抑癌基因p53异构体delta113p53/delta133p53促进DNA双链断裂修复基因表达分子机制研究

Organisms have developed a number of efficient DNA damage repair pathways to deal with the frequent challenge of endogenous and exogenous DNA insults.If damaged DNA is not properly repaired,it will result genomic instability, which can influence the organism's development and ageing process greatly. More importantly, loss of genomic integrity predisposes the organism to immunodeficiency, neurological disorders, and cancer. Tumor suppressor p53 plays a central role in the DNA damage response. It is a transcription factor and can be activated by DNA damage signals.The activation of p53 results expression and repression of a large number downstream genes,which will not only lead cell-cycle arrest or cause cell apoptosis,but also participate DNA damage repair, to guard organismal genomic stability. Surprisingly p53 only possitively regulates DNA damage repair pathways such as: the Base Excision Repair (BER), the Mismatch Repair (MMR) and the Nucleotide Excision Repair(NER). However, on the other hand, it negatively regulates DNA double-strand break (DSB) repair pathways including: the Non-Homologous End Joining (NHEJ), the Homologous Recombination (HR) and the Single Strand Annealing (SSA), which seems to contradict its tumor suppressor roles. One of recent important developments in the p53 field is the discovery of various p53 isoforms in vivo. However, information on the functions of these p53 isoforms in the DNA damage repair is lacking. Our previous studies showed that an N-terminal truncated p53（lack transcriptional activation domain） delta113p53 is a p53 target gene, which is strongly induced by DNA damage signals to antaganize p53 mediated cell apoptosis. Our latest discovery showed that delta113p53 is only uniquely induced by DNA double strand break (gamma irradiation), but not by other stress signals such as UV and heat shock, at meanwhile the fulllength p53 was activated upon all of the treatments（unpublished data）. More importantly, we found that delta113p53 not only suppresses p53 negative role in DNA DSB prepare, but also significantly enhances DNA DBS repair independent of p53(unpublished data). The aims of this project are as follow: 1. To identify genes of DNA DSB repair which are regulated by delta113p53/delta133p53 independent of full length p53; 2. Does delta113p53/delta133p53 regulate DNA DSB repair gene transcription dependend on p63 or p73？ This research will be first one to uncover the different roles of p53 family members and their isoforms in DNA double strand break repair and how delta113p53 (lack transcriptional activation domain) regulates repair gene transcription which is independent of p53. It will be useful for chemotherapy and radiotherapy.

抑癌基因p53可被DNA损伤激活从而诱导细胞周期停滞或细胞凋亡，同时调节DNA损伤修复，确保遗传物质的稳定性。意外的是p53仅对碱基修饰，错配，嘧啶二聚体等损伤修复起促进作用，但对DNA双链断裂修复起抑制作用。近年我们发现N端转录激活域缺失的p53异构体?113p53，它是p53目标基因，在DNA损伤下大量表达，起着抑制p53诱导细胞凋亡的作用。我们最新结果显示?113p53仅在DNA双链断裂γ-射线照射下大量表达，不仅拮抗p53抑制DNA双链断裂修复作用，而且能单独显著促进DNA双链断裂修复。在此项目中我们将： 1） 确定?113p53单独促进的DNA双链断裂修复是否取决于调控修复基因的转录。 2） 研究?113p53单独调控修复基因转录是否与p63或p73相关。 研究将厘清p53家族在修复途径中作用认识的困惑，揭示缺少转录激活域的?113p53激活基因表达的分子机理。

抑癌基因p53在DNA损伤、癌基因激活等胁迫下激活，抑制细胞周期进行损伤修复，或诱导细胞凋亡，确保机体遗传物质的稳定性。DNA最大的损伤就是DNA双链断裂，DNA双链断裂修复有三种途径：1）重组修复；2）非重组末端修复;3）单链复合修复。令人奇怪的是p53对DNA双链断裂的三种修复起着抑制作用，这与它的抑癌作用相矛盾。在此研究项目中，利用我们在斑马鱼和人类细胞系中建立可视化可定量化分析三种DNA双链断裂修复途径的体系，研究发现p53异构体∆113p53/∆133p53仅被DNA双链断裂胁迫激活，p53蛋白积累在DNA损伤的早期（4小时），促进损伤严重的细胞走向凋亡；而∆113p53/∆133p53蛋白积累在24小时才达到高峰，起着与p53完全相反作用，促进损伤轻的细胞进行DNA双链断裂修复，从而维持基因组DNA的稳定性，∆113p53/∆133p53促进DNA双链断裂修复是通过提高损伤修复关键基因如：RAD51,RAD52和LIG4转录来进行的，并且此种转录功能不依赖于p53（Cell Research, 2015）。这个发现又提出新的问题，及∆133p53自身缺少转录激活域，那它又是怎样提高修复基因转录的呢？我们进一步研究发现，p53家族基因p73(具有转录激活域)在DNA双链断裂胁迫的后期与∆133p53共同激活，同样具有促进DNA双链断裂修复功能， p73和∆133p53促进DNA双链断裂修复功能是相互依赖的，两个蛋白在体内能够形成复合体，∆133p53是p73结合到修复基因RAD51,RAD52和LIG4的启动子上所必需的。非常有意义的是我们在COSMIC癌症数据网中发现在∆133p53的启动子中存在着高频率的突变，这些突变并不改变全长p53,我们证明这些突变会降低∆133p53的表达（Cell Death & Differetiation,in revision）。我们的这些发现不仅证明缺少∆133p53有诱导肿瘤发生的作用，并且可能为癌症病人的放疗和化疗提供新的启示。.此外我们还发现∆133p53在细胞重编程中同样激活表达，不仅促进诱导性多功能干细胞形成频率，而且能够提高诱导性多功能干细胞的遗传稳定性。将会大大推动iPS细胞在再生医学上的应用(Scientific Reports,2016)。