DNA damage-induced regulatory interplay between DAXX, p53, ATM kinase and Wip1 phosphatase

Abstract

Death domain-associated protein 6 (DAXX) is a histone chaperone, putative regulator of apoptosis and transcription, and candidate modulator of p53-mediated gene expression following DNA damage. DAXX becomes phosphorylated upon DNA damage, however regulation of this modification, and its relationship to p53 remain unclear. Here we show that in human cells exposed to ionizing radiation or genotoxic drugs etoposide and neocarzinostatin, DAXX became rapidly phosphorylated in an ATM kinase-dependent manner. Our deletion and site-directed mutagenesis experiments identified Serine 564 (S564) as the dominant ATM-targeted site of DAXX, and immunofluorescence experiments revealed localization of S564-phosphorylated DAXX to PML nuclear bodies. Furthermore, using a panel of human cell types, we identified the p53-regulated Wip1 protein phosphatase as a key negative regulator of DAXX phosphorylation at S564, both in vitro and in cells. Consistent with the emerging oncogenic role of Wip1, its DAXX-dephosphorylating impact was most apparent in cancer cell lines harboring gain-of-function mutant and/or overexpressed Wip1. Unexpectedly, while Wip1 depletion increased DAXX phosphorylation both before and after DNA damage and increased p53 stability and transcriptional activity, knock-down of DAXX impacted neither p53 stabilization nor p53-mediated expression of Gadd45a, Noxa, Mdm2, p21, Puma, Sesn2, Tigar or Wip1. Consistently, analyses of cells with genetic, TALEN-mediated DAXX deletion corroborated the notion that neither phosphorylated nor non-phosphorylated DAXX is required for p53-mediated gene expression upon DNA damage. Overall, we identify ATM kinase and Wip1 phosphatase as opposing regulators of DAXX-S564 phosphorylation, and propose that the role of DAXX phosphorylation and DAXX itself are independent of p53-mediated gene expression.

Keywords: ATM; ATM, ataxia telangiectasia mutated; CHX, cycloheximide; DAXX, Death domain-associated protein 6; IR, ionizing radiation; NCS, neocarzinostatin; p21, p21WAF1/Cip1 cyclin-dependent kinase inhibitor 1; PML, promyelocytic leukemia protein; VP16, etoposide; Wi; DAXX; DNA damage; Wip1; p53.

Figure 1.

(See previous page) DAXX is phosphorylated on S564 rapidly after DNA damage. (A) HEK 293T cells were transfected with a vector expressing FLAG-DAXX^WT and treated or not, as indicated, with 8 nM NCS or10 μM VP16 for 1 hour. Cells were lysed and FLAG-tagged proteins immunoprecipitated using anti-FLAG M2 beads, prior to analysis by SDS-PAGE and western blotting with antibodies against phospho-(S/T) ATM/ATR substrate, FLAG M2, phospho-Chk2 (T68) and DAXX. (B) HEK 293T cells were transfected with expression constructs encoding FLAG-tagged wild-type DAXX, single (DAXX^S564A), triple or quadruple mutant as indicated and treated with 10 μM VP16 for 1 hour. Cells were lysed, immunoprecipitated using anti-FLAG M2 beads and analyzed by protein gel blotting with indicated antibodies. (C) BJ fibroblasts were exposed to 10 Gy of IR, collected at the indicated time points and subjected to western blotting analysis using specific phospho-DAXX (S564), DAXX, phospho-p53 (S15) and phospho-Chk2 (T68) antibodies. GAPDH was used as a loading control. (D) BJ fibroblasts were transfected with control (siCTRL) or DAXX siRNA and 3 d later treated or not as indicated with 10 μM VP16 for 1 hour. Immunofluorescence analysis using α-PML (red) and α-P-DAXX (S564) (green) showed that upon DNA damage DAXX is preferentially phosphorylated at PML nuclear bodies.

Figure 2.

DAXX depletion or S564A mutation does not affect Mdm2/p53 stability or p53‑mediated gene expression. (A) BJ fibroblasts stably transduced with empty lentiviral pCDH vector or either pCDH-DAXX^WT or pCDH-DAXX^S564A were treated with 40 μM VP16 for 0, 2, 4 or 6 hours and RNA expression of the indicated p53-dependent genes was analyzed by quantitative RT‑PCR. Expression values were normalized to the average of 3 reference genes (β-actin, SDH and ALAS). (B) Transduced BJ fibroblast as in (A) were exposed to 40 μM VP16 for the indicated times and subjected to protein gel blotting analysis using add p53 - antibodies against DAXX, phospho-p53 (S15), p53 or p21. (C) U2OS cells transfected with pXJ41 Hdm2 (human Mdm2) together with empty FLAG-CMV, FLAG-DAXX^WT or FLAG-DAXX^S564A were treated with 50 μl/ml CHX alone or together with 10 μM VP16 for the specified time points. Cell were harvested and lysates separated by SDS–PAGE and probed with indicated antibodies. (D) BJ fibroblasts were depleted by control siRNA (siLuc) or siRNA against Wip1 and 3 d after transfection treated with 4 nM NCS. Cells were lysed at the indicated time points after DNA damage and analyzed by western blotting using labeled antibodies. GAPDH was used as a loading control.

Figure 3.

DAXX deletion does not affect Mdm2/p53 stability or p53-mediated gene expression. (A) Mdm2 and p53 protein stability was examined in control U2OS cells, 2 independent DAXX^+/+ clones (0–4, 0–18), 3 independent DAXX^−/- clones (17-7, 17-18, 17–42) and in 17-7 DAXX^−/- clone stably transduced with pCDH empty vector (EV) or pCDH-DAXX^WT. Cells were collected after the treatment with 50 μl/ml CHX at the indicated time and subjected to protein gel blotting analysis using labeled antibodies. THIIF was used as a loading control. (B) DAXX^+/+ (clone 0–18) and DAXX^−/- (clones 17-18) cells were exposed to 4 nM NCS for the specified time points. Lysed cells were then separated by SDS–PAGE and immunoblotted with indicated antibodies. GAPDH was used as a loading control. (C) RNA expression of p53-dependent genes 8 hours after the treatment with 10 μM VP16 (expressed as fold change after VP16) in U2OS clones. RNA was analyzed by quantitative RT‑PCR and the expression values were normalized to the average of 3 reference genes (β-actin, SDH and ALAS). (D) U2OS cells transfected with control non-targeting siRNA and siRNA against p53 were treated with 10 μM VP16 for 8 hours and RNA expression of p53 and indicated p53-dependent genes was analyzed by quantitative RT‑PCR. The expression values were normalized to the average of 3 reference genes (β-actin, SDH and ALAS).

Figure 4.

Overview of ATM-dependent phosphorylation of DAXX at S564 after DNA damage. (A) U2OS cells were pretreated with DMSO or 10 μM ATM inhibitor KU-55933 (ATMi) for 30 min, exposed to 2 mM hydroxyurea (HU), 4 nM neocarzinostatin (NCS) or 1 μM camptothecin (CPT) for the indicated times and subjected to western blotting analysis using labeled antibodies. (B) Table showing the level of phosphorylated S564 on DAXX and the activity of ATM/ATR kinases after different types of DNA damage. (C) Sequence-based predicted modular organization of DAXX according to Escobar-Cabrera et al. with indicated putative ATM/ATR phosphorylation motifs at C-terminus of DAXX (S564, S707, S712 and T726). SUMO-Interaction Motif (SIM), DAXX Helix Bundle (DHB) domain, segments rich in Ser/Pro/Glu residues (SPE) and in Ser/Pro/Thr (SPT) residues.

Figure 5.

DAXX is a substrate of Wip1 phosphatase. (A) BJ fibroblasts were exposed to 10 Gy or 2 Gy of IR and lysed at the indicated time points after DNA damage. Western blotting analysis using antibodies against phospho-DAXX (S564), DAXX, Wip1 or GAPDH showed that the DAXX S564 dephosphorylation coincides with increased expression of Wip1. (B) In vitro phosphatase assay was performed with recombinant wild-type Wip1 or phosphatase-dead Wip1-D314A mutant on FLAG-DAXX^WT immunopurified from transfected U2OS cells exposed to DNA damage. Samples were separated by SDS–PAGE and probed with indicated antibodies. As control, phosphatase buffer without Mg²⁺ was used or treatment with lambda protein phosphatase (λPP). (C) Wip1 was depleted by siRNA in U2OS cells stably expressing FLAG-DAXX^WT. Western blotting analysis using indicated antibodies showed that after DNA damage more phosphorylated DAXX is present in Wip1 siRNA treated cells compared to control GAPDH siRNA transfected cells.

Figure 6.

Wip1 affects DNA damage-induced phosphorylation of endogenous DAXX. MCF7 or U2OS cells were transfected with control (siLuc) or siRNA targeting Wip1 and 3 d later exposed to 5 Gy of IR or 4 nM NCS. Cells were lysed at the indicated time points after DNA damage and analyzed by protein gel blotting with the indicated antibodies (* unspecific band). α‑tubulin was used as a loading control.