Wip1 directly dephosphorylates gamma-H2AX and attenuates the DNA damage response

Abstract

The integrity of DNA is constantly challenged throughout the life of a cell by both endogenous and exogenous stresses. A well-organized rapid damage response and proficient DNA repair, therefore, become critically important for maintaining genomic stability and cell survival. When DNA is damaged, the DNA damage response (DDR) can be initiated by alterations in chromosomal structure and histone modifications, such as the phosphorylation of the histone H2AX (the phosphorylated form is referred to as gamma-H2AX). gamma-H2AX plays a crucial role in recruiting DDR factors to damage sites for accurate DNA repair. On repair completion, gamma-H2AX must then be reverted to H2AX by dephosphorylation for attenuation of the DDR. Here, we report that the wild-type p53-induced phosphatase 1 (Wip1) phosphatase, which is often overexpressed in a variety of tumors, effectively dephosphorylates gamma-H2AX in vitro and in vivo. Ectopic expression of Wip1 significantly reduces the level of gamma-H2AX after ionizing as well as UV radiation. Forced premature dephosphorylation of gamma-H2AX by Wip1 disrupts recruitment of important DNA repair factors to damaged sites and delays DNA damage repair. Additionally, deletion of Wip1 enhances gamma-H2AX levels in cells undergoing constitutive oncogenic stress. Taken together, our studies show that Wip1 is an important mammalian phosphatase for gamma-H2AX and shows an additional mechanism for Wip1 in the tumor surveillance network.

Figure 1. Wip1 reduced IR-induced γ-H2AX signal

A, immunocytochemistry of either sham-irradiated (No IR) or IR-exposed H1299 cells induced to express Wip1 showed reduced γ-H2AX foci [+Dox, Wip1 induction; −Dox, no Wip1 induction; green, Wip1; red, γ-H2AX; blue, 4′,6-diamidino-2-phenylindole (DAPI)]. The white arrowhead points to a single non–Wip1-expressing cell that showed elevated γ-H2AX levels. B, immunoblot analysis showed decreased IR-induced γ-H2AX levels in H1299 cells expressing Wip1 (+Dox). p38 immunoblot is used as a loading control. C, immunocytochemistry of IR-exposed HCT116 cells that overexpress Wip1 showed reduced γ-H2AX foci (white arrows). D, immunoblotting of HCT116 cells showed that depletion of Wip1 by siRNA (Wip1i) or overexpression of Wip1 (Wip1) by transient transfection of a Flag-tagged Wip1 construct resulted in increased or decreased levels of γ-H2AX, respectively, basally after IR exposure compared with the control (GFPi). Tubulin is used as a loading control.

Figure 2. Wip1 did not block phosphorylation of H2AX after stress

A) Immunocytochemistry of H1299 cells induced to express Wip1 either sham irradiated (“No IR”) or harvested at the indicated time points after IR exposure showed reduced γ̃H2AX foci at later time points. Arrows designate cells that do not express Wip1. B) Immunoblot analysis of the cell lysates from A) showed decreased IR-induced γ̃H2AX levels at later time points in H1299 cells expressing Wip1 (“+dox”) compared to the control (“-dox”). C) Immunocytochemistry showed reduced γ̃H2AX foci in Wip1 expressing H1299 cells compared to control cells (white arrows) after UVR exposure. (Wip1, green; γ̃H2AX, red; DAPI, blue) D) Immunoblot analysis of the cell lysates from C) showed similar results. The p38 immunoblot is used as a loading control.

Figure 3. Induction of Wip1 after IR exposure reduces IR-induced γ̃H2AX levels

A) Schematic of the experimental design. H1299 cells were exposed to IR, and either untreated (“-Dox”) or treated with doxycycline to induce Wip1 expression (“+Dox”) 30 minutes after IR. Cells were harvested at the indicated time points after IR to measure levels of γ̃H2AX and Wip1. B) Immunoblot analysis and C) immunofluorescence of Wip1 and γ̃H2AX levels showed that cells expressing Wip1 had reduced γ̃H2AX levels after IR. β-actin is used as a loading control. White arrows indicate Wip1 expressing cells. (Wip1, green; γ̃H2AX, red; DAPI, blue)

Figure 4. Deletion of Wip1 enhanced basal and IR-induced γ̃H2AX levels in cells undergoing oncogenic stress

A) Immunocytochemistry of Wip1-/- E1A/Ras MEFs showed enhanced basal γ̃H2AX levels compared to wt E1A/Ras MEFs in the absence of p-ATM staining. B) Immunoblot analysis of γ-H2AX showed that Wip1-/- E1A/Ras MEFs had elevated γ-H2AX levels basally and after IR at the indicated time points compared to the wild type control (“wt E1A/Ras MEFs”). ERK2 is used as a loading control.

Figure 5. Wip1 directly dephosphorylates γ̃H2AX

A) Immunoblot analysis demonstrated reduced or enhanced γ̃H2AX levels in UVR exposed H1299 cells stably overexpressing Wip1 (“Wip1”) or a phosphatase dead mutant of Wip1 (“Wip1DA”), respectively. β-actin is used as a loading control. B) γ̃H2AX was detected by immunoblot (“IB”) in Flag-immunoprecipitates (“IP”) from sham (“-”) and IR exposed (time points indicated) HCT116 cells transfected with a Flag-Wip1 construct (“Wip1”), but not in cells transfected with an empty vector (“mock”). C) In vitro phosphatase reactions analyzed by immunoblot showed an increasing loss of γ̃H2AX levels with the indicated increasing concentrations of recombinant Wip1 protein. γ̃H2AX levels were restored with a Wip1 inhibitor (“I”, Compound M from {Belova et al., 2005, Cancer Biol Ther, 4, 1154-8}, 0.5 μM).

Figure 6. Wip1 reduced foci formation of several DNA damage response proteins

Immunocytochemistry showed decrease foci of A) γ̃H2AX, NBS1 and Rad50 (red) or B) Mdc1 and 53BP1 (red) in irradiated H1299 cells expressing Wip1 (white arrows) compared to the controls (white arrowheads). In B), cells that showed reduced γ̃H2AX foci are indicated as Wip1 expressing cells. (A, Wip1-green, DAPI-blue; B, γ̃H2AX-green, DAPI-blue). C) Decreased levels of NBS1 were detected by immunoblot (“IB”) in Mdc1 immunoprecipitates from H1299 cells expressing Wip1 (“+Dox”) compared to the controls (“-Dox”) at the indicated time points after IR exposure.

Figure 7. Wip1 delayed DNA repair after IR

H1299 cells showed comet tails ten minutes after IR regardless of Wip1 expression. Comet tails of Wip1 expressing cells (“Dox”) persisted 5 hours after irradiation, unlike the control cells (“Control”). B) Quantification of comet-positive cells in Wip1 expressing (“Dox”) and control H1299 cells (“Control”) at ten minutes, two hours, and five hours after IR. (Percentage of comet-positive cells verses time after irradiation).