∆113p53/∆133p53特异性促进编码区中的DNA双链断裂进行同源重组修复的分子机制研究

Induced Pluripotent Stem cells (iPSCs) have great potential in regenerative medicine, but this depends on the integrity of their genomes. A number of studies have shown that the reprogramming process can induce genetic abnormalities in human iPSCs. The most possible reason for generation of genetic variants in iPSCs is that early reprogramming of iPSCs generates DNA double-stand breaks (DSBs). DNA DSBs are the most catastrophic forms of genotoxic insult a cell can encounter. Improper repair of such damage results in genomic instability which predisposes an organism to immunodeficiency, neurological damage and cancer. As such, organisms have evolved three efficient systems to repair such damage. They are Homologous Recombination (HR), Non-Homologous End Joining (NHEJ) and Single Strand Annealing (SSA) pathways. While NHEJ and SSA promote the potentially inaccurate relegation of DSBs, HR precisely restores the genomic sequence of the broken DNA ends by utilizing sister chromatids as template for repair..A central part of the DNA damage response is the activation of the tumour repressor p53 resulting in cell cycle arrest, DNA damage repair, apoptosis, or senescense to provide genome stability. p53 has been reported to inhibit DNA double-strand break (DSB) repair pathways. However, Δ133p53, an N-terminal truncated isoform of p53, a p53 target gene, not only functions to inhibit p53 apoptotic activity, but also promotes DNA DSB repair pathways to protect cells from death and DNA damages upon DNA DSBs..p53 plays a dual role in iPSC reprogramming. The DNA damage response in early reprogramming activates p53. The activated p53 prevents the reprogramming of cells carrying various types of DNA damage by promoting apoptosis and senescence of these cells. Although the knockdown of p53 allows high reprogramming efficiency, the generated iPSCs have a high risk of carrying DNA aberrations. .Our recent study demonstrates that Δ133p53 is also induced in iPSC reprogramming. The expression of Δ133p53 during reprogramming not only increases reprogramming efficiency by its anti-apoptotic activity, but also reduces the chromosomal aberrations by promoting DNA DSB repair. To further investigate whether the overexpression of Δ133p53 can improve the genetic quality of the resultant iPSCs, a whole-genome sequencing approach was applied. A total of ten independent reprogrammed iPSC clones at passage four (including 5 iPSC lines induced by 4 Yamanaka factors and five iPSC lines induced by the co-expression of 4 Yamanaka factors and Δ133p53) and the parental CDD-1079sk cell line have been subjected to whole-genome sequencing with an Illumina Hiseq Xten sequencer. When compared to the human reference genome sequence (hg19), we identified approximately 3.0-4.2 million Single nucleotide variants (SNVs including SNPs and Indels) in each of the iPSC lines as well as in their parental CDD-1079sk cells. The total and de novo SNVs were only slightly decreased in the iPSCs with overexpression of Δ133p53, compared to the iPSCs induced with Yamanaka 4 factors only. Strikingly, the total and de novo SNVs in coding region were significantly reduced by the expression of Δ133p53. .Single SNV in coding regions may lead complete loss of function in the encoded protein. However, the chance for SNVs in non-coding regions to completely change the gene function is not as high as those in coding regions. Thus, it raises an important scientific question: does Δ133p53 promote cell to preferentially repair DNA DSBs in coding region with HR pathway? The aims of this project are as follow: .1. To determine whether HR pathway is encoded for preferentially repairing DNA DSBs in coding region; .2. To explore the molecular mechanisms for Δ133p53 to specifically reduce the SNVs in coding region of iPSCs;.3. At organism level, to investigate the roles of Δ113p53 and genes related for recruitment of HR to coding region on zebrafish genetic stability.

体外重编程会造成DNA双链断裂，因此诱导型多功能干细胞（iPSC）的基因组中会发生高频率的简单核苷酸变异（SNVs）。我们前期发现重编程可激活Δ133p53表达，Δ133p53通过抑制细胞凋亡来提高重编程频率，同时又可以通过促进DNA双链断裂修复来维持遗传稳定性。近期我们将过表达Δ133p53所获得的iPSC克隆进行全基因组测序意外发现，Δ133p53特异性显著降低全基因组的编码区SNVs。鉴于编码区的重要性，那么体内是否存在着编码区DNA双链断裂优先采用同源重组修复（HR）呢？此研究将阐明：.1. 编码区和非编码区DNA双链断裂是否存在着HR修复的差异；.2. ∆133p53特异性降低iPSC编码区SNVs的分子机制；.3. 有机体水平上，探讨斑马鱼∆113p53以及参与选择性修复的基因在维持编码区稳定性中的作用。

DNA双链断裂(DSB)修复主要有两种途径：1）同源重组修复（HR），以未断裂的正确拷贝为模板进行重组修复，是完全正确修复方式；2）非同源末端连接修复(NHEJ)，简单的将断裂的地方连接起来。如果断裂的地方发生核苷酸丢失，那么NHEJ修复通常会带来突变。体外重编程会造成DNA双链断裂（DSB），因此诱导型多功能干细胞（iPSC）的基因组中会发生高频率的基因变异。我们前期发现在细胞重编程过程中，Δ133p53会激活表达，促进DNA双链断裂修复，减少iPSC中染色体畸变的频率。.在此项目中，我们首先通过全基因组测序比较5个过表达Δ133p53 iPSC克隆和5个对照组iPSC克隆的基因组新增突变频率，发现Δ133p53没有显著减少全基因组新增突变频率，但是特异性减少全基因组外显子新增突变的发生频率。并且不论是在亲本细胞中还是iPSC中编码区的突变频率都显著低于内含子以及其它非编码区域。鉴于外显子及编码区的重要性，并且现已有研究证明基因组外显子和内含子以及其它非编码区的组蛋白修饰是有差异的，如H3K36me3更多的标记于外显子，此外参与和识别此种修饰的基因如PSIP1都在HR修复中起作用。我们提出外显子DNA双链断裂优先采用同源重组修复（HR）的假设。.利用细胞系和斑马鱼为实验体系，我们在此项目中继续证明此种假设。具体结果如下：1. 证明Δ133p53可以促进PSIP1（编码H3K36me3识别蛋白）的转录；2. 采用CRISPR/dCas9定点成像和CRISPR/Cpf1定点切割双基因编辑体系，结合RAD51和53BP1免疫荧光，成功地构建了一个可视化定点检测DNA双链断裂修复途径体系；3.根据H3K36me3修饰，计算损伤位点HR/NHEJ的比例，结果证明，H3K36me3修饰水平高的DNA区域双链断裂倾向于选择HR修复，证明外显子优先选择同源重组修复的假设；4.证明PSIP1是DNA双链断裂选择性进行HR修复所必需的；5. 成功构建斑马鱼psip1a和psip1b单、双突变体，尽管突变体能够正常存活和繁殖，但这些突变体对DNA双链断裂胁迫更加敏感。突变体全基因组测序已经完成，数据在分析中。.该项研究首次揭示编码区DNA双链断裂会优先选择HR修复，对于与DNA损伤修复相关疾病的认识有很重要的意义。