Δ133p53 Coordinates With P73 To Promote DNA Double-Strand Break Repair

The p53 family members are of high importance in differentiation and development, tumorigenesis, cell cycle regulation, apoptosis, and so on. The p53 family members (p53, p63, p73) are modular proteins with a similar basic structure, the highest degree of homology among the three members is observed within the core DNA binding domain (DBD), the DBD of p63 and p73 are 60% and 63% identical with p53 respectively. It indicates that both p63 and p73 can bind to canonical p53 DNA-binding sites and activate transcription from p53-responsive promoters. Tumor suppressor p53 is activated and accumulated in the nuclelous upon DNA damage to induce cell cycle arrest for DNA damage repair or to promote cell apoptosis to maintain the stability of genomic DNA. However, previous studies show that p53 suppress DNA double strands break (DSB) repair.The inhibitory role of p53 in DSB repair seems contradictory to its tumor-suppressing. Recent research demonstrates that p53 gene could express multiple protein isoforms, in theory, they might exert their effects by two mechanisms:the modulation of full length p53 activity and/or the autonomous functional properties while their function and biological significance still needed further research. The p53 isoform zebrafish Δ113p53 and its human human ortholog Δ33p53 lacking the transactivation domain of full-length p53 is a p53 target gene that antagonizes p53 apoptotic activity. Strikingly, Δ113p53/Δ133p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment, hence what function on earth does Δ133p53 have in DNA DSB repair?The first part of my subject was investigating this question following the two sub-parts of detailed results:First, Δ133p53 promoted DNA DSB repair pathways, including non-homologous end joining (NHEJ), homologous recombination (HR), and single-strand annealing(SSA).We observed that Δ133p53 was strongly induced by DNA DSB signal and we used three Egfp-repairing-aided visual-plus-quantitative analysis reporter systems to measure HR, NHEJ and SSA repairs, we demonstrated that Δ113p53/Δ133p53 promotes all three DNA DSB repair pathways in both zebrafish and human cells in a p53-independent manner. The mechanism analysis revealed that Δ113p53/Δ133p53 upregulates the expression of the DNA DSB repair key genes Lig4,Rad51 and Rad52 to promote DNA DSB repair.Second, Δ133p53 coordinated with 73 to promote DNA DSB repair. It was currently unclear how Al 13p53/Δ133p53 lacking the transactivation domain of full-length p53 exerts a transcriptional activity independent of full-length p53.P53 family members showed high similarities among their carboxy-terminal oligomerization domain (OD), it indicated that p53 family members isoform may form heteropolymer. Thus we checked the expression level of p53 family members upon DNA damage response and we demonstrated that Δ133p53 and p73 were both strongly accumulated at later stage in response to DNA DSB signals.The further research revealed that Δ33p53 promoting DNA DSB repair was depended on p75.In response of DNA DSB signal, knocking down p73 could aggravate DNA damage extent and accelerate the cell senescence. We performed a co-immunoprecipitation (co-IP) experiment to verify that p73 could form a complex with Δ133p53. A chromatin immunoprecipitation (ChIP) assay was performed to verify that Δ133p53 promote p73 bind to their respective Response Elements in the promoters of Lig4,Rad51 and Rad52 genes in vivo upon y-irradiation. We firstly revealed the functional mechanism of Δ133p53 and p73 in DNA DSB repair and figured out the puzzle that full length p53 repress DNA DSB repair.Secondary part of my subject was the project of zebrafish 22 Genes Knock Out (KO) for chromosome 1. The technology development of CRISPR-Cas9 decreased the cost of large-scale knocking out zebrafish. In order to establish the Chinese library of zebrafish mutants and enhance the international influence of Chinese zebrafish scientist, the Chinese scientist set up a zebrafish all genes KO consortium for chromosome 1.1 have already got 22 zebrafish mutants by CRISPR-Cas9 technology. Upon knocking out these genes could help us to reveal their function and to establish many development and disease models.