Abstract 19566: Transactivation Domain of P53 Regulates DNA Integrity in Human Cardiac Fibroblast Derived iPS Cells
Introduction: Retaining DNA integrity is essential to induced-pluripotent stem cells. p53 regulates a wide range of cellular processes including cell death and cell cycle progression, cell metabolism and DNA damage and repair. p53 is composed of transactivation domain (TAD), proline-rich region, DNA-binding domain, tetramerization domain and extreme carboxyl domain. The role of p53 in DNA damage and repair mechanisms has been studied extensively in various somatic cells. However, the role of p53 TAD, a multifunctional and dynamic domain, on DNA damage repair and retaining DNA integrity in human cardiac fibroblast derived induced-pluripotent stem cells (hiPS) cells has not been studied.
Methods: The transactivation domain of p53 was knocked out using CRISPR/Cas9 system. Both, hiPS cells expressing wild type p53 (p53WT) and p53 TAD knockout (p53TADKO) were exposed to low dose of doxorubicin to induce oxidative stress and DNA damage. DNA damage was assessed by comet assay utilizing OxiSelect Comet Assay kit and DNA damage response (DDR) was analyzed by staining for γH2A.X protein.
Results and Discussion: The transactivation domain of p53 was knocked out using CRISPR/Cas9 technology and confirmed by sequencing and western blot analysis. mRNA transcripts of pluripotent stem cell markers such as OCT4, SOX2 and c-MYC were not affected in p53TADKO compared to p53WT cells. Exposure to oxidative stress induced a significant amount of DNA damage and DDR in both p53WT and p53TADKO hiPS cells. Following removal of oxidative stress p53WT hiPS cells corrected their damaged DNA and restored DNA integrity. However, p53TADKO hiPS cells did not undergo a complete DNA damage repair and failed to restore DNA integrity. Whether failure to repair DNA damage leads to cellular senescence and activating p53TAD using small molecule promotes DNA repair will be studied and discussed at the meeting.
Conclusion: Our data clearly shows that silencing transactivation domain of p53 did not affect the pluripotency of human iPS cells but affected the DNA damage response and repair processes. Therefore, transactivation domain of p53 plays a major role in DNA repair and specifically activating this domain using small molecules may promote DNA integrity in cardiac fibroblast derived iPS cells.
Author Disclosures: R. Kannappan: None. S. Mattapally: None. W. Zhu: None. J. Zhang: None.
- © 2016 by American Heart Association, Inc.