Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015;14(9):1413-25.
doi: 10.1080/15384101.2015.1007785.

USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168

Qianzheng Zhu  1 , Nidhi Sharma, Jinshan He, Gulzar Wani, Altaf A Wani
Affiliations

USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing RNF168

Qianzheng Zhu et al. Cell Cycle. 2015.

Abstract

During DNA damage response (DDR), histone ubiquitination by RNF168 is a critical event, which orchestrates the recruitment of downstream DDR factors, e.g. BRCA1 and 53BP1. Here, we report USP7 deubiquitinase regulates the stability of RNF168. We showed that USP7 disruption impairs H2A and ultraviolet radiation (UVR)-induced γH2AX monoubiquitination, and decreases the levels of pBmi1, Bmi1, RNF168 and BRCA1. The effect of USP7 disruption was recapitulated by siRNA-mediated USP7 depletion. The USP7 disruption also compromises the formation of UVR-induced foci (UVRIF) and ionizing radiation-induced foci (IRIF) of monoubiquitinated H2A (uH2A) and polyubiquitinated H2AX/A, and subsequently affects UVRIF and IRIF of BRCA1 as well as the IRIF of 53BP1. USP7 was shown to physically bind RNF168 in vitro and in vivo. Overexpression of wild-type USP7, but not its interaction-defective mutant, prevents UVR-induced RNF168 degradation. The USP7 mutant is unable to cleave Ub-conjugates of RNF168 in vivo. Importantly, ectopic expression of RNF168, or both RNF8 and RNF168 together in USP7-disrupted cells, significantly rescue the formation of UVRIF and IRIF of polyubiquitinated H2A and BRCA1. Taken together, these findings reveal an important role of USP7 in regulating ubiquitin-dependent signaling via stabilization of RNF168.

Keywords: 53BP1; BRCA; DNA damage response; RNF168; USP7; deubiquitinating enzyme; histone modification.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
USP7 regulates H2A and γH2AX ubiquitination in DNA damage response. (A) HCT116 and HCT116-USP7−/− cells were exposed to 20-J/m2 UV and the cell extracts were prepared at indicated time points following UVR. The uH2A and ubiquitinated γH2AX were examined by Western blotting using anti-uH2A and γH2AX antibodies. (B) The cells were treated as in Figure 1A. Ring1B and Bmi1 were examined by Western blotting. (C) The cells were subjected to UVR and then treated with or without proteasome inhibitor MG132 and the cell extracts were examined by Western blotting. “Long Exp." indicates longer exposure for chemiluminescent detection (D) Anti-γH2AX blots were overexposed to show low level of γH2AX ubiquitination in HCT116-USP7−/− cells. (E) HeLa cells were transfected with USP7 siRNA or control siRNA for 2 rounds. The transfected cells were irradiated with UV and processed as in Figure 1B. Ubiquitinated γH2AX, uH2A, USP7 and other DDR factors were examined by Western blotting. (F) HCT116 cells were exposed to UVR, and then incubated with USP7 inhibitor HBX 411108 at indicated concentration for 8 h. The Ub-γH2AX/γH2AX ratio is calculated based on gray scale of the blots examined by ImageJ.
Figure 2.
Figure 2.
USP7 disruption compromises the formation of UVRIF of uH2A, FK2 and BRCA1 (A) HCT116 and HCT116-USP7−/− cells were exposed to micropore UV irradiation at 100 J/m2. Sub-nuclear spot accumulations of indicated DDR factors were visualized by immunofluorescence using specific antibodies. Calibration bar is 10 μm. Left panel: UVRIF of uH2A and γH2AX; right panel: UVRIF of CPD and γH2AX. (B) UVRIF of FK2 and γH2AX. (C) UVRIF of BRCA1 and γH2AX. (D) The quantitative data of γH2AX, uH2A, FK2 and BRCA1 foci from HCT116 and HCT116-USP7−/− cells. Mean ± SD were calculated from 4–6 microscopic fields of 3 independent experiments.
Figure 3.
Figure 3.
USP7 disruption compromises the formation of IRIF of uH2A and FK2, thereby affecting the BRCA1 and to a lesser extent the 53BP1 recruitment to DNA strand breaks. HCT116 and HCT116-USP7−/− cells were exposed to IR at 10 Gy. Nuclear accumulations of uH2A, γH2AX, FK2, BRCA1 and 53BP1 were visualized by immunofluorescence using specific antibodies. Calibration bar is 10 μm. (A) IRIF of uH2A and γH2AX. (B) IRIF of FK2 and γH2AX. (C) IRIF of BRCA1 and γH2AX. (D) IRIF of 53BP1 and γH2AX. (E) The quantitative data of IRIF of γH2AX, uH2A, FK2, BRCA1 and 53BP1 from HCT116 and HCT116-USP7−/− cells. Only the cells with 10 or more distinctive IRIF were considered IRIF positive and scored for quantitation. Mean ± SD of IRIF vs. γH2AX positive cell ratio was calculated from 4–6 microscopic fields of 3 independent experiments. (F) Magnified views of IRIF of 53BP1 in HCT116 and HCT116-USP7−/− cells exposed to IR at 10 Gy.
Figure 4.
Figure 4.
USP7 binds to RNF168, deubiquitinates RNF168 ubiquitin (Ub)-conjugates and protects RNF168 from UV-induced degradation. (A) Diagram of structural domains of USP7. TRAF represents tumor necrosis factor-receptor associated factor (TRAF) domain. UBL represents Ub-like domain. (B) USP7 interacts with RNF168 in vitro. GST pull-down assay were conducted using whole cell lysates of HCT 116 in RIPA buffer. The GST fusion proteins loaded on beads were verified as described in materials and methods. (C) Interaction between RNF168 and USP7 in vivo. FLAG-tagged USP7 and M1 mutant were transiently expressed in HCT116 cells. Immunoprecipitation was performed using anti-FLAG agarose gels and cells lysates made in E1A buffer, followed by Western blot analysis for presence of RNF168. Non-transfected HCT116 cells were used as control. (D) Overexpression of WT-USP7 protects RNF168 from UV-induced degradation. Construct for expressing FLAG-tagged USP7 was transfected into HCT116 cells for 48 h. The transfected cells were UV irradiated at 20 J/m2 and allowed to repair DNA for 2 h. RNF168 and USP7 were detected by Western blotting. Anti-Lamin B blots served as loading control. (E) Overexpression of M1 mutant USP7. (G) USP7 deubiquitinates RNF168 Ub-conjugates in vivo. HCT116 cells were transfected with expressing constructs for RNF168, HA-tagged Ub and FLAG-tagged USP7 in combination as illustrated in the figure. The transfected cells were treated with proteasome inhibitor MG132 or vehicle DMSO for 8 h. Expression of transfected RNF168 and USP7 was examined by Western blotting (Input); RNF168 Ub-conjugates were examined by immunoprecipitation followed by Western blotting.
Figure 5.
Figure 5.
Adenovirus-mediated expression of RNF168 and RNF8 or168 partially rescues the formation of UVRIF of uH2A, FK2 and BRCA1 in HCT116-USP7−/− cells. (A) HCT116-USP7−/− cells were infected with the indicated adenoviral vectors expressing HA-tagged RNF8 and RNF168. Expression of RNF8 and RNF168 was examined by anti-HA Western blotting. (B) HCT116-USP7−/− cells were infected with indicated adenoviral vector or vector combination. The infected cells were harvested 2 h after UV exposure. The cell lysates from infected cells were examined by Western blotting for γH2AX and BRCA1 with anti-Actin blot as loading control. (C) The adenoviral vector infected cells were exposed to micropore UV irradiation at 100 J/m2. Two hour after UV irradiation, UVRIF of uH2A and γH2AX were visualized by immunofluorescence using specific antibodies. (D) UVRIF of FK2 and γH2AX. (E) UVRIF of BRCA1 and γH2AX. (F) Bar graph illustrates quantitative data of UVRIF. Mean ± SD of UVRIF vs. γH2AX positive cell ratio was calculated from 4–6 microscopic fields of 3 independent experiments. The p values were results from Student's t-test. Symbol * indicates P ≤ 0.05; Symbol ** indicates P ≤ 0.01. Calibration bar is 10 μm.
Figure 6.
Figure 6.
Adenovirus-mediated expression of RNF168 and RNF8 or168 partially rescues the formation of IRIF of uH2A, FK2 and BRCA1 in HCT116-USP7−/− cells. (A) HCT116-USP7−/− cells were infected with the indicated adenoviral vectors as in Figure 5. The infected cells were exposed to IR at 10 Gy. One hour after IR, sub-nuclear foci formations as IRIF of uH2A, γH2AX, FK2 and BRCA1 were visualized by immunofluorescence using specific antibodies. (A) IRIF of uH2A and γH2AX. (B) IRIF of FK2 and γH2AX. (C) IRIF of BRCA1 and γH2AX. (D) Bar graph illustrates quantitative data of IRIF of γH2AX, uH2A, FK2 and BRCA1. Mean ± SD of IRIF vs. γH2AX positive cell ratio was calculated from 4–6 microscopic fields of 3 independent experiments. Symbol * indicates P ≤ 0.05; Symbol ** indicates P ≤ 0.01. Calibration bar is 10 μm. (E) Graphic illustration of regulation of DDR by USP7. Upon DNA damage, phosphorylation of H2AX is triggered by DNA strand-breaks and accumulated at damage sites. γH2AX is subsequently ubiquitinated by the concerted action of RNF168 and RNF8. The ubiquitinated γH2AX in turn facilitates the accumulation of repair factor BRCA1 and 53BP1. USP7 (green hue) regulates polyubiquitination of γH2AX, mono-ubiquitination of γH2AX and H2A through modulating the stability of RNF168, Ring1B and Bmi1 E3 ligases (red hue). USP7 may also directly regulate stability of BRCA1.
See this image and copyright information in PMC

Comment in

  • USP7 saves RIDDLE for the end.
    Pandita TK. Pandita TK. Cell Cycle. 2015;14(13):1999. doi: 10.1080/15384101.2015.1049087. Epub 2015 May 27. Cell Cycle. 2015. PMID: 26017280 Free PMC article. No abstract available.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature 2009; 461:1071-8; PMID:19847258; http://dx.doi.org/10.1038/nature08467 - DOI - PMC - PubMed
    1. Harper JW, Elledge SJ. The DNA damage response: ten years after. Mol Cell 2007; 28:739-45; PMID:18082599; http://dx.doi.org/10.1016/j.molcel.2007.11.015 - DOI - PubMed
    1. Celeste A, Fernandez-Capetillo O, Kruhlak MJ, Pilch DR, Staudt DW, Lee A, Bonner RF, Bonner WM, Nussenzweig A. Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat Cell Biol 2003; 5:675-9; PMID:12792649; http://dx.doi.org/10.1038/ncb1004 - DOI - PubMed
    1. Stucki M, Clapperton JA, Mohammad D, Yaffe MB, Smerdon SJ. and Jackson SP. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 2005; 123:1213-26; PMID:16377563; http://dx.doi.org/10.1016/j.cell.2005.09.038 - DOI - PubMed
    1. Lukas C, Falck J, Bartkova J, Bartek J, Lukas J. Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat Cell Biol 2003; 5:255-60; PMID:12598907; http://dx.doi.org/10.1038/ncb945 - DOI - PubMed

Publication types

MeSH terms

  NODES
chat 1
twitter 2