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. 2020 Jul 10;6(28):eaba7822.
doi: 10.1126/sciadv.aba7822. eCollection 2020 Jul.

TIP60 K430 SUMOylation attenuates its interaction with DNA-PKcs in S-phase cells: Facilitating homologous recombination and emerging target for cancer therapy

Shan-Shan Gao  1 , Hua Guan  1 , Shuang Yan  1   2 , Sai Hu  1 , Man Song  1 , Zong-Pei Guo  1 , Da-Fei Xie  1 , Yike Liu  3 , Xiaodan Liu  1 , Shimeng Zhang  1 , Ping-Kun Zhou  1   3
Affiliations

TIP60 K430 SUMOylation attenuates its interaction with DNA-PKcs in S-phase cells: Facilitating homologous recombination and emerging target for cancer therapy

Shan-Shan Gao et al. Sci Adv. .

Abstract

Nonhomologous end joining (NHEJ) and homologous recombination (HR) are major repair pathways of DNA double-strand breaks (DSBs). The pathway choice of HR and NHEJ is tightly regulated in cellular response to DNA damage. Here, we demonstrate that the interaction of TIP60 with DNA-PKcs is attenuated specifically in S phase, which facilitates HR pathway activation. SUMO2 modification of TIP60 K430 mediated by PISA4 E3 ligase blocks its interaction with DNA-PKcs, whereas TIP60 K430R mutation recovers its interaction with DNA-PKcs, which results in abnormally increased phosphorylation of DNA-PKcs S2056 in S phase and marked inhibition of HR efficiency, but barely affects NHEJ activity. TIP60 K430R mutant cancer cells are more sensitive to radiation and PARP inhibitors in cancer cell killing and tumor growth inhibition. Collectively, coordinated regulation of TIP60 and DNA-PKcs facilitates HR pathway choice in S-phase cells. TIP60 K430R mutant is a potential target of radiation and PARPi cancer therapy.

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Figures

Fig. 1
Fig. 1. The interaction of DNA-PKcs and TIP60 is abated in S phase.
(A) Representative flow cytometric histograms of the synchronized HeLa cells by double blockage of thymidine method. (B) Co-IP assays were performed with TIP60 antibody to test the interaction between TIP60 and DNA-PKcs or ATM. Cyclin A2 was used as an S-phase marker. The efficiency of synchronization was monitored by flow cytometry. (C) Co-IP assays were performed with DNA-PKcs antibody to check the interaction between TIP60 and DNA-PKcs in different cell cycle phases of HeLa cells. (D) Co-IP was performed to determine the essential region of DNA-PKcs for its interaction with TIP60. Upper panel: Schematic representation of different DNA-PKcs truncated mutants. Lower panel: HEK-293T cells were transiently transfected with the indicated constructs for 30 hours, then cell lysates were immunoprecipitated with anti-Flag affinity gel, and Western blotting was performed with indicated antibodies. (E) GST pull-down assay of DNA-PKcs H domain (AA3540-4128) for detecting its interaction with TIP60. (F) GST pull-down assay of TIP60 for detecting its interaction with DNA-PKcs antibody. (G) GST pull-down assay of DNA-PKcs H domain for detecting its interaction with TIP60 in different phases of the cell cycle.
Fig. 2
Fig. 2. Identification of TIP60 K430 site SUMO2 modification is responsible for the attenuation of TIP60–DNA-PKcs interaction in S phase.
(A) Schematic representation of different TIP60 mutants and the minimum interaction region. (B) Co-IP assay was performed to determine the essential regions of TIP60 protein to interact with DNA-PKcs. HEK-293T cells were transiently transfected with the indicated constructs for 30 hours. Cell lysates were immunoprecipitated with anti-Flag affinity gel, and Western blot was performed with indicated antibodies. (C) GST pull-down assay of TIP60 AA404–471 to detect its interaction with DNA-PKcs. (D) Co-IP was performed to determine the sites of TIP60 protein essential for the TIP60–DNA-PKcs interaction. HEK-293T cells were transiently transfected with the indicated Flag-tagged TIP60 mutant constructs for 30 hours. Cell lysates were immunoprecipitated with anti-Flag affinity gel, and Western blot was performed with indicated antibodies. (E) GST pull-down assay of DNA-PKcs H domain and TIP60 K430R mutant using the indicated proteins expressed in bacteria. (F) HEK-293T cells were transiently transfected with indicated plasmids and synchronized to different phases of the cell cycle. Cells were lysed, and SUMOylated TIP60 proteins were pulled down using Flag beads and detected by Western blotting. (G) HEK-293T cells were transiently transfected with indicated plasmids for 36 hours. Cells were lysed, and SUMOylated TIP60 proteins were pulled down and detected by Western blotting.
Fig. 3
Fig. 3. Identification of PIAS4 as the E3 ligase and SENP3 as the deSUMOylation enzyme of TIP60 SUMO2 modification.
(A) 293T cells were transiently transfected with the indicated plasmids and siRNAs against PISA1 to PISA4 or HDAC4 or HDAC7. Cells were lysed, and SUMOylated TIP60 proteins were pulled down using Flag beads and detected by Western blotting. (B) 293T cells were transiently transfected with indicated plasmids expressing Flag-TIP60, HA-SUMO2, and SENP1 or SENP2/SENP3/SENP4/SENP5/SENP6. Cells were lysed, and SUMOylated TIP60 proteins were pulled down using Flag beads and detected by Western blotting. (C and D) 293T cells were transiently transfected with indicated plasmids. Thirty-six hours after transfection, cells were lysed, and Co-IP assays were performed with indicated antibodies and then detected by Western blotting. (E) 293T cells were transiently transfected with indicated plasmids and siRNAs against PISA4. SUMOylated TIP60 proteins were pulled down from cellular lysates using Flag beads and detected by Western blotting. (F) 293T cells were transiently transfected with indicated plasmids and siRNAs against SENP3. SUMOylated TIP60 proteins were pulled down from cellular lysates using Flag beads and detected by Western blotting.
Fig. 4
Fig. 4. TIP60 K430 SUMOylation blocks its interaction with DNA-PKcs in S-phase cells both in vitro and in vivo.
(A) PIAS4 SUMOylates and SENP3 deSUMOylates TIP60 in vitro. Workflow of the in vitro assay (upper panel). Briefly, His-TIP60 WT and His-TIP60 K430R expressed and purified from E. coli were used as substrates, and His-PIAS4 and His-SENP3 expressed and purified from E. coli were used as enzymes. The in vitro SUMOylation assay was performed, followed by in vitro deSUMOylation assay, as described in Materials and Methods. Samples were separated by SDS-PAGE and blotted with indicated antibodies. (B) PIAS4 mediates SUMOylation and SENP3 mediates deSUMOylation of TIP60 K430, which is essential for the binding of TIP60 and DNA-PKcs. Workflow of the in vitro assay (upper panel). Briefly, after in vitro SUMOylation and deSUMOylation assay, the samples were incubated with GST or GST–DNA-PKcs H domain for pull-down assay, and then samples were detected by Western blotting with indicated antibodies. (C and D) 293T cells were transiently transfected with indicated plasmids and siRNAs. Then, the synchronized or unsynchronized S-phase cells were lysed. Co-IP assay was performed with indicated antibodies. Then, samples were detected by Western blotting with indicated antibodies.
Fig. 5
Fig. 5. TIP60 K430 SUMO2 modification facilitates HR repair.
(A) TIP60 K430R mutation decreases the efficiency of DNA DSB repair as shown by the increased residual γH2AX foci after 4-Gy irradiation. (B) Quantification of γH2AX foci. Data are means ± SD from three independent experiments (100 cells for each point were scored in each experiment). *P < 0.05, **P < 0.01. two-tailed Student’s t test. Scale bar, 40 μm. (C) HR efficiency was determined using the direct repeat GFP (DR-GFP) reporter assay. (D) The NHEJ efficiency was determined using the EJ5-GFP reporter assay. BRCA1 or 53BP1 siRNAs were used as a positive or negative control, respectively. Data are means ± SD from three independent experiments. *P < 0.01, two-tailed Student’s t test. (E and F) TIP60 K430R mutation sensitizes cancer cells to the PARP inhibitor olaparib, measured by colony formation ability assay (E) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay (F). SR50 represents the concentration for 50% cell growth inhibition (μM). Data are means ± SEM from three independent experiments. *P < 0.05, two-way analysis of variance (ANOVA). (G) RPA2 accumulation at sites of 4-Gy–induced DNA damage. One hour after irradiation, cells were subjected to immunostaining of RPA2 antibody. (H) Quantification of γH2AX and RPA foci in the cells 1 hour after 4-Gy irradiation. Data are means ± SD from three independent experiments (50 cells in each experiment). *P < 0.05, two-tailed Student’s t test.
Fig. 6
Fig. 6. TIP60 K430 SUMO2 modification leads to DNA-PKcs activity inhibition in S phase.
(A) Effect of TIP60 K430R mutation and cell cycle on the acetylation of DNA-PKcs, ATM, and H4K16ac. The cells were synchronized or not to S phase and irradiated with 4 Gy. The cells were lysed 1 hour after irradiation and subjected to Co-IP assay with indicated antibodies and then to Western blotting with indicated antibodies. (B) Effect of TIP60 K430R mutation and cell cycle on the phosphorylation of DNA-PKcs S2056, T2609, and ATM S1981. The cells were synchronized or not to S phase and irradiated with 4 Gy. Cells were lysed 1 hour after irradiation and detected by Western blotting with indicated antibodies. (C and D) TIP60 WT and TIP60 K430R mutant HeLa cells were irradiated with 4 Gy, and 1 hour after irradiation, cells were harvested and immunostained with indicated antibodies. Quantification (D) is the mean ± SD from three independent experiments (50 cells in each experiment). *P < 0.05, two-tailed Student’s t test.
Fig. 7
Fig. 7. TIP60 K430 SUMO2 modification is a new potential target for cancer therapy of both DNA damage drugs and irradiation.
(A) Flow cytometric histograms of apoptosis detection. The TIP60 WT and K430R mutant HeLa cells were treated with 1 μM olaparib or 4-Gy γ-ray irradiation or the combination. Apoptosis was detected at 24 hours after treatments. (B) Quantification of apoptosis induction. Data are means ± SD from three independent experiments. P < 0.05, as compared with the wild-type group TIP60 WT group. (C and D) Survival of TIP60 WT and TIP60 K430R mutant HeLa (C) and MBA-MD231 (D) cells exposed to γ-rays with or without the combination of olaparib treatment. Data are means ± SD from three independent experiments. *P ≤ 0.05. (E and F) Sensitivity of TIP60 WT and TIP60 K430R mutant HeLa (E) and MBA-MD231 (F) cells to DNA damage or replication stress–inducing agents was determined by MTS assays. Data are means ± SD from three biological triplicates. *P < 0.05, **P < 0.01. (G to I) Tumorigenicity of TIP60 WT and TIP60 K430R mutant HeLa cells in nude mice. TIP60 WT (0.1 ml; 2 × 106 cells) or TIP60 K430R mutant HeLa cells were injected into each nude mouse. Three days later, mice were treated as indicated in Materials and Methods. Tumor growth was measured and compared every 3 days. Four weeks after injection, the mice were sacrificed, and the tumor samples were collected and weighed. Data are means ± SD. *P < 0.05. Photo credit: Shanshan Gao, Department of Radiation Biology, Beijing Institute of Radiation Medicine.
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References

    1. Ciccia A., Elledge S. J., The DNA damage response: Making it safe to play with knives. Mol. Cell 40, 179–204 (2010). - PMC - PubMed
    1. Chatterjee N., Walker G. C., Mechanisms of DNA damage, repair, and mutagenesis. Environ. Mol. Mutagen. 58, 235–263 (2017). - PMC - PubMed
    1. Downs J. A., Jackson S. P., A means to a DNA end: The many roles of Ku. Nat. Rev. Mol. Cell Biol. 5, 367–378 (2004). - PubMed
    1. Radhakrishnan S. K., Lees-Miller S. P., DNA requirements for interaction of the C-terminal region of Ku80 with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA Repair 57, 17–28 (2017). - PMC - PubMed
    1. Rouhani M., Modeling the interplay between DNA-PK, Artemis, and ATM in non-homologous end-joining repair in G1 phase of the cell cycle. J. Biol. Phys. 45, 147 (2019). - PMC - PubMed

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