p53-dependent upregulation of PIG3 transcription by γ-ray irradiation and its interaction with KAP1 in responding to DNA damage

Abstract

PIG3 (p53-inducible gene 3), originally identified as one of a set of genes induced by p53 before the onset of apoptosis, was assumed to contribute to early cellular response to DNA damage. Here, we studied the relation between p53 status and the increased expression of PIG3 by ionizing radiation (IR), and the related clues regarding the involvement of PIG3 in the cellular response to IR-induced DNA damage signaling. We demonstrated that the pentanucleotide microsatellite sequence was responsible for the p53-dependent induction of PIG3 transcription after irradiation, while sequence upstream of PIG3 promoter could maintain the basal level of expression which was not inducible by irradiation. The interaction of PIG3 and the KRAB-ZFP-associated protein 1 (KAP1), a DNA damage response protein, was revealed. PIG3 nucleus foci were formed 15 min after γ-ray irradiation, and which were found to partially colocalize with the phospho-KAP-1 foci as well as γ-H2AX foci. Although the lac operator tagged EGFP based reporter system revealed that PIG3 does not remodel chromatin in large scale in the cells under normal growing condition, it indeed prompted the chromatin relaxation in the cellular response to DNA damage signaling. All these data suggest that PIG3 is involved in IR-induced DNA damage response, and which maybe partially attribute to its interaction with KAP1. The p53-inducible gene 3 (PIG3 or TP53I3) was originally identified as one of a set of genes induced by p53 before the onset of apoptosis [1]. The closest relative of PIG3 in mammals is zeta-Crystallin, an NADPH-quinone oxidoreductase and a potent generator of reactive oxygen species (ROS) [2]. As consistent with its classification in the quinine oxidoreductase (QOR) family, PIG3 was confirmed an activity of NADPH dependent reductase with orthoquinones [3]. In addition, in vitro activation and in vivo overexpression of PIG3 was demonstrated to accumulate reactive oxygen species (ROS), while an inactive PIG3 mutant (Ser151Val) did not produce ROS in cells, indicating that enzymatic active protein is necessary for its function [3]. PIG3 was also recently demonstrated to contribute to early cellular response to DNA damage induced by UV radiation and the DNA damaging-chemical neocarzinostatin through recruiting 53BP1, Mre11, Rad50 and Nbs1 to the sites of DNA break lesions and modulating intra-S and G2/M phase checkpoint [4]. A p53 dependent upregulation of PIG3 way demonstrated during p53-mediated growth arrest or under genotoxic stress [5]. Szak et al. [6] reported that increased p53 binding to PIG3, p21 and MDM2 promoters occurred in the human colorectal carcinoma cell line RKO within 2 h after p53 activation, but a lower p53 affinity was demonstrated for the consensus binding site in the PIG3 promoters compared to

PIG3 Functions in DNA Damage Response through Regulating DNA-PKcs Homeostasis

“…This was the first evidence suggesting the involvement of PIG3 in the IR-induced DNA damage response. We also observed that PIG3 formed specific ionizing radiation-induced foci (IRIF) colocalized with γ-H2AX foci and partially with pKAP-17. J-H Lee et al extensively investigated the participation of PIG3 in DNA damage checkpoints after UV irradiation or after treatment using the radiomimetic drug neocarzinostatin (NCS) 8.…”

“…PIG3 has been recently reported to participate in DNA damage response induced by UV radiation and the radiomimetic drug neocarzinostatin 8, and by ionizing radiation 7. One of the mechanistic pathways for PIG3 to function in DDR is to prompt a large scale chromatin relaxation in response to DNA damage signaling 7. It is well known that PIKK family members, including DNA-PKcs, ATM and ATR, play important roles in this DNA damage signaling process.…”

“…Our previous study demonstrated that PIG3 was induced in a dose-dependent manner by ionizing radiation (IR) 6. The IR-increased expression of PIG3 is p53 dependent 7. This was the first evidence suggesting the involvement of PIG3 in the IR-induced DNA damage response.…”

“…Although it is clear that PIG3 participates in the regulation of H2AX phosphorylation and DNA damage response 7, 8, it remains unknown how this oxidoreductase PIG3 functions in DNA damage signaling. In the present study, small interference RNA (siRNA) and lentivirus-mediated shRNA expression strategies were used to knock down PIG3 transiently or stably, and the participation of PIG3 in the DNA damage response induced by ionizing radiation was further confirmed.…”

DNA damage, signaling and repair: Protecting genomic integrity and reducing the risk of human disease

“…KRAB-associated protein (KAP-1), also known as tripartite motif-containing 28 (TRIM28), is a critical effector in ATM-dependent chromatin relaxation in response to DNA DSBs induced by ionizing radiation (IR). Qin et al [5] report that p53-inducible gene 3 (PIG3), also known as tumor protein p53-inducible protein 3 (TP53I3), formed foci in the nucleus after IR-induced DNA DSB, and that this protein interacted and partially colocalized with phospho-KAP-1 foci. PIG3 must play a role in large-scale chromatin relaxation in DNA damage signaling.…”