CDC20 protein level. (D) HepG2 cells were treated with 0, 10, 25 and 50 µM concentrations of Etoposide for 24 h followed by western blot analysis. It was observed that, with increasing drug, the endogenous p53 protein level increased with subsequent decrease in CDC20 protein level. (E) HepG2 cells expressing higher levels of p53 due to 5-FU treatment showed no increase in CDC20 protein level when treated with the proteasomal inhibitor MG115 indicating that the decrease in CDC20 protein with increasing p53 protein level was not due to proteasomal-mediated protein degradation. (F) Western blot analysis of HepG2 cells treated with MG115 in the absence of 5-FU showed inhibition of degradation of endogenous p53.

Figure 2.

(A) 1 kb DNA sequence upstream of the start site of Cdc20 transcription was cloned into the pGL3 basic vector such that it transcribes the luciferase gene. (B) HCT116 p53^−/− cells were transiently co-transfected with pTB1 vector along with 0, 50,100, 250, 500 and 1000 ng of pCMVp53 vector followed by luciferase assay after 48 h. With increasing p53 expression the luciferase expression driven by the Cdc20 promoter reduces. (C) HepG2 cells transfected with pTB1 were treated with 0, 1 and 10 µg/ml of 5FU followed by luciferase assay after 48 h. The luciferase activity decreased subsequently with increasing drug dose. (D) HepG2 cells transfected with pTB1 were treated with 0, 10, 25 and 50 µM of Etoposide followed by luciferase assay after 48 h. The Cdc20 promoter driven luciferase activity decreased with increasing drug dose.

Figure 3.

(A) Cis elements on the Cdc20 promoter and the nucleotide sequence of the identified p53 direct binding site. (B) Electrophoretic mobility shift assay done with the oligonucleotide sequence corresponding to the p53-binding site and nuclear extract from HepG2 cells treated with 5FU. The first three lanes of the left panel show binding of the oligonucleotide and 1, 3 and 5 µg of the nuclear extract, the fourth lane shows the supershift with anti-p53 antibody and the last lane is the chase with unlabeled probe (100× molar excess). In the right panel, the first three lanes correspond to the binding of p53 from 5FU treated HepG2 nuclear extracts with Cdc20 promoter oligo and chase with 100× molar concentration of the non-radiolabeled Cdc20 promoter oligo and p21 promoter oligo respectively. The next three lanes correspond to the binding of p53 from 5FU-treated HepG2 nuclear extract with the well characterized p21 promoter oligo and chase with 100× molar concentration of non-radiolabeled p21 promoter oligo and Cdc20 promoter oligo respectively. (C) ChIP in MCF7 cells treated with 5FU. Anti-p53 antibody was used to precipitate the chromatin–protein complex and Cdc20 promoter-specific primers were used to amplify the precipitated DNA. (D) ChIP in HCT116 p53^−/− cells transfected with 1 µg of pCMVp53 vector. Anti-p53 antibody was used for immunoprecipitation and Cdc20 promoter-specific primers were used for amplification of the precipitated DNA. A fraction of the input was loaded.

Figure 4.

(A) The NFY and E2F response elements and putative p53-binding site on the Cdc20 promoter have been mutated by site directed mutagenesis. The crossed boxes refer to the mutated responsive elements. (B) Luciferase assays done in HCT116 p53^−/− cell with different mutants (as indicated) in presence or absence of wild-type p53 overexpression. (C) Luciferase assay performed in HCT116 p21^−/− cells with the different mutants along with ectopic expression of wild-type p53. (D) Luciferase assay performed in HCT116 p21^−/− cells with the different mutants in presence of the DNA damaging drugs 5FU and Etoposide.

Figure 5.

(A) HCT116 p53^−/− cells cotransfected with pTB1 and pCMVp53 expression vectors were treated with increasing doses of the HDAC inhibitor Trichostatin A (TSA) and luciferase assay was performed after 48 h. Luciferase acivity is expressed as Relative Light Unit (RLU) per microgram of total protein. (B) HCT116 p53^−/− cells cotransfected with hTERT prom-luc and pCMVp53 expression vectors were treated with increasing doses of TSA and luciferase assay was performed after 48 h. Luciferase activity is expressed as RLU per microgram of total protein. (C) ChIP assay was done in 5-FU treated HepG2 cells with anti-p53, anti-HDAC1 and anti-mSin3A antibodies and the precipitated chromatin amplified using primers specific for the Cdc20 promoter. (D) ChIP assay was done in 5FU treated HCT116 p53^−/−with anti-p53, anti-HDAC1, anti-mSin3A and anti-RNA pol II antibodies and the precipitated DNA was amplified using primers specific for the Cdc20 promoter. (E) ChIP done in HCT116 cells treated or untreated with 5-FU. Amplification with the Cdc20 promoter specific primer was observed in chromatin precipitated with anti-p53 and anti-HDAC1 antibodies only in the treated cells but not in the untreated cells. (F) ChIP done in HCT116 cells treated with or without 5-FU. Immunoprecipitation was done using antibody against Histone3 lysine 9 tri-methylation and the precipitated DNA amplified using CDC20 promoter-specific primers. (G) Western blot analysis of HCT 116 cells treated with or without 5FU using anti-p53, anti-CDC20 and anti- β-actin antibodies showed an increase in endogenous p53 level with consequent decrease in CDC20 level upon drug treatment.

Figure 6.

Schematic representation of the proposed model. DNA damage induces p53 within cells which directly bind to the Cdc20 promoter and represses its transcription. HDAC1 and mSin3A are recruited causing heterochromatinization of the Cdc20 promoter. The CDE element on the promoter seems to participate in p53 dependent repression only when there is a very high concentration of p53 present within the cell. This phenomenon is p21 independent.