Dataset Information

RNF8 promotes efficient DSB repair by inhibiting the pro-apoptotic activity of p53 through regulating the function of Tip60.

ABSTRACT:

Objectives

RING finger protein 8 (RNF8) is an E3 ligase that plays an essential role in DSB repair. p53 is a well-established tumour suppressor and cellular gatekeeper of genome stability. This study aimed at investigating the functional correlations between RNF8 and p53 in DSB damage repair.

Materials and methods

In this article, wild-type, knockout and shRNA-depleted HCT116 and U2OS cells were stressed, and the roles of RNF8 and p53 were examined. RT-PCR and Western blot were utilized to investigate the expression of related genes in damaged cells. Cell proliferation, apoptosis and neutral cell comet assays were applied to determine the effects of DSB damage on differently treated cells. DR-GFP, EJ5-GFP and LacI-LacO targeting systems, flow cytometry, mass spectrometry, IP, IF, GST pull-down assay were used to explore the molecular mechanism of RNF8 and p53 in DSB damage repair.

Results

We found that RNF8 knockdown increased cellular sensitivity to DSB damage and decreased cell proliferation, which was correlated with high expression of the p53 gene. RNF8 improved the efficiency of DSB repair by inhibiting the pro-apoptotic function of p53. We also found that RNF8 restrains cell apoptosis by inhibiting over-activation of ATM and subsequently reducing p53 acetylation at K120 through regulating Tip60.

Conclusions

Taken together, these findings suggested that RNF8 promotes efficient DSB repair by inhibiting the pro-apoptotic activity of p53 through regulating the function of Tip60.

SUBMITTER: Chen H

PROVIDER: S-EPMC7106964 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundActivation of p53-mediated gene transcription is a critical cellular response to DNA damage and involves a phosphorylation-acetylation cascade of p53. The discovery of differences in the response to different agents raises the question whether some of the p53 oncosuppressor functions might be exerted by different posttranslational modifications. Stress-induced homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates p53 at serine-46 (Ser46) for p53 apoptotic activity; p53 acetylation at different C-terminus lysines including p300-mediated lysine-382 (Lys382) is also required for full activation of p53 transcriptional activity. The purpose of the current study was to evaluate the interplay among HIPK2, p300, and p53 in p53 acetylation and apoptotic transcriptional activity in response to drug by using siRNA interference, p300 overexpression or deacetylase inhibitors, in cancer cells.ResultsKnockdown of HIPK2 inhibited both adriamycin-induced Ser46 phosphorylation and Lys382 acetylation in p53 protein; however, while combination of ADR and zinc restored Ser46 phosphorylation it did not recover Lys382 acetylation. Chromatin immunoprecipitation studies showed that HIPK2 was required in vivo for efficient p300/p53 co-recruitment onto apoptotic promoters and that both p53 modifications at Ser46 and Lys382 were necessary for p53 apoptotic transcription. Thus, p53Lys382 acetylation in HIPK2 knockdown as well as p53 apoptotic activity in response to drug could be rescued by p300 overexpression. Similar effect was obtained with the Sirt1-inhibitor nicotinamide. Interestingly trichostatin A (TSA), the inhibitor of histone deacetylase complexes (HDAC) did not have effect, suggesting that Sirt1 was the deacetylase involved in p53 deacetylation in HIPK2 knockdown.ConclusionThese results reveal a novel role for HIPK2 in activating p53 apoptotic transcription. Our results indicate that HIPK2 may regulate the balance between p53 acetylation and deacetylation, by stimulating on one hand co-recruitment of p300 and p53Lys382 on apoptotic promoters and on the other hand by inhibiting Sirt1 deacetylase activity. We attempted to reactivate p53 apoptotic transcriptional activity by rescuing both Ser46 and Lys382 modification in response to drug. Our data propose combination strategies for the treatment of tumors with dysfunctional p53 and/or HIPK2 that include classical chemotherapy with pharmacological or natural agents such as Sirt1-deacetylase inhibitors or zinc, respectively.