Programmed cell death 6, a novel p53‐responsive gene,targets to the nucleus in the apoptotic response to DNA damage

The cellular response to genotoxic stress is multifaceted in nature. Following DNA damage, the tumor suppressor gene p53 activates and plays critical roles in cell cycle arrest, activation of DNA repair and in the event of irreparable damage, induction of apoptosis. The breakdown of apoptosis causes the accumulation of mutant cells. The elucidation of the mechanism for the p53‐dependent apoptosis will be crucial in applying the strategy for cancer patients. However, the mechanism of p53‐dependent apoptosis remains largely unclear. Here, we carried out ChIP followed by massively parallel DNA sequencing assay (ChIP‐seq) to uncover mechanisms of apoptosis. Using ChIP‐seq, we identified PDCD6 as a novel p53‐responsive gene. We determined putative p53‐binding sites that are important for p53 regulation in response to DNA damage in the promoter region of PDCD6. Knockdown of PDCD6 suppressed p53‐dependent apoptosis. We also observed that cytochrome c release and the cleavage of PARP by caspase‐3 were suppressed by depletion of PDCD6. We further observed that PDCD6 localizes in the nucleus in response to DNA damage. We identified the nuclear localization signal of PDCD6 and, importantly, the nuclear accumulation of PDCD6 significantly induced apoptosis after genotoxic stress. Therefore, we conclude that a novel p53‐responsive gene PDCD6 is accumulated in the nucleus and induces apoptosis in response to DNA damage.     

In vivo analyses of UV-irradiation-induced p53 promoter binding using a novel quantitative real-time PCR assay

The p53 tumor suppressor protein mediates cell cycle arrest and apoptosis through transactivation of downstream target genes. While many target genes have been identified to date, the mechanisms and time course of their induction are still unclear. We investigated the kinetics of p53 binding to the p21CIP1, MDM2, BAX and PIG3 promoters in vivo using a novel quantitative real-time chromatin immunoprecipitation-PCR assay. Our results demonstrate distinct kinetics of p53 promoter binding dependent on the target gene promoters. The timed induction of target genes due to genotoxic stress is likely to play a pivotal role for the divergent functions of p53.     

Proteasome-mediated degradation of Tob is pivotal for triggering UV-induced apoptosis

Eukaryotic cells respond to genotoxic stress by inducing cell growth arrest or apoptosis. Although the p53 tumor suppressor largely contributes to the response by regulating antiproliferative or pro-apoptotic genes, some genotoxic stresses including ultraviolet (UV) light induce apoptosis even in the absence of p53. The molecular mechanisms by which cells respond to UV in the p53-independent manner remain to be established. Here, we show that UV-induced stress promotes proteasome-dependent degradation of Tob, triggering an apoptotic signal. We found that Tob with either short deletion or a tag sequence at the C terminus was resistant to UV-induced degradation. Introduction of the degradation-resistant Tob impaired UV-induced apoptosis. Reciprocally, suppression of Tob by small interfering RNA (siRNA) resulted in frequent induction of apoptosis irrespective of the presence of functional p53 even at UV doses that do not promote Tob degradation. Finally, tob-deficient (tob(-/-)) mice and primary embryonic fibroblasts (MEFs) from tob(-/-) mice exhibit increased sensitivity to UV irradiation. Thus, proteasomal clearance of Tob provides a novel p53-independent pathway for UV-induced apoptosis.