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. 2012 Feb 1;11(2):167-76.
doi: 10.1016/j.dnarep.2011.10.016. Epub 2011 Nov 6.

TAp63γ enhances nucleotide excision repair through transcriptional regulation of DNA repair genes

Juan Liu  1 , Meihua Lin, Cen Zhang, Duoduo Wang, Zhaohui Feng, Wenwei Hu
Affiliations

TAp63γ enhances nucleotide excision repair through transcriptional regulation of DNA repair genes

Juan Liu et al. DNA Repair (Amst). .

Abstract

p63 and p73, two p53 family members, play crucial roles in development and tumor suppression. p63 and p73 have multiple isoforms, which have similar or distinct biological functions. Transactivation (TA) isoforms of p63 and p73 have high similarity with p53 and often have biological functions similar to p53. p53 plays an important role in nucleotide excision repair (NER) through transcriptional regulation of target genes involved in NER, including DDB2, XPC and GADD45. To investigate whether TAp63 and TAp73 play a similar role in NER, Saos2 cells with inducible expression of specific isoforms of TAp63 and TAp73, including TAp63α/β/γ and TAp73α/β/γ isoforms, were employed. Overexpression of TAp63γ significantly enhances NER of ultraviolet (UV)-induced DNA damage, including cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, and enhances cell survival after UV irradiation in Soas2 cells. The enhancement of NER of UV-induced DNA damage by TAp63γ was also confirmed in H1299 cells with overexpression of TAp63γ. Consistently, knockdown of endogenous TAp63 decreases NER of UV-induced DNA damage in H1299 cells. TAp63α/β and TAp73α/β/γ isoforms do not have a clear effect on NER in Saos2 or H1299 cells. TAp63γ overexpression clearly induces the expression of DDB2, XPC and GADD45 at both RNA and protein levels. Furthermore, luciferase reporter assays show that TAp63γ transcriptionally activates DDB2, XPC and GADD45 genes through the regulation of the p53 binding elements in these genes. These results demonstrate that TAp63γ enhances NER to remove UV-induced DNA damage and maintain genomic stability through transcriptional induction of a set of NER proteins, which provides an additional important mechanism that contributes to the function of TAp63 in tumor suppression.

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Figures

Fig. 1
Fig. 1
The expression of α, β and γ isoforms of TAp63 and TAp73 proteins in Saos2-TAp63α/β/γ and Saos2-TAp73α/β/γ cells. (A) Schematic structure of α, β and γ isoforms of TAp63 and TAp73 proteins. TA: transactivation domain; PR: prolin-rich domain, DBD: DNA binding domain; OD: oligomerization domain. (B) The expression of α, β, and γ isoforms of TAp63 and TAp73 proteins in Saos2-TAp63α/β/γ and Saos2-TAp73α/β/γ cells treated with Dox. Saos2 cells, which are deficient for p53, p63 and p73, were stably transfected with Tet-on expression vectors for TAp63α, TAp63β, TAp63γ, TAp73α, TAp73β, or TAp73γ (Saos2-TAp63α/β/γ and Saos2-TAp73α/β/α cells). These 6 cell lines were treated Dox (2 μg/ml) for 18 or 24 h before Western-blot assays. (C) The regulation of Perp and p21 by α, β and γ isoforms of TAp63 and TAp73, respectively, in Saos2 cells. Perp and p21 are known target genes for TAp63 and TAp73, respectively. Saos2-TAp63α/β/γ and Saos2-TAp73α/β/γ cells were treated with or without Dox (2 μg/ml) for 18 or 24 h. mRNA levels of Perp and p21 were determined by Taqman real-time PCR assays and normalized with the levels of β-actin in cells. Data are represented as mean ± SD (n = 3).
Fig. 2
Fig. 2
TAp63γ enhances the repair of CPD and 6–4 PP in Saos2 cells. Saos2-TAp63α/β/γ and Saos2-TAp73α/β/γ cells were treated with Dox (2 μg/ml) for 24 h to induce the expression of α, β, or γ isoforms of TAp63 and TAp73 proteins. Cells without Dox treatment were used as controls. Cells were then irradiated with UV (20 J/m2), and collected immediately (0 h) or cultured in fresh medium with Dox (2 μg/ml) for different hours (4, 6, 9, or 24 h) before assays. (A) TAp63γ enhances the repair of CPD in cells. The levels of CPD in genomic DNA of cells were analyzed by a cellular CPD DNA-damage detection ELISA kit. (B) TAp63γ enhances the repair of 6–4 PP in cells. The levels of 6–4 PP in genomic DNA of cells were analyzed by a cellular 6–4 PP DNA-damage detection ELISA kit. Data are represented as mean ± SD (n = 3). *p < 0.05.
Fig. 3
Fig. 3
TAp63γ promotes host cell reactivation of UV-irradiated luciferase reporter plasmids in cells. (A) TAp63γ promotes host cell reactivation of luciferase reporter plasmids damaged by UV irradiation in Saos2-TAp63γ cells. The pGL3 luciferase reporter plasmids irradiated with different doses of UV (300 and 1000 J/m2) were transfected into Saos2-TAp63α/β/γ cells which were treated with Dox (2 μg/ml) for 24 h before transfection. Non-irradiated pRL-SV40 plasmid was co-transfected as an internal control to normalize the transfection efficiency. After transfection, cells were cultured in fresh medium with Dox (2 μg/ml) for 24 h and luciferase activities were then measured. (B and C) TAp63γ promotes host cell reactivation of luciferase reporter plasmids damaged by UV in Saos2 (B) and H1299 (C) cells. The pGL3 luciferase reporter plasmids irradiated with UV (1000 J/m2) were transfected into Saos2 cells (B) and H1299 (C), respectively, together with pcDNA-TAp63α, pcDNA-TAp63β, or pcDNA-TAp63γ expression plasmids. The pRL-SV40 plasmid was co-transfected as an internal control. (D) Knockdown of endogenous TAp63 decreases host cell reactivation of luciferase reporter plasmids damaged by UV in H1299 cells. H1299 cells were transfected with siRNA against TAp63 or TAp73. Twenty-four hours after transfection, the pGL3 luciferase reporter plasmids irradiated with UV (1000 J/m2) were then transfected into cells. Relative mRNA expression levels of TAp63 and TAp73 were determined by real-time PCR assays and normalized with β-actin. The pRL-SV40 plasmid was co-transfected as an internal control. The luciferase activities were measured at 24 h after transfection. The relative luciferase activity, which represents the relative extent of NER repair of UV-induced DNA damage, was determined as the percentage of luciferase activity expressed from UV-irradiated plasmids to that from untreated plasmids. The data are represented as mean ± SD (n = 3). *p < 0.05. (E) UV induces the TAp63γ protein accumulation in cells. Saos2-TAp63γ cells were pretreated with Dox (50 ng/ml) for 8 h before they were exposed to UV (20 J/m2). The cells were collected at different time points after UV irradiation (0–24 h), and the TAp63γ protein levels were determined by Western-blot assays.
Fig. 4
Fig. 4
TAp63γ increases cell survival after UV irradiation. Saos2-TAp63α, Saos2-TAp63β, Saos2-TAp63γ, Saos2-TAp73α, Saos2-TAp73β, Saos2-TAp73γ cells with (closed triangle) or without (closed circle) Dox treatment (2 μg/ml) were irradiated with different doses of UV (2, 4, and 6 J/m2), and their colony formation abilities were measured. The survival (%) was calculated as a percentage of colony formation ability of UV-irradiated cells compared with non-irradiated cells. The data are represented as mean ± SD (n = 3). *p < 0.05.
Fig. 5
Fig. 5
Differential transcriptional regulation of DDB2, XPC and GADD45 by TAp63 and TAp73 isoforms. Saos2-TAp63α/β/γ and Saos2-TAp73α/β/γ cells were cultured in medium containing Dox (2 μg/ml) for different hours (0, 18, and 24 h) to induce the expression of specific isoforms of TAp63 and TAp73. The expression of TAp63α/β/γ and TAp73α/β/γ isoforms were shown in Fig. 1B. The mRNA and protein expression levels of DDB2, XPC and GADD45 expression levels were measured by real-time PCR and Western-blot assays, respectively. (A) The protein levels of DDB2, XPC and GADD45 in Saos2-TAp63α/β/γ and Saos2-TAp73α/β/γ cells treated with Dox. (B) The relative mRNA expression levels of DDB2, XPC and GADD45 in Saos2-TAp63β, Saos2-TAp63γ, Saos2-TAp73β and Saos2-TAp73γ cells treated with Dox. (C) The relative mRNA expression levels of DDB2, XPC and GADD45 in H1299 cells with ectopic TAp63γ expression. H1299 cells were transiently transfected with TAp63γ expression plasmids. The mRNA levels of genes were normalized with β-actin. Data are presented as mean ± SD (n = 3).
Fig. 6
Fig. 6
TAp63γ transactivates the luciferase reporter genes containing p53-consensus DNA binding elements in human DDB2 and XPC genes. (A) p53-consensus DNA binding elements in human DDB2, XPC and GADD45 genes. N, any nucleotide; Pu, purine; Py, pyrimidine. Positions of p53-consensus DNA binding elements relative to the ATG site are indicated. (B) TAp63γ transactivates the luciferase reporter genes containing p53-consensus binding elements in human DDB2, XPC and GADD45 genes. Saos2 and Saos2-TAp63γ cells were treated with or without Dox (2 μg/ml) for 24 h before being transfected with pGL2 luciferase reporter plasmids containing one copy of p53-consensus binding elements in human DDB2, XPC or GADD45 genes. pRL-SV40 plasmid was co-transfected as an internal control. Cells were then cultured in medium with or without Dox (2 μg/ml) for another 24 h after transfection, and the luciferase activities were measured. The reporter activity was calculated as luciferase activity of reporter plasmids in cells induced with Dox compared with that in cells without Dox treatment. Data are presented as mean ± SD (n = 3).
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References

    1. Dotsch V, Bernassola F, Coutandin D, Candi E, Melino G. p63 and p73, the ancestors of p53. Cold Spring Harb Perspect Biol. 2010;2:a004887. - PMC - PubMed
    1. Yang A, Kaghad M, Caput D, McKeon F. On the shoulders of giants: p63, p73 and the rise of p53. Trends Genet. 2002;18:90–95. - PubMed
    1. De Laurenzi V, Costanzo A, Barcaroli D, Terrinoni A, Falco M, Annicchiarico-Petruzzelli M, Levrero M, Melino G. Two new p73 splice variants, gamma and delta, with different transcriptional activity. J Exp Med. 1998;188:1763–1768. - PMC - PubMed
    1. De Laurenzi VD, Catani MV, Terrinoni A, Corazzari M, Melino G, Costanzo A, Levrero M, Knight RA. Additional complexity in p73: induction by mitogens in lymphoid cells and identification of two new splicing variants epsilon and zeta. Cell Death Differ. 1999;6:389–390. - PubMed
    1. Bourdon JC, Fernandes K, Murray-Zmijewski F, Liu G, Diot A, Xirodimas DP, Saville MK, Lane DP. p53 isoforms can regulate p53 transcriptional activity. Genes Dev. 2005;19:2122–2137. - PMC - PubMed

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