The nuclear function of p53 is required for PUMA-mediated apoptosis induced by DNA damage

Abstract

The tumor suppressor p53 can induce apoptosis by activating gene expression in the nucleus, or by directly permeabilizing mitochondria in the cytoplasm. It has been shown that PUMA, a downstream target of p53 and a BH3-only Bcl-2 family member, plays an essential role in apoptosis induced by both nuclear and cytoplasmic p53. To understand how PUMA does so, we used homologous recombination to delete the binding sites of p53 in the promoter of PUMA in human colorectal cancer cells. As a result, the induction of PUMA and apoptosis in response to p53 and DNA-damaging agents were abrogated. Transcription coactivator recruitment and histone modifications in the PUMA promoter were suppressed. However, induction of PUMA and apoptosis in response to non-DNA-damaging stimuli were unaffected. These results indicate that the binding of nuclear p53 to the specific sites within the PUMA promoter is essential for its ability to induce apoptosis and is likely to be required for its tumor suppressive capacity.

Fig. 1.

Targeted deletion of the p53-binding sites in the PUMA promoter in HCT116 cells. (A) PUMA genomic locus and the targeting construct. The targeting construct consists of two homologous arms and the neomycin-resistance gene (Neo). Boxes 1a–4 represented exons 1a–4 of PUMA. Homologous recombination resulted in a deletion of 92 base pairs, including a part of both p53-binding sites (p53 BS1 and BS2). The same construct was used in the second round of gene targeting after the Neo, flanked by two LoxP sites, was excised from the heterozygous cells by Cre recombinase. The positions of the primers (P1 and P2) for PCR screening were indicated. (B) Identifying BS-KO clones. PCR was used to analyze HCT116 clones with different p53-binding site (BS) genotypes after removal of Neo. WT and the recombinant (BS-KO) alleles were indicated. (C) DNA sequences of the genomic region containing the p53-binding sites in the parental and BS-KO HCT116 cells. PCR and subsequent sequencing were performed after Neo was excised from both alleles. Two p53-binding sites and overlapping sequences between the WT and BS-KO alleles were indicated.

Fig. 2.

Induction of PUMA by DNA-damaging agents requires direct binding of p53 to the PUMA promoter. (A) The binding of p53 to the PUMA promoter. WT and BS-KO HCT116 cells were treated with 5-FU for 12 h. The bindings of p53 to the PUMA and p21 promoters were analyzed by ChIP, followed by PCR. Amplified region in the PUMA promoter is 5′ to the deleted region in the BS-KO cells. Amplified region in the p21 promoter is across the p53-binding sites. (B–F) Isogenic HCT116 cells with different p21, PUMA, BS, and p53 genotypes were subjected to the indicated treatments. Expression of p53, PUMA, and p21 was analyzed by Western blotting. α-Tubulin was used as the loading control. (B) Cells were infected with an adenovirus expressing p53 (Ad-p53) for 24 h. (C) Cells were treated with 5-FU (50 μg/ml) or adriamycin (Adr; 0.4 μg/ml) for 24 h. (D) Cells were treated with adriamycin (Adr; 0.4 μg/ml) or etoposide (Etp; 40 μM) for 24 h. (E) Cells were exposed to γ-irradiation (IR; 12 Gy), and cell lysates were prepared 24 h after the exposure. (F) Cells were transfected with HA-tagged p73 (HA-p73) or the control empty pCDNA3 vector. Cell lysates were prepared 24 h after transfection.

Fig. 3.

Recruitment of p300 and acetylation of histone H4 after DNA damage depend on the binding of p53 to the PUMA promoter. Parental, BS-KO, and p53-KO HCT116 cells were treated with adriamycin (Adr, 0.4 μg/ml) for 12 h. Chromatin complexes were cross-linked and isolated for ChIP analysis. (A) Map of the PCR primers for ChIP analysis. The positions of the primers that amplify a proximal, middle, or distal region relative to the p53-binding sites were indicated. (B) Bindings of p300 and E2F1 to the PUMA promoter. Immunoprecipitation (IP) was performed by using antibodies specific for p300 and E2F1. PCR was performed to quantify the amount of p300 and E2F1 bound to the indicated regions within the PUMA promoter. (C) Acetylation of histones H3 and H4. Antibodies specific for the acetyl-H3 and acetyl-H4 were used for ChIP. For “-Ab” controls, normal mouse or rabbit IgG was used. “Input” represented 1% of the starting materials for IP amplified by the proximal primers.

Fig. 4.

PUMA-dependent apoptosis induced by p53 and DNA-damaging agents was abrogated in the BS-KO cells. HCT116 cells with the indicated genotypes were infected with Ad-p53 or treated with DNA-damaging agents, including 5-FU (50 μg/ml), oxaliplatin (50 μM), etoposide (40 μM), adriamycin (0.4 μg/ml), camptothecin (100 nM), or γ-irradiation (IR; 12 Gy). (A) Apoptosis determined by nuclear staining. After the indicated treatments for 48 h, attached and floating cells were fixed and analyzed by fluorescence microscopy after Hoechst 33258 staining. Those with fragmented and condensed nuclei were counted as apoptotic cells. (B) Colony formation assay for long-term cells survival. Approximately 500 cells treated with adriamycin (0.1 μg/ml) or etoposide (5 M) for 48 h were plated into 12-well plates. Colonies were visualized by crystal violet staining 2 weeks later. (C) Caspase activation. After adriamycin (Adr) and etoposide (Etp) treatments for 48 h, caspases 3, 8, and 9 were analyzed by Western blotting. The arrow indicated cleaved caspases. α-Tubulin was used as the loading control.

Fig. 5.

Induction of PUMA and apoptosis by non-DNA-damaging agents does not depend on the binding of p53 to the PUMA promoter. Cells with the indicated p21, PUMA, and BS genotypes were treated with non-DNA-damaging agents, including microtubule-targeting agent taxol (5 nM), kinase inhibitor staurosporine (60 nM), ER stress-inducers brefeldin A (10 nM) and thapsigargin (12 nM), or serum starvation. (A) Activation of PUMA. After the indicated treatments for 24 h, PUMA, p53, and α-tubulin were analyzed by Western blotting. (B) Apoptosis determined by nuclear staining. After the treatments for 48 h, apoptotic cells were counted after nuclear staining.

Fig. 6.

Targeted deletion of PUMA or p53-binding sites abrogated p53-induced apoptosis in DLD1 cells. (A) Targeting PUMA in DLD1 cells. Cells with indicated PUMA genotypes were infected by Ad-p53 for 24 h. p53, PUMA and α-tubulin were analyzed by Western blotting. (B) Targeting the BS in DLD1 cells. DLD1 cells with the indicated BS genotypes were infected with Ad-p53 for 24 h. p53, PUMA and α-tubulin were analyzed by Western blotting. (C) Induction of PUMA by p73. DLD1 cells with the indicated genotypes were transfected with HA-tagged p73 (HA-p73) or the control empty pCDNA3 vector. The expression of the indicated proteins at 24 h after transfection was analyzed by Western blotting. (D) Induction of PUMA by non-DNA-damaging agents. WT and BS-KO DLD1 cells were treated with the indicated non-DNA-damaging agents for 24 h. PUMA, p53, and α-tubulin were analyzed by Western blotting. (E) Apoptosis in the PUMA-KO and BS-KO DLD1 cells. WT, PUMA-KO, and BS-KO DLD1 cells were infected with Ad-p53 or treated with staurosporine. Apoptosis was analyzed by nuclear staining 48 h after the treatment.