P53 Plays An Essential Role On The Chromatin Remodeling In Response To Radiation-induced DNA Damage

Eukaryotic genomic DNA is packaged into condensed chromatin, which creates a natural barrier for functional factors to access to DNA during replication, transcription, repair and recombination. Interaction of these factors with DNA requires relaxation of local chromatin structure, a type of chromatin dynamic alterations called chromatin remodeling. Increasing evidences have indicated that chromatin remodeling plays key role in DNA damage repair by facilitating the recruitment of DNA damage response proteins to DNA lesions to initiate the DNA repair process. However, the mechanism regarding the association between chromatin remodeling and DNA damage repair is poorly understood. To investigate the association between chromatin remodeling and DNA repair and its coupling mechanism, we firstly estabolished and employed a lac-repressor and lac-operator based fluorescent chromatin remodeling detection system. Through this system, the lac repressor-target gene-EGFP fusion protein can bind to the lac-operator element integrated in the genome of AO3-1 cells, and potentially reflects the process of chromatin remodeling after DNA damage. The main purpose of our study was to explore the association between the DNA damage repair and large-scale chromatin remodeling through the reporting system and to identify potential proteins involved in DNA damage repair.PIG3 (p53-inducible gene 3), originally identified as one of a set of genes induced by p53 before the onset of apoptosis, was found to contribute to early cellular response to DNA damage. Here, we will use the lac repressor-based system to investigate the potential effect of PIG3 on large-scale chromatin dynamics.p53 has been called a"cellular gatekeeper"or"the guardian of the genome"because of its central role in coordinating the cellular responses to a broad range of cellular stress factors. P53 functions as a node for organizing whether the cell responds to various types and levels of stress with apoptosis, cell cycle arrest, senescence, DNA repair, or cell metabolism. p53-controlled transactivation of target genes is an essential feature of each stress response pathway and p53 is regulated by an array of posttranslational modifications both during normal homeostasis and in stress-induced responses. For the important role of p53 involved in the DNA damage repair, its potential role in large-scale chromatin remodeling has been investigated, and which could open a novel prospective for understanding the mechanism of cellular DNA damage response.In this study, an advanced and practical large-scale chromatin remodeling fluorescent technology has been estabolished based on a lac repressor (LacR) -operator interaction system. In this system, multiple copies of the lac operator were engineered into the genome of CHO cells, and together with the surrounding genomic sequences, were amplified to produce a 90-Mb heterochromatic region. By inserting the test gene into the lac-repressor-GFP to construct recombinant plasmid, the lac repressor-test gene-EGFP fusion protein can bind to the lac-operator elements integrated in the genome of AO3-1 cells, and potentially drives the process of chromatin remodeling after DNA damage. Followng results have been obtained:(1) To delineate the role of P53 on the ionizing radiation-induced upregulation of PIG3 expression, we detected the alterations of PIG3 protein levels in A549 cells (wild-type p53) and H1299 cells (p53 deficient) after irradiation. Result showed that the 4Gy-increased PIG3 expression was started 2h post-irradiation and persisted in at least until 48h in A549 cells. Although a relative high constitutive expression of PIG3 was detected in H1299 cells, no further inducible expression was observed in this p53 deficient cell line after 4Gy irradiation.(2) Using the lac repressor-based large scale chromatin relaxation detection system, we have observed that PIG3 expression seems not to induce large scale chromatin decondensation during normal growing status, but PIG3 can promote the large scale chromatin relaxation in the cellular response to DNA damage induced by 10Gyγ-ray irradiation. It suggests there is a linking between large scale chromatin relaxation and DNA damage repair reaction.(3) A strong direct activivity of large scale chromatin relaxation for P53 have been revealed, especially in the cells after 10Gyγ-ray irradiation. Further study shows both anacardic acid (an inhibitor of acetyltransferase) and Nu7026 (a specific inhibitor of DNA-PKcs kinase) can markably inhibit P53-mediated large-scale chromatin relaxation. In addition, we observed the entire process of P53-mediated large-scale chromatin relaxation using Perkin Elmer UltraView Vox Spinning Disk Confocal. And microspheres nuclease digestion experiment also demonstrates the function of P53 in the large-scale chromatin relaxation. (4) A series of P53 mutants have been constructed, and the large scale chromatin relaxation activity has been investigated. The results demonstrated that S46 site plays a negative regulation on P53 large scale chromatin relaxation activity, and the S15, S20, S37 and K120 play the positive role. Among them, K120 is an acetylation site, and others are phosphorylation sites.In this study, we have focused on the large scale chromatin relaxation linking to DNA damage repair response. P53 is in the first time domenstrated a direct chromatin remodling fucntion.