Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells

• Published in: American journal of physiology. Heart and circulatory physiology Vol. 315; no. 3; pp. H512 - H521
• Main Authors: Kannappan, Ramaswamy, Mattapally, Saidulu, Wagle, Pooja A, Zhang, Jianyi
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 01.09.2018
• Series: Signaling and Stress Response
• Subjects: Acid Anhydride Hydrolases - metabolism; BAX protein; Bcl-2 protein; Cell culture; Cell cycle; Cell Cycle Checkpoints; Cell proliferation; Cells; Cells, Cultured; Checkpoint Kinase 2 - metabolism; CHK2 protein; Continuous culture; CRISPR; Cyclin-Dependent Kinase Inhibitor p16 - metabolism; Deoxyribonucleic acid; DNA; DNA damage; DNA polymerase; DNA Repair; DNA sequencing; DNA-binding protein; DNA-Binding Proteins - metabolism; DNA-directed DNA polymerase; Doxorubicin; Doxorubicin - toxicity; Gene expression; Genomic Instability; Growth factors; Humans; Induced Pluripotent Stem Cells - drug effects; Induced Pluripotent Stem Cells - metabolism; INK4a protein; Insulin; Integrity; MDM2 protein; MRE11 Homologue Protein - metabolism; MRE11 protein; p16 Protein; p53 Protein; Protein Domains; Proteins; Repair; Senescence; Silence; Stem cells; Transcription; Tumor Suppressor Protein p53 - chemistry; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism; Xeroderma pigmentosum; human induced pluripotent stem cells; cellular senescence; DNA repair; p53
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.00160.2018

The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human induced pluripotent stem cells (hiPSCs) has never been studied. p53-TAD was knocked out in iPSCs using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD knockout (KO) cells were characterized by accelerated proliferation, decreased population doubling time, and unaltered Bcl-2, Bcl-2-binding component 3, insulin-like growth factor 1 receptor, and Bax and altered Mdm2, p21, and p53-induced death domain transcript expression. In p53-TAD KO cells, the p53-regulated DNA repair proteins xeroderma pigmentosum group A, DNA polymerase H, and DNA-binding protein 2 expression were found to be reduced compared with p53 wild-type cells. Exposure to a low dose of doxorubicin (Doxo) induced similar DNA damage and DNA damage response (DDR) as measured by RAD50 and MRE11 expression, checkpoint kinase 2 activation, and γH2A.X recruitment at DNA strand breaks in both cell groups, indicating that silence of p53-TAD does not affect the DDR mechanism upstream of p53. After removal of Doxo, p53 wild-type hiPSCs underwent DNA repair, corrected their damaged DNA, and restored DNA integrity. Conversely, p53-TAD KO hiPSCs did not undergo complete DNA repair and failed to restore DNA integrity. More importantly, continuous culture of p53-TAD KO hiPSCs underwent G /M cell cycle arrest and expressed the cellular senescent marker p16 . Our data clearly show that silence of the TAD of p53 did not affect DDR but affected the DNA repair process, implying the crucial role of p53-TAD in maintaining DNA integrity. Therefore, activating p53-TAD domain using small molecules may promote DNA repair and integrity of cells and prevent cellular senescence.