Journal: Nucleic Acids Research

Article Title: ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

doi: 10.1093/nar/gkv957

Figure Lengend Snippet: Replication-associated activation of ATM by FA. ( A ) FA-induced phospho-ATM and phospho-CHK2 localized to EdU-incorporating cells. H460 were treated for 3 h with 300 μM FA. ( B ) Absence of phospho-ATM and phospho-CHK2 immunostaining in H460 cells with shRNA-depleted ATM. ( C ) S-phase specificity of FA-induced phospho-CHK2 in H460 and ( D ) IMR90 cells (300 μM FA, 3 h). Labels: p-CHK2—percentage of cells displaying immunostaining for p-CHK2, p-CHK2/EdU+—percentage of cells that showed both p-CHK2 and EdU staining. Data are Means ± SD for three experiments with each scoring >100 cells. ( E ) Lack of ATM and CHK2 phosphorylation by FA in growth-arrested IMR90 cells. Data for cycling and confluent cells are from the same blots after removal of intervening lanes. ( F ) Inhibition of FA-induced ATM signaling in IMR90 cells by aphidicolin (2 μM, added 60 min before FA exposure for 1.5 h). ( G ) Independence of ATM signaling on the ATR kinase in IMR90 cells. ATRi1 (3 mM caffeine) was added for 1 h before FA exposure for 3 h. (H) Normal ATM and KAP1 phosphorylation in H460 cells in the presence of the ATR inhibitor VE821 (ATRi2, 10 μM). Cells were preincubated for 1 h with ATRi2 and then treated with FA for 3 h. ( I ) ATM phosphorylation in IMR90 and Seckel syndrome fibroblasts treated for 3 h with FA. ( J ) ATR inhibitors did not restore ATM activation by FA in the presence of aphidicolin (ATRi1 - 3 mM caffeine, ATRi2 - 10 μM VE821, ATRi3 - 0.5 μM AZ20). Cells were preincubated with 2 μM aphidicolin and ATR inhibitors for 1 h before FA exposure for 2 h.

Article Snippet: Primary antibodies used were pT68-CHK2 (2661), CHK2 (2662), pS317-CHK1 (2344) and pS15-p53 (9284) from Cell Signaling; pS1981-ATM (ab81292), pT21-RPA (ab109394) and KAT5 (ab23886) from Abcam; pS824-KAP1 (A300–767A) and pS4/8-RPA (A300–245A) from Bethyl; γ-tubulin (T6557) from Sigma; ATM (sc-23921), cyclin A (H-432) and lamin B (sc-6216) from Santa Cruz; MRE11 (611366) and MSH2 (556349) from BD Biosciences; RPA32 (NA19L) from Calbiochem; AcK (acetyl-lysine antibody; 05–515) and γ-H2AX (05–636) from Millipore.

Techniques: Activation Assay, Immunostaining, shRNA, Staining, Phospho-proteomics, Inhibition

Journal: Nucleic Acids Research

Article Title: ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

doi: 10.1093/nar/gkv957

Figure Lengend Snippet: ATM signaling by FA is unrelated to DSB or replication stress. IMR90 cells were treated with FA and other stressors for 3 h. ( A ) MRE11 depletion did not affect ATM signaling by FA. ( B ) Inhibition of bleomycin-induced ATM autophosphorylation by MRE11 depletion (5 μg/ml bleomycin, 20 min). ( C ) Detection of DSB by PFGE in cells treated with FA, HU or bleomycin (Bleo, 30 μg/ml). ( D ) Protein phosphorylation in IMR90 treated with FA and bleomycin (Bleo, 30 μg/ml). ( E ) Amount of chromatin-bound ATM in cells treated with 200 μM FA or 2 μM CPT. ( F ) RPA32-S4/8 phosphorylation in cells treated with bleomycin (Bleo, 30 μg/ml) or FA. ( G ) RPA32-T21 phosphorylation in IMR90 treated with FA, HU, CPT (1 μM) and bleomycin (Bleo, 30 μg/ml). pp-RPA32—hyperphosphorylated form of RPA32. ( H ) Effect of the proteasome inhibitor MG132 on ATM, KAP1 and p53 phosphorylation by FA.

Article Snippet: Primary antibodies used were pT68-CHK2 (2661), CHK2 (2662), pS317-CHK1 (2344) and pS15-p53 (9284) from Cell Signaling; pS1981-ATM (ab81292), pT21-RPA (ab109394) and KAT5 (ab23886) from Abcam; pS824-KAP1 (A300–767A) and pS4/8-RPA (A300–245A) from Bethyl; γ-tubulin (T6557) from Sigma; ATM (sc-23921), cyclin A (H-432) and lamin B (sc-6216) from Santa Cruz; MRE11 (611366) and MSH2 (556349) from BD Biosciences; RPA32 (NA19L) from Calbiochem; AcK (acetyl-lysine antibody; 05–515) and γ-H2AX (05–636) from Millipore.

Techniques: Inhibition, Phospho-proteomics

Journal: BMC cancer

Article Title: Functional deficiency of NBN, the Nijmegen breakage syndrome protein, in a p.R215W mutant breast cancer cell line.

doi: 10.1186/1471-2407-14-434

Figure Lengend Snippet: Figure 2 Assessment of cellular radiosensitivity in the colony formation assay. A. Cellular radiosensitivity of p.R215W mutant cells (HCC1395) compared with wild type breast epithelial cells (MCF10A) as measured by the colony formation assay after irradiation at doses of 0, 0.1, 0.25, 0.5 or 1Gy. The surviving fraction is presented as the mean value with SEM from at least 3 independent experiments. B. Induction of PARP1 cleavage after irradiation with 2 Gy in p.R215W mutant cells (HCC1395) compared with wild type breast epithelial cells (MCF10A) as measured by immunoblotting of cleaved PARP1 (89 kDa) and total PARP1 (116 kDa). C. Immunoblot analysis of radiation-induced ATM signalling in HCC1395 cells compared with MCF10A. Cells were untreated or irradiated with 0.5, 1, 2, 4 or 6 Gy as indicated. Protein extracts were prepared 30 min after irradiation and were analysed through Western blotting for their immunoreactivity towards the phosphorylated forms of SMC1 (pSer966, top panel), KAP1 (p824, middle panel), and CHEK2 (pSer19, bottom panel). β-actin served as the loading control in each experiment.

Article Snippet: Antibodies to NBN, SMC1pS966, KAP1-pS824 were obtained from Novus Biologicals (rabbit polyclonal); anti CHEK2-pS19 from Cell Signaling (rabbit polyclonal); anti RAD50 (mouse monoclonal) from Abcam; anti MRE11 (mouse monoclonal 12D7) from GeneTex; PARP1 and cleaved PARP1 (rabbit polyclonal) from Cell Signaling, and anti β-Actin (mouse monoclonal) from Sigma.

Techniques: Colony Assay, Mutagenesis, Irradiation, Western Blot, Control

Journal: The EMBO Journal

Article Title: Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

doi: 10.1038/emboj.2012.86

Figure Lengend Snippet: PP4-mediated dephosphorylation of KAP-1 on S824 impacts its role in the DNA damage response. (A) PP4-mediated regulation of KAP-1 impacts the expression of Gadd45α and p21. HeLa cells were transfected with PP4R3β siRNA, or endogenous KAP-1 was replaced with WT, mutant forms A (KAP1-S824A) or D (KAP1-S824D). After IR, cells were harvested and RNA purified at indicated times and quantitative real-time PCR (qRT–PCR) was performed. Data represent average and s.d. of four independent experiments. (B, C) PP4R3β depletion and KAP1 phosphomimetic mutant (S824D) prolong damage-induced chromatin relaxation. HeLa cells were transfected with control or PP4R3β siRNA, or endogenous KAP-1 was replaced with WT or KAP1-S824D mutant (D). Cells were irradiated and harvested at indicated times. Nuclei were purified, treated with micrococcal nuclease; DNA isolated and analyzed as described in experimental procedures. The relative intensity of each nucleosomic form (mono, bi, tri or poly) is expressed as the percentage of the total signal (for a given lane). Bar graphs represent average and s.d. of three independent experiments. The KAP-1 replacement has been shown in the immunoblot with endogenous and Myc-tagged KAP-1 indicated. (D) The ‘return' of CHD3 to sites of slow-repairing DSBs following ATMi addition is significantly attenuated in PP4R3β-depleted cells. Primary human fibroblasts were transfected with either control or PP4R3β siRNA. After 72 h, cells were irradiated with 8-Gy IR. At 24 h post IR, cells were treated with ATMi and harvested 10, 30, 60 and 120 min later. To examine the retention of CHD3 at sites of late-repairing damage, cells were pre-extracted with PBS containing 0.1% (v/v) Triton × 100 for 30 s before being fixed and immunostained for CHD3, γH2AX and DAPI (left panel). The signal intensity of CHD3 at regions of γH2AX foci (as determined by computer analysis) was measured (∼200 foci per sample). The data represent the mean and s.d. of multiple experiments. siRNA-mediated knock-down efficiency was independently verified for each experiment and was >80% (of cells with good knock-down) (right panel). It shows representative images at 60 min time point after ATMi addition from quantified data. In the zoomed-in and three-dimensional plotted images, note the relative difference in red–green overlap (yellow signal), indicative of changes in CHD3 abundance at sites of ongoing DSB repair, following the addition of ATMi for 1 h.

Article Snippet: Antibodies used were against KAP-1 (BD Transduction Laboratories), pS824-KAP-1 (Bethyl), pS473-KAP-1 (BioLegend), 53BP1 (Cell signaling), CHD3 (Bethyl), CHD4 (Bethyl), RPA2 (Cell Signaling), PP4R1 (Bethyl), PP4R2 (Bethyl), PP4R3α (Bethyl), PP4R3β (Bethyl), PP4C (Bethyl), PP1α (Bethyl), PP1β (Bethyl), CHK1 (Cell Signaling), CHK2 (Cell Signaling), Flag-tag (Sigma), α-tubulin (Sigma) and Phoshphoserine-agarose conjugate (Abcam).

Techniques: De-Phosphorylation Assay, Expressing, Transfection, Mutagenesis, Purification, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Irradiation, Isolation, Western Blot

Journal: The EMBO Journal

Article Title: Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

doi: 10.1038/emboj.2012.86

Figure Lengend Snippet: PP4C influences the phosphorylation status of multiple DDR proteins. (A) Schematic for phosphoproteomics-based identification of putative PP4C substrates. (B) Identification of regulated phosphorylation in response to PP4C depletion. The geometric mean of iTRAQ spectral peak height ratios for replicate samples was plotted as a function of the sum of the geometric means of all iTRAQ spectral peak heights. Maximum approximate conditional likelihood (MACL) was used to determine an intensity-based variance function from which the 95% acceptance region was calculated (grey curve and Supplementary Table 1). Several DDR proteins, including KAP1, CHD4 and TP53BP1, exhibited hyperphosphorylation in response to deplection of PP4C. (C) Validation of the PP4 targets. (Left panel) HeLa S3 cells transfected with PP4C or scrambled siRNAs were exposed to IR, lysed after 2 h and immunoprecipitated (IP) using a pan-phosphoSer antibody and probed for DDR proteins as indicated. The relative band intensities are provided below each immunoblot. (Right panel) Proteins identified as putative PP4 substrates were confirmed using Phos–tag. HeLa S3 cells were transfected with PP4C siRNA and irradiated with 10-Gy IR. Lysates were subjected to SDS–PAGE containing 20 μM Phos–tag and immunobloted with indicated antibodies. Lysates treated with λ protein phosphatase (λPP) served as control for the Phos–tag-induced mobility shift.

Article Snippet: Antibodies used were against KAP-1 (BD Transduction Laboratories), pS824-KAP-1 (Bethyl), pS473-KAP-1 (BioLegend), 53BP1 (Cell signaling), CHD3 (Bethyl), CHD4 (Bethyl), RPA2 (Cell Signaling), PP4R1 (Bethyl), PP4R2 (Bethyl), PP4R3α (Bethyl), PP4R3β (Bethyl), PP4C (Bethyl), PP1α (Bethyl), PP1β (Bethyl), CHK1 (Cell Signaling), CHK2 (Cell Signaling), Flag-tag (Sigma), α-tubulin (Sigma) and Phoshphoserine-agarose conjugate (Abcam).

Techniques: Transfection, Immunoprecipitation, Western Blot, Irradiation, SDS Page, Mobility Shift

Journal: The EMBO Journal

Article Title: Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

doi: 10.1038/emboj.2012.86

Figure Lengend Snippet: IR-induced phosphorylation of KAP1 on S473 is regulated by a combination of ATM/CHK2 and PP4C/R3β. (A) Alignment of region surrounding S473 of KAP1 across different organisms. Sequence alignment was performed with ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2) and consensus sequence is shown in color-shaded boxes (Upper panel). Phosphorylation of KAP-1 on S473 after IR is CHK2-mediated. HeLa cells after transfection with CHK1 or CHK2 siRNAs were irradiated after 3 days and harvested at indicated times (lower panel). Immunoblotting was performed with the indicated antibodies. (B) HeLa cells transfected with siRNAs for PP4C or PP4R3β were irradiated and harvested at the indicated times and pS473-KAP-1 was assessed by immunoblot using phospho-KAP-1 antibody (phosphoSerine 473). (C) PP4R3β depletion attenuates pS473-KAP-1 turnover after IR independent of Chk2 or ATM activity. HeLa cells transfected with siRNAs were irradiated after 3 days. At 0.5 h post IR, Chk2i (left panel) and ATMi (right panel) were added. Cells were harvested at the indicated times and immunoblotting was performed with p-KAP-1 antibody (S473). Figure source data can be found with the Supplementary data.

Article Snippet: Antibodies used were against KAP-1 (BD Transduction Laboratories), pS824-KAP-1 (Bethyl), pS473-KAP-1 (BioLegend), 53BP1 (Cell signaling), CHD3 (Bethyl), CHD4 (Bethyl), RPA2 (Cell Signaling), PP4R1 (Bethyl), PP4R2 (Bethyl), PP4R3α (Bethyl), PP4R3β (Bethyl), PP4C (Bethyl), PP1α (Bethyl), PP1β (Bethyl), CHK1 (Cell Signaling), CHK2 (Cell Signaling), Flag-tag (Sigma), α-tubulin (Sigma) and Phoshphoserine-agarose conjugate (Abcam).

Techniques: Sequencing, Transfection, Irradiation, Western Blot, Activity Assay

Journal: The EMBO Journal

Article Title: Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

doi: 10.1038/emboj.2012.86

Figure Lengend Snippet: KAP-1 phosphonull mutants reverse the phenotype induced by silencing PP4R3β. (A) In cells expressing phosphonull KAP-1 mutants, PP4 depletion does not affect the expression of Gadd45α, p21. In HeLa cells, endogenous KAP-1 was replaced with WT, mutant forms A (S824A or/and S473A), and either control siRNA or PP4R3β siRNA was transfected. After IR, cells were harvested and RNA purified at indicated times and quantitative real-time PCR (qRT–PCR) was performed. Data represent average and s.d. of four independent experiments. (B) KAP-1 S473A mutant, not S824A, reverses G2/M checkpoint phenotype induced by PP4R3β depletion. The G2/M checkpoint assay was performed as described in Figure 6D after silencing PP4R3β with U2OS cells expressing wild-type or phosphonull KAP-1 mutants. The results from three independent experiments are graphically represented (WT/PP4R3β siRNA, P<0.041; S824A/PP4R3β siRNA, P<0.011; S473A/Ctrl siRNA, P<0.019; S473A/PP4R3β siRNA, P<0.009). (C) Working model. In response to IR, ATM directly phosphorylates KAP-1 at S824 and indirectly induces the phosphorylation at S473 via CHK2. Phosphorylation of both S824 and S473 regulates chromatin structure and the expression of KAP-1 target genes, whereas only S473 is involved in the checkpoint response. A PP4 complex dephosphorylates KAP-1 at S824 and at S473 impacting the role of KAP-1 in DDR.

Article Snippet: Antibodies used were against KAP-1 (BD Transduction Laboratories), pS824-KAP-1 (Bethyl), pS473-KAP-1 (BioLegend), 53BP1 (Cell signaling), CHD3 (Bethyl), CHD4 (Bethyl), RPA2 (Cell Signaling), PP4R1 (Bethyl), PP4R2 (Bethyl), PP4R3α (Bethyl), PP4R3β (Bethyl), PP4C (Bethyl), PP1α (Bethyl), PP1β (Bethyl), CHK1 (Cell Signaling), CHK2 (Cell Signaling), Flag-tag (Sigma), α-tubulin (Sigma) and Phoshphoserine-agarose conjugate (Abcam).

Techniques: Expressing, Mutagenesis, Transfection, Purification, Real-time Polymerase Chain Reaction, Quantitative RT-PCR