H2AX is a histone variant that plays a major role in the DNA damage response (DDR) and maintenance of genomic stability. A well-established function of H2AX is the recognition and repair of double-strand breaks (DSBs) created by genotoxic stress, and monitoring the removal of H2AX phosphorylation has been shown to be a useful tool in detecting the completion of DNA repair (1
). However, agents that induce other types of DNA damage, such as ultraviolet radiation (UVR), also induce the phosphorylation of H2AX (3
). Furthermore, an early action of some oncogenes is thought to be to trigger DNA damage by inducing stalling and collapse of DNA replication forks, which triggers the DDR and leads to phosphorylation of H2AX (4
). Thus, H2AX phosphorylation may have a variety of roles in genomic stability.
H2AX phosphorylation is a critical event during the DDR [reviewed in (1
)]. Briefly, when a DSB is formed by ionizing radiation (IR) exposure, for example, the Mre11-Rad50-NBS1 (MRN) complex recognizes the free DNA ends at the break and recruits the PI3K-like kinases, such as ataxia telangiectasia mutated (ATM), which phosphorylate H2AX on serine 139. This phosphorylated form of H2AX (γ-H2AX) then serves as a platform and facilitates the recruitment of further DNA repair factors necessary to mend the lesion. First, mediator of DNA damage checkpoint protein 1 (MDC1) binds to γ-H2AX and coordinates with the MRN complex to activate ATM. ATM then phosphorylates H2AX, resulting in a cyclical process that leads to as large as a two megabase pair spreading of γ-H2AX molecules from the DSB site. The γ-H2AX molecules surrounding the damage site then act as a scaffold for DNA repair factors that repair the damage, and other DDR response proteins that arrest the cell cycle until the DNA is repaired (1
). However, the mechanism of γ-H2AX formation is different depending on the type of genotoxic stress. For example, where as H2AX is primarily phosphorylated by ATM at DSBs after IR exposure, the mechanism of UVR-induced γ-H2AX formation is different and depends on the cell cycle and can occur in G1-phase cells presumably in the absence of DSBs (3
Once DNA damage is repaired, the cell must have a means of terminating DDR signaling, and protein modifications like γ-H2AX that occurred during the DDR need to be removed. γ-H2AX could be removed by dephosphorylation or by histone exchange, and currently there is evidence that both occur. The phosphatases PP2A and PP4C have been shown to dephosphorylate γ-H2AX, which is required for checkpoint recovery (12
). Additionally, several groups have shown that replacement of γ-H2AX with unmodified H2AX is induced by nucleosomal conformational changes (15
), can occur after IR exposure (16
), and occurs in the yeast Saccharomyces cerevisiae
Due to the presence of γ-H2AX in pre-cancerous lesions and its role in the DDR (1
), it is not surprising that H2AX has tumor suppressor properties. The gene encoding H2AX, H2AFX
, has a chromosomal location of 11q23, which is a region that is often altered in many types of cancers (20
). Furthermore, deletion of the H2AFX
gene leads to genomic instability in mice (21
), and H2AX has been shown to maintain genomic stability by inhibiting the production of chromosomal aberrations by DNA breaks after IR exposure and DNA replication stress (22
). Although the H2AFX-/-
mice do not develop cancers in higher frequency than wild type mice, even a heterozygous deletion of this gene in a p53-null background renders mice susceptible to the accelerated development of cancerous lesions (20
). Additionally, H2AX has been shown to inhibit lymphomagenesis by synchronizing DNA repair with the cell cycle and proliferation (26
), which indicates that H2AX can function as a tumor suppressor in certain cellular environments.
Wild-type p53-induced phosphatase 1 (Wip1) is a nuclear, oncogenic type 2C protein phosphatase (PP2C). Wip1 has been found to be overexpressed and in some cases amplified in many types of human cancers such as breast cancer, ovarian clear cell carcinoma, and medulloblastomas (27
). Further work using various cancer mouse models has validated the oncogenic properties of Wip1. Specifically, Wip1 has been shown to act as an oncogene by inhibiting tumor suppressors, such as p53, and complementing other oncogenes, such as H-Ras1 (34
). The involvement of Wip1 in tumorigenesis makes it an attractive drug target for the treatment of cancers, and recently this notion was demonstrated in ovarian clear cell carcinoma with Wip1 pharmacological inhibitor (35
). Therefore, unraveling the molecular functions of Wip1 is necessary.
Wip1 was discovered as a protein induced after ionizing radiation (IR) in a p53-dependent manner (36
). Investigation of Wip1 molecular functions indicates that it facilitates in returning the cell to homeostasis after stress, and the literature to date focuses on the role of Wip1 after genotoxic stress such as IR and UVR. Once it is induced after DNA damage, Wip1 is then responsible for terminating stress-induced signaling pathways by dephosphorylating a number of proteins involved in these pathways such as p38, p53, ATM and MDM2 (34
). The functional consequences of these dephosphorylation events all support the survival and potential onset of genomic instability and tumorigenesis if Wip1 is overexpressed by reversing cell cycle arrest, inhibiting DNA repair and inhibiting apoptotic signaling (34
In this study, we identified Wip1 as a novel phosphatase for γ-H2AX. Phosphorylation of H2AX, caused by IR and UVR, is significantly reduced by ectopic expression of Wip1 in vivo
. Furthermore, deletion of Wip1 in oncogene-transformed mouse embryonic fibroblasts (MEF) results in heightened basal and IR-induced γ-H2AX levels. Wip1 constitutively associates with H2AX and dephosphorylates γ-H2AX in an in vitro
phosphatase assay. We also show that premature dephosphorylation of γ-H2AX after IR by Wip1 expression results in failure to recruit various DNA repair molecules such as the MRN components and MDC1 to damaged foci, which slows DNA repair and is consistent with the H2AXF
-/- phenotype (21
). The present study extends our understanding of γ-H2AX regulation and of Wip1 as a modulator of the DDR and an important regulator of the tumor surveillance network.