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

Abstract

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.

Fasudil Attenuates Adriamycin-Induced Cardiac Damage by Modifying Oxidative Stress and Apoptosis Mediated Cellular Signaling

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Targeting tumor suppressor p53 for organ fibrosis therapy

“…TAD1 and TAD2 involve several phosphorylation sites, which can modulate degradation and activity of p53 in response to stress stimulus [ 13 ]. In addition, the inactivation of TAD1 and TAD2 has been shown to play a role in directing p53 target gene selection, suppressing the growth of tumor, and promoting cellular senescence [ 14 , 15 ]. Mutations or deletions in the PRD can regulate p53 degradation, transactivation function, and growth suppression [ 16 , 17 ].…”

At the Crossroads of Life and Death: The Proteins That Influence Cell Fate Decisions

“…Its domain structure is critical to its ability to participate in the different molecular mechanisms [ 4 ]. The N-terminal tandem transcription activation domains (TADs) are required for its gene induction activity in response to DNA damage and oncogene activation [ 5 ]. Adjacent to the TADs is a proline-rich domain, which contributes to p53s transcriptional activity but is also essential for restricting cell growth [ 6 ].…”