WSTF regulates the H2A.X DNA damage response via a novel tyrosine kinase activity

Abstract

DNA double-stranded breaks present a serious challenge for eukaryotic cells. The inability to repair breaks leads to genomic instability, carcinogenesis and cell death. During the double-strand break response, mammalian chromatin undergoes reorganization demarcated by H2A.X Ser 139 phosphorylation (gamma-H2A.X). However, the regulation of gamma-H2A.X phosphorylation and its precise role in chromatin remodelling during the repair process remain unclear. Here we report a new regulatory mechanism mediated by WSTF (Williams-Beuren syndrome transcription factor, also known as BAZ1B)-a component of the WICH complex (WSTF-ISWI ATP-dependent chromatin-remodelling complex). We show that WSTF has intrinsic tyrosine kinase activity by means of a domain that shares no sequence homology to any known kinase fold. We show that WSTF phosphorylates Tyr 142 of H2A.X, and that WSTF activity has an important role in regulating several events that are critical for the DNA damage response. Our work demonstrates a new mechanism that regulates the DNA damage response and expands our knowledge of domains that contain intrinsic tyrosine kinase activity.

Figure 1. Y142 of H2A.X is a novel phosphorylation mark regulated by DNA damage signals

(a) Comparison of the extreme C-terminal sequence of H2A.X demonstrates that Tyr142 is conserved in metazoans (mammals, frogs, and fruit flies) but not in unicellular eukaryotes. (b) Primary MEF cells were treated with 10 Gy of ionizing radiation (IR) and recovered for a period of time as indicated. Acid-extracted histones were separated by SDS-PAGE and subjected to immunoblotting. H2A.X Y142 phosphorylation levels in MEFs gradually decline, reaching minimum at 8 hr. γ-H2A.X signal is initiated upon damage and maintained up to 16 hours post IR.

Figure 2. The WSTF-SNF2H chromatin remodeling complex is specifically associated with H2A.X nucleosomes in vivo

(a) Purification scheme of H2A.X-containing mononucleosomes. MEFs reconstituted with Flag-H2A.X (WT or Y142F) were treated with or without IR. (b) Two polypeptides migrating at 145 and 171 KDa were associated with undamaged WT (left), but not the Y142F mutant (right) H2A.X-mononucleosomes in a silver-stained gel. Mass spectrometry (MS) analyses revealed these polypeptides as WSTF (171KDa) and SNF2H (145 KDa). The third band at 60 KDa was identified as β–actin in the MS analyses. Staining and MS of the lower molecular weight bands (<20 KDa) revealed similar levels of H2A.X and other core histones. (c) The association between the WICH complex and undamaged WT H2A.X mononucleosomes was confirmed by IP-western experiments. (d) In WSTF RNAi cells, the expression level of WSTF was significantly diminished; and H2A.X Y142 phosphorylation level was significantly reduced.

Figure 3. WSTF contains a novel kinase domain that phosphorylates Y142 of H2A.X

(a) Schematic demonstration of the domain architecture of the human WSTF protein and a series of recombinant proteins representing portions of WSTF. (b)-(f) recombinant WSTF proteins were generated in insect cells; and the N- and C- motifs (g) were generated in E. Coli. (b) Y142 in the nucleosome is phosphorylated by recombinant full length WSTF protein. (c) The specific phosphorylation of H2A.X at Y142 was detected by immunoblotting with Y142ph antibodies. In (d)-(g), free H2A.X proteins were used as substrates in in vitro kinase assays. (d) N-WSTF, but not C-WSTF, had kinase activity. (e) The WSTF 1-340 construct had much reduced kinase activity (<50 fold) in comparison to the other constructs. (f) The C338A point mutant (derived from WT 1-359) had much reduced kinase activity. This mutation does not change the global folding properties of the kinase domain (Supplementary Figure S6). (g) The co-expressed N-motif and C-motif had significant kinase activity towards H2A.X, while the N-motif alone had a minimal kinase activity. Note: The C-motif construct, when expressed alone, was unstable and partially degraded from its C-terminus, however, it was protected when co-expressed with the N-motif.

Figure 3. WSTF contains a novel kinase domain that phosphorylates Y142 of H2A.X

(a) Schematic demonstration of the domain architecture of the human WSTF protein and a series of recombinant proteins representing portions of WSTF. (b)-(f) recombinant WSTF proteins were generated in insect cells; and the N- and C- motifs (g) were generated in E. Coli. (b) Y142 in the nucleosome is phosphorylated by recombinant full length WSTF protein. (c) The specific phosphorylation of H2A.X at Y142 was detected by immunoblotting with Y142ph antibodies. In (d)-(g), free H2A.X proteins were used as substrates in in vitro kinase assays. (d) N-WSTF, but not C-WSTF, had kinase activity. (e) The WSTF 1-340 construct had much reduced kinase activity (<50 fold) in comparison to the other constructs. (f) The C338A point mutant (derived from WT 1-359) had much reduced kinase activity. This mutation does not change the global folding properties of the kinase domain (Supplementary Figure S6). (g) The co-expressed N-motif and C-motif had significant kinase activity towards H2A.X, while the N-motif alone had a minimal kinase activity. Note: The C-motif construct, when expressed alone, was unstable and partially degraded from its C-terminus, however, it was protected when co-expressed with the N-motif.

Figure 4. WSTF is critical for the maintenance of γ-H2A.X phosphorylation after DNA damage

(a) γ-H2A.X maintenance is defective in WSTF RNAi cells after DNA damage treatment. The time points labeled indicate the recovery time following the IR treatment. (b) Immunofluorescent staining experiments were performed on control and WSTF RNAi cells fixed at different time points (as labeled) following 10 Gy of IR treatment. (c & d) Mdc1 and phos-ATM recruitment are also defective in WSTF deficient cells. Immunofluorescent staining experiments were performed with α-ATM S1981 phos and α-Mdc1 antibodies on control or WSTF RNAi cells, 8-hour post 10 Gy of IR treatment. (e) WSTF RNAi cells were complemented with the WT or mutant (C338A) kinase domain constructs (1-359, Myc epitope-tagged at N-terminus) of WSTF. Eight hours after treated with 10 Gy of IR, cells were fixed and co-stained with γ-H2A.X (green) and α-myc antibodies (red). (f) The kinase activity of WSTF (red) is also critical for phos-ATM foci maintenance (green).

Figure 4. WSTF is critical for the maintenance of γ-H2A.X phosphorylation after DNA damage

(a) γ-H2A.X maintenance is defective in WSTF RNAi cells after DNA damage treatment. The time points labeled indicate the recovery time following the IR treatment. (b) Immunofluorescent staining experiments were performed on control and WSTF RNAi cells fixed at different time points (as labeled) following 10 Gy of IR treatment. (c & d) Mdc1 and phos-ATM recruitment are also defective in WSTF deficient cells. Immunofluorescent staining experiments were performed with α-ATM S1981 phos and α-Mdc1 antibodies on control or WSTF RNAi cells, 8-hour post 10 Gy of IR treatment. (e) WSTF RNAi cells were complemented with the WT or mutant (C338A) kinase domain constructs (1-359, Myc epitope-tagged at N-terminus) of WSTF. Eight hours after treated with 10 Gy of IR, cells were fixed and co-stained with γ-H2A.X (green) and α-myc antibodies (red). (f) The kinase activity of WSTF (red) is also critical for phos-ATM foci maintenance (green).

Figure 4. WSTF is critical for the maintenance of γ-H2A.X phosphorylation after DNA damage

(a) γ-H2A.X maintenance is defective in WSTF RNAi cells after DNA damage treatment. The time points labeled indicate the recovery time following the IR treatment. (b) Immunofluorescent staining experiments were performed on control and WSTF RNAi cells fixed at different time points (as labeled) following 10 Gy of IR treatment. (c & d) Mdc1 and phos-ATM recruitment are also defective in WSTF deficient cells. Immunofluorescent staining experiments were performed with α-ATM S1981 phos and α-Mdc1 antibodies on control or WSTF RNAi cells, 8-hour post 10 Gy of IR treatment. (e) WSTF RNAi cells were complemented with the WT or mutant (C338A) kinase domain constructs (1-359, Myc epitope-tagged at N-terminus) of WSTF. Eight hours after treated with 10 Gy of IR, cells were fixed and co-stained with γ-H2A.X (green) and α-myc antibodies (red). (f) The kinase activity of WSTF (red) is also critical for phos-ATM foci maintenance (green).