As the functions of the WICH complex, especially that of WSTF, in the DNA damage response have not been explored, we investigated the role of WSTF in the DNA damage response. Upon DNA damage treatment, the initial γ-H2A.X phosphorylation of H2A.X and its foci formation in WSTF RNAi cells (as described in ) were similar to control cells (up to 1-hour post IR, ). However, in control cells, the γ-H2A.X level remained unchanged until 16-hour post IR. In contrast, the level of γ-H2A.X phosphorylation rapidly declined in WSTF RNAi cells 4 hours after IR treatment (). It is well-established that the number and morphology of γ-H2A.X foci undergo significant changes during the DNA damage response. Initially, a larger number of small foci are formed, while at late stages of the DNA damage response, only a few large foci are usually observed32
. As expected, large γ-H2A.X foci were formed in control cells starting 4-hours post IR (, upper panels
) and persisting until 12-hours after IR, while the overall level of γ-H2A.X phosphorylation remains relatively constant. However, this morphological progression was not observed in WSTF RNAi cells despite the initial formation of small γ-H2A.X speckles as in the controls (, lower panels
). Instead, the amount and intensity of the γ-H2A.X foci were significantly less than in controls (only 16% of the WSTF RNAi retain γ-H2A.X foci 4 hours post IR), and no large foci were observed in WSTF RNAi cells during the entire experiment, even 12 hours post IR (). Since the major kinases for γ-H2A.X phosphorylation are ATM and ATR, and γ-H2A.X foci maintenance is dependent on sustained recruitment of active ATM to the damage foci via Mdc117
, we asked if the relocalization of ATM and Mdc1 were defective in WSTF deficient cells. In control cells, large Mdc1 foci were observed at a late stage of the DNA damage response (8-hour post 10 Gy of IR) (), while similar foci were not observed in the WSTF deficient cells, indicating that the recruitment of Mdc1 is defective at a late stage of the DNA damage response in these cells. Autophosphorylation at Ser 1981 (referred to as phos-ATM hereafter) is one of the indicators of ATM activation during the DNA damage response33,34,35
. Foci formation of phos-ATM (and potentially other targets of ATM) was also impaired in WSTF deficient cells (). These data strongly suggest that WSTF plays a critical role in the recruitment of active ATM and Mdc1 to the damage sites, both of which are critical for γ-H2A.X foci formation.
We next investigated if the kinase activity of WSTF is required for its function during the DNA damage response, namely, the maintenance of phos-ATM and γ-H2A.X foci. Since the WSTF mRNA sequences targeted by the short hairpin RNAi constructs are different between human and mouse (see above), we complemented WSTF RNAi cells (derived from mouse 3T3 cells) with WT or C338A mutant kinase domain constructs of human WSTF. This approach successfully restored the expression of kinase domain of WSTF (). The expression of the WT WSTF kinase domain rescued the γ-H2A.X and phos-ATM foci formation defects in the WSTF RNAi cells (). In nearly 100% of the cells complemented with the WT construct, γ-H2A.X and phos-ATM foci were observed at least 8 hours after DNA damage treatment (). On the other hand, the phos-ATM and γ-H2A.X foci formation were as defective in cells expressing the C338A mutant as in WSTF RNAi cells containing vector alone (). Taken together, these results demonstrate that WSTF plays an important role during the DNA damage response via its novel kinase activity, an activity that targets H2A.X Y142 and possibly other yet unknown substrates.
We wondered, however, if S139 phosphorylation is required for the steady–state balance of H2A.X phosphorylation at Y142 or vice versa. As previously reported15,18
, the maintenance of γ-H2A.X phosphorylation is affected in the Mdc1-/- MEF cells (Supplementary Figure S8
). Y142, however, becomes gradually dephosphorylated in the Mdc1-/- cells as in the WT control cells (Supplementary Figure S8
), indicating that sustained γ-H2A.X phosphorylation is not required for Y142 dephosphorylation. On the other hand, S139 phosphorylation level and foci formation were greatly reduced upon DNA damage in both Y142L and Y142F mutant cells (Supplementary Figure S8
). In addition, mutations on Y142 affect Mdc1 binding to H2A.X C-tail phosphorylated at S139 (Supplementary Figure S8
). These data suggests that these phosphorylation events may be coordinated during the DNA damage response (see Discussion