Δ113p53/Δ133p53 converts P53 from a repressor to a promoter of DNA double-stand break repair

ABSTRACT

In response to DNA damage, p53 (TP53, best known as p53) is quickly activated leading to cell cycle arrest or apoptosis to ensure genomic integrity; however, this represses DNA double-strand break (DSB) repair. Our recent work revealed that Δ113p53/Δ133p53 protein is accumulated at a later stage upon DNA DSB stress to switch p53 signaling from repression to promotion of DNA DSB repair.

DNA double-strand breaks (DSBs) can be generated by exogenous factors, such as ionizing radiation (IR), as well as by endogenous factors such as free radicals.¹ DSBs also occur as normal intermediates during mitotic and meiotic recombination, DNA replication, and V(D)J recombination. Unrepaired and incorrectly repaired DSBs can give rise to genomic instability, which is the driving force behind cancer development. There are 3 main pathways of DSB repair: homologous recombination (HR), non-homologous end joining (NHEJ), and single-strand annealing (SSA).¹

The central component of the DNA damage response is the activation of tumor repressor p53 (TP53, best known as p53).² Activated p53 induces or represses the expression of several sets of downstream genes leading to cell cycle arrest, DNA damage repair, apoptosis, and/or senescence to maintain genetic stability. However, it has been well documented that p53 represses DNA DSB repair, including the HR, NHEJ, and SSA pathways, which seems to be contradictory to its role as “guardian of the genome.”³

To date, 14 p53 isoforms have been identified in human cells: p53, p53β, p53γ, Δ40p53, Δ40p53β, Δ40p53γ, Δ133p53, Δ133p53β, Δ133p53γ, Δ160p53, Δ160p53β, Δ160p53γ, Δp53, and p53ψ.^4,5 An abundance of evidence has demonstrated that p53 isoforms play an important role in regulating cell fate in response to different stresses by differentially regulating gene expression.⁴ However, whether these isoforms play a role in DNA damage repair is poorly understood.

The fact that p53 inhibits DNA DSB repair remained perplexing until our recent article entitled “p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage” was published.⁶ Both zebrafish Δ113p53 and its human counterpart Δ133p53 are transcribed from an alternative p53 promoter in intron 4.^7,8 Our previous study demonstrated that Δ113p53 is a p53 target gene and is strongly induced by γ-irradiation and DNA damaging drugs such as camptothecin and roscovitine. Moreover, Δ113p53 functions to inhibit p53-mediated apoptosis.⁷ The original aim of our recent study was to investigate whether Δ113p53 is induced in response to other stresses.⁶ Interestingly, we found that zebrafish Δ113p53 was strongly induced only by γ-irradiation, but not by ultraviolet (UV) irradiation or heat shock treatment, whereas full-length p53 was activated by all 3 stresses. Upon γ-irradiation, the patterns of p53 and Δ113p53 protein accumulation were different. Levels of full-length p53 protein peaked as early as 4 hours post irradiation (hpi), whereas Δ113p53 protein peaked later, at 24 hpi. Similar results were obtained in human cells. These observations promoted us to investigate whether Δ113p53/Δ133p53 has functions in DNA DSB repair.

To achieve this, we used 3 enhanced green fluorescent protein-based visual and quantitative reporter systems to measure HR, NHEJ, and SSA repair, respectively, and performed comet assays and repair foci analysis on γ-irradiated zebrafish and human cells. We demonstrated that Δ113p53/Δ133p53 promotes all 3 DNA DSB repair pathways in a p53-independent manner. Furthermore, in γ-irradiated zebrafish embryos, the proportion of apoptotic cells reached the highest level at around 8 hpi and decreased to basal level at 24 hpi, which correlated nicely with the level of p53 protein, whereas the extent of DNA damage decreased rapidly after 28 hpi, corresponding to the level of Δ113p53 protein. Hence, we revealed that the p53 signaling pathway protects genomic stability in response to DNA DSB stress by accumulating p53 protein to a high level at the early stage to induce cells with severe DNA damage to undergo apoptosis and by accumulating Δ113p53 protein at a later stage to inhibit apoptosis and promote DNA DSB repair in cells with less DNA damage (Fig. 1).

Figure 1.

p53 signaling in response to DNA double-strand breaks (DSBs). To minimize the toxicity of DSBs induced by ionizing irradiation at the organismal level, p53 protein is quickly accumulated to a high level in cells with severe DNA damage to inhibit DNA DSB repair and guide cells to undergo apoptosis; in cells with less and repairable DNA damage, p53 protein is accumulated to a low level for transcription of its target genes including mdm2 (encoding an E3 ligase) and Δ113p53. The expression of mdm2 can promote protein degradation of p53 but not of Δ113p53, which lacks the mdm2-interacting motif. Therefore, Δ113p53 protein can accumulate to higher levels at a later stage as p53 protein decreases to the basal level. Subsequently, Δ113p53 promotes DNA DSB repair in the remaining surviving cells by upregulating the expression of rad51, ligaseIV (lig4), and rad52. HR, homologous recombination; NHEJ, non-homologous end joining; SSA, single strand annealing.

To evaluate the biological significance of Δ113p53/Δ133p53 functions in DNA DSB repair, we generated a zebrafish Δ113p53 knockout mutant by deleting one p53-responsive element in its promoter without any effects on the expression of full-length p53. Upon γ-irradiation, the extent of DNA damage and the amount of apoptosis were significantly increased in the mutant embryos, resulting in high mortality. Next, we injected mRNA for bcl2L (an antiapoptotic protein) to block apoptosis in order to determine the contribution of Δ113p53 function in DSB repair to embryo viability. Although embryo viability in wild type (WT) and Δ113p53^M/M zebrafish was significantly increased by blocking apoptosis upon γ-irradiation, the difference in viability between WT and Δ113p53^M/M embryos remained. We further demonstrated that knockdown of Δ133p53 in human cells coupled with γ-irradiation increased the extent of DNA damage and resulted in cell growth arrest at the G2 phase that in turn led to enhanced cell senescence at the later stage. Our findings demonstrate that Δ113p53/Δ133p53 is a prosurvival factor in conditions of DNA DSB stress.

In our study of molecular mechanisms, we found that Δ113p53 enhanced expression of the DNA DSB repair genes rad51, ligaseIV (lig4), and rad52, independent of full-length p53. We further demonstrated that Δ113p53 binds to a novel type of p53 response element in the promoters of these 3 genes by promoter function analysis, gel shift, and chromatin immunoprecipitation (ChIP) assays. Taken together, these findings demonstrate that Δ113p53/Δ133p53 promotes DNA DSB repair by upregulating the expression of DNA DSB repair genes.

Several questions remain unanswered. For instance, how is cell fate decided at the early stage upon DNA DSB stress at the organismal level? Is p53 differentially activated in different cells according to the level of DNA damage within cells to decide the cell fate? Why is Δ113p53/Δ133p53, a p53 target gene, induced only by γ-irradiation, but not by UV irradiation and heat shock treatment? How does Δ113p53/Δ133p53, which lacks the transcriptional domain, transcribe DNA DSB genes independent of full-length p53? In addition, the study also implies that Δ113p53/Δ133p53 may play a role in other biological processes in which DNA DSBs are induced. One such process is cell reprogramming. Many genetic abnormalities are found in human induced pluripotent stem (iPS) cells because early reprogramming of iPS cells triggers the DNA DSB response.^9,10 It will be very interesting to know whether Δ133p53 not only increases reprogramming efficiency by its antiapoptotic function, but also ensures genomic integrity in iPS cells by its ability to promote DNA DSB repair. All of these questions must be further explored.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.