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The Dynamic Alterations of H2AX Complex during DNA Repair Detected by a Proteomic Approach Reveal the Critical Roles of Ca2+/Calmodulin in the Ionizing Radiation-induced Cell Cycle Arrest*S

      By using DNA nuclease digestion and a quantitative “dual tagging” proteomic approach that integrated mass spectrometry, stable isotope labeling, and affinity purification, we studied the histone H2AX-associating protein complex in chromatin in mammalian cells in response to ionizing radiation (IR). In the non-irradiated control cells, calmodulin (CaM) and the transcription elongation factor facilitates chromatin transcription (FACT) were associated with H2AX. Thirty minutes after exposing cells to IR the CaM and FACT complexes dissociated, whereas two DNA repair proteins, poly(ADP-ribose) polymerase-1 and DEAH box polypeptide 30 isoform 1, interacted with H2AX. Two hours and 30 min after exposure, none of the above proteins were in the complex. H2B, nucleophosmin/B23, and calreticulin were associated with H2AX in both non-irradiated and irradiated cells. The results suggest that the H2AX complex undergoes dynamic changes upon induction of DNA damage and during DNA repair. The genuine interactions between H2AX and H2B, nucleophosmin/B23, calreticulin, poly(ADP-ribose) polymerase-1, and CaM under each condition were validated by immunoprecipitation/Western blotting and mammalian two-hybrid assays. Because multiple Ca2+-binding proteins were found in the H2AX complex, the roles of Ca2+ were examined. The results indicate that Ca2+/CaM plays important roles in regulating IR-induced cell cycle arrest, possibly through mediating chromatin structure. The dataset presented here demonstrates that sensitive profiling of the dynamics of functional cellular protein-protein interactions can successfully lead to the dissection of important metabolic or signaling pathways.
      The basic structural unit of chromatin is the nucleosome, which consists of a histone octamer (two copies of each H2A, H2B, H3, and H4) around which is coiled 1.7 turns of 168-bp DNA stabilized by a fifth histone, H1. Controlled nuclease digestion results in a stable subnucleosomal “core particle,” which contains 146-bp DNA coiled around the histone octamer. Except for histone H4, each histone is in a family of subtypes. Of particular interest are the histone subtypes called “replacement histones.” Unlike the major histone subtypes, which are synthesized in S phase of the cell cycle, replacement histones are synthesized through the cell cycle and in terminally differentiated cells (
      • Bradbury E.M.
      • van Holde K.E.
      ). H2AX is such a replacement histone, which exists in all cell types, and accounts for 2–25% of the total cellular H2A (
      • Rogakou E.P.
      • Pilch D.R.
      • Orr A.H.
      • Ivanova V.S.
      • Bonner W.M.
      DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.
      ,
      • Bradbury E.M.
      Chromatin structure and dynamics: state-of-the-art.
      ). Accumulating evidence has shown that H2AX is a key factor in the repair of DNA double strand breaks (DSBs)
      The abbreviations used are: DSB, DNA double strand break; CaM, calmodulin; FACT, facilitates chromatin transcription; γ-H2AX, phosphorylated H2AX; IR, ionizing radiation; PARP-1, poly(ADP-ribose) polymerase-1; DMEM, Dulbecco’s modified Eagle’s medium; Gy, gray; BD, binding domain; AD, activation domain; GFP, green fluorescent protein.
      1The abbreviations used are: DSB, DNA double strand break; CaM, calmodulin; FACT, facilitates chromatin transcription; γ-H2AX, phosphorylated H2AX; IR, ionizing radiation; PARP-1, poly(ADP-ribose) polymerase-1; DMEM, Dulbecco’s modified Eagle’s medium; Gy, gray; BD, binding domain; AD, activation domain; GFP, green fluorescent protein.
      (
      • Paull T.T.
      • Rogakou E.P.
      • Yamazaki V.
      • Kirchgessner C.U.
      • Gellert M.
      • Bonner W.M.
      A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.
      ). Recent studies suggest that H2AX may be a major source of genome instability (
      • Bassing C.H.
      • Suh H.
      • Ferguson D.O.
      • Chua K.F.
      • Manis J.
      • Eckersdorff M.
      • Gleason M.
      • Bronson R.
      • Lee C.
      • Alt F.W.
      Histone H2AX: a dosage-dependent suppressor of oncogenic translocations and tumors.
      ). H2AX is rapidly phosphorylated (γ-H2AX) following exposure of cells to IR, which induces DSBs, and forms IR-induced foci at the damage sites (
      • Rogakou E.P.
      • Pilch D.R.
      • Orr A.H.
      • Ivanova V.S.
      • Bonner W.M.
      DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.
      ,
      • Paull T.T.
      • Rogakou E.P.
      • Yamazaki V.
      • Kirchgessner C.U.
      • Gellert M.
      • Bonner W.M.
      A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.
      ). It was proposed that γ-H2AX may serve as a docking site for other DNA damage repair/signaling proteins to bind in the vicinity of DNA lesions (
      • Celeste A.
      • Fernandez-Capetillo O.
      • Kruhlak M.J.
      • Pilch D.R.
      • Staudt D.W.
      • Lee A.
      • Bonner R.F.
      • Bonner W.M.
      • Nussenzweig A.
      Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks.
      ). However, despite major advances achieved primarily by immunofluorescence and immunochemical techniques, important questions regarding the role of H2AX in chromatin in DNA damage/repair remain to be elucidated. For example, what are the factors involved in the formation of H2AX IR-induced foci prior to and after the induction of the DSBs?
      Extraction of nuclear proteins from mammalian cells with buffers containing high salt and detergent has become a standard procedure. However, a recent study designed to profile RNA polymerase II-interacting proteins has failed to identify any known transcription or elongation factors and illustrates the limitations of the salt extraction approach in characterizing chromatin-related protein complexes (
      • Jeronimo C.
      • Langelier M.F.
      • Zeghouf M.
      • Cojocaru M.
      • Bergeron D.
      • Baali D.
      • Forget D.
      • Mnaimneh S.
      • Davierwala A.P.
      • Pootoolal J.
      • Chandy M.
      • Canadien V.
      • Beattie B.K.
      • Richards D.P.
      • Workman J.L.
      • Hughes T.R.
      • Greenblatt J.
      • Coulombe B.
      RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.
      ). To obtain H2AX-interacting proteins from insoluble chromatin, we used nuclease to digest chromatin. The advantage of this approach is that soluble intact protein complexes containing histone H2AX can be obtained without using high salt conditions.
      Due to the high sensitivity, high specificity, and the capability of high throughput, mass spectrometry has emerged as a powerful tool to efficiently and systematically identify proteins in biological samples (
      • Ranish J.A.
      • Yi E.C.
      • Leslie D.M.
      • Purvine S.O.
      • Goodlett D.R.
      • Eng J.
      • Aebersold R.
      The study of macromolecular complexes by quantitative proteomics.
      ,
      • Tian Q.
      • Feetham M.C.
      • Tao W.A.
      • He X.C.
      • Li L.
      • Aebersold R.
      • Hood L.
      Proteomic analysis identifies that 14-3-3ζ interacts with β-catenin and facilitates its activation by Akt.
      ). To identify the components of H2AX complexes, we used a mass spectrometry based dual-tagging proteomic approach (Fig. 1). The bait protein H2AX is epitope-tagged for affinity isolation of the complex (epitope tagging), and in parallel the whole proteome of the cells expressing the epitope-tagged H2AX at physiologically relevant levels is labeled with deuterium-labeled heavy amino acids (isotope tagging). In mass spectrometric measurements, the heavy amino acids incorporated in the cellular proteins provide “in-spectra” quantitative markers so that proteins in a complex with H2AX can be unambiguously identified after a single step affinity purification (
      • Wang T.
      • Gu S.
      • Ronni T.
      • Du Y.C.
      • Chen X.
      In vivo dual-tagging proteomic approach in studying signaling pathways in immune response.
      ). In the present study, by using DNA nuclease digestion of nuclei and the dual tagging strategy, we identified the proteins that associate with H2AX in chromatin in mammalian cells both before and after IR to monitor the dynamics of the complex during DNA repair.
      Figure thumbnail gr1
      Fig. 1Identification of H2AX-interacting proteins by a quantitative dual tagging proteomic approach.A, strategy to identify H2AX-interacting proteins. The control cells are cultured in regular DMEM (green dish, unlabeled), and the FLAG-H2AX cells are cultured in the Leu-d3-containing medium (red dish, labeled). Equal numbers of unlabeled and labeled cells are mixed, and their nuclei are isolated from the total lysate. After digestion and lysis of the nuclei, the resulting soluble nuclear proteins are affinity-purified with anti-FLAG beads. Proteins eluted from the beads are separated by SDS-PAGE and digested with trypsin, and the resulting peptides are analyzed by mass spectrometry. Whereas the ratios of the labeled:unlabeled peak intensity for the nonspecific binding proteins are around 1, the ratios for the proteins that specifically bind to H2AX are significantly larger than 1. B, representative MS spectra showing the nonspecific and specific binding to H2AX: panel a, nonspecific binding; panel b, specific binding.

      EXPERIMENTAL PROCEDURES

       Plasmids, Stable Cell Line, and Cell Culture—

      The coding sequence of H2AX (
      • Rogakou E.P.
      • Pilch D.R.
      • Orr A.H.
      • Ivanova V.S.
      • Bonner W.M.
      DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.
      ) was cloned into the BamHI and XhoI sites of a retroviral vector, pMIR-DFT, with a double FLAG tag at the N terminus. The plasmids containing the tag alone or FLAG-H2AX were transfected into 293T cells with the calcium phosphate method, and the cells were selected in DMEM supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, and 1.2 μg/ml puromycin. 293T is an SV40 T antigen-expressing variant of the 293 adenovirus-transformed human embryonic kidney cell line. The cell line offers many advantages such as high transfection yield, can be easily grown in suspension culture, and is widely used for various biological studies (
      • Tian Q.
      • Feetham M.C.
      • Tao W.A.
      • He X.C.
      • Li L.
      • Aebersold R.
      • Hood L.
      Proteomic analysis identifies that 14-3-3ζ interacts with β-catenin and facilitates its activation by Akt.
      ,
      • Bouwmeester T.
      • Bauch A.
      • Ruffner H.
      • Angrand P.O.
      • Bergamini G.
      • Croughton K.
      • Cruciat C.
      • Eberhard D.
      • Gagneur J.
      • Ghidelli S.
      • Hopf C.
      • Huhse B.
      • Mangano R.
      • Michon A.M.
      • Schirle M.
      • Schlegl J.
      • Schwab M.
      • Stein M.A.
      • Bauer A.
      • Casari G.
      • Drewes G.
      • Gavin A.C.
      • Jackson D.B.
      • Joberty G.
      • Neubauer G.
      • Rick J.
      • Kuster B.
      • Superti-Furga G.
      A physical and functional map of the human TNF-α/NF-κB signal transduction pathway.
      ). Whereas the cells containing the tag alone (control cells) were cultured in regular DMEM, the cells containing FLAG-tagged H2AX (FLAG-H2AX cells) were maintained in DMEM containing Leu-d3 to isotopically label the proteome (Fig. 1A).

       Irradiation of Cells, Nuclei Isolation, and Nuclease Digestion—

      Cells were irradiated with a γ-ray 137Cs source (Mark I model 68A) at 3.7 Gy/min. Approximately 8 × 108 FLAG-H2AX cells as well as the control cells were harvested and washed twice with cold PBS, and then equal numbers of each cell were mixed. The mixed cells were lysed in 5 packed cell pellet volumes of lysis buffer (10 mm Hepes-NaOH, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, and 0.5 mm β-mercaptoethanol) supplemented with protease inhibitor mixture (Roche Applied Science) and phosphatase inhibitors (1 mm sodium orthovanadate, 10 mm sodium fluoride, and 10 mm β-glycerophosphate). The nuclei were isolated by centrifuging the lysate at 1000 × g for 10 min at 4 °C and washed twice with 10 ml of digestion buffer (10 mm Tris-HCl, pH 8.0, 7.5 mm MgCl2, 0.75 mm CaCl2, and 0.5 mm β-mercaptoethanol) plus the protease and phosphatase inhibitors. The nuclei were then digested with 500 units/ml DNase I (Roche Applied Science) at 37 °C for 50 min, lysed in nuclei lysis buffer (10 mm Tris-HCl, pH 7.6, 5 mm EDTA, 0.05% Nonidet P-40, and 0.5 mm β-mercaptoethanol) plus the protease and phosphatase inhibitors, and cleared by centrifugation. Completion of the digestion was checked with a 2% agarose gel.

       Protein Purification—

      Following completion of the chromatin digestion, glycerol and NaCl were added to the cleared lysate to the final concentrations of 20% and 150 mm, respectively, and the nuclear extract was incubated with 200 μl of M2 anti-FLAG beads (Sigma) at 4 °C for 2 h. The beads were then washed four times with 4 ml of washing buffer (10 mm Tris-HCl, pH 7.6, 150 mm NaCl, 20% glycerol, 1 mm EDTA, 0.05% Nonidet P-40, and 0.5 mm β-mercaptoethanol) plus the protease and phosphatase inhibitors. The bound proteins were eluted with a buffer containing 250 μg/ml 3× FLAG peptide (Sigma). The eluted proteins were concentrated with trichloroacetic acid precipitation and then separated by 4–20% SDS-PAGE. After staining with Coomassie Brilliant Blue, the whole lane of gel was cut into 25 slices for LC-MS/MS analysis.

       LC-MS/MS Analysis and Database Searching—

      In-gel digestion and LC-MS/MS analysis were performed as described previously (
      • Gu S.
      • Liu Z.
      • Pan S.
      • Jiang Z.
      • Lu H.
      • Amit O.
      • Bradbury E.M.
      • Hu C.A.
      • Chen X.
      Global investigation of p53-induced apoptosis through quantitative proteomic profiling using comparative amino acid-coded tagging.
      ). The MS/MS data for each sample were searched against the NCBInr (January 20, 2004) protein sequence database downloaded from the National Center for Biotechnology Information under the species restriction of Homo sapiens using the in-house licensed Mascot searching program (version 2.0). The Leu-d3 modification was added in the configuration file and selected as variable modifications in the database searching. The parameters for database searching were as follows: (i) 0.2-Da mass error tolerance for both MS and MS/MS, (ii) tryptic enzyme specificity with a maximum of two missed cleavages, and (iii) the following variable modifications: acetylation at peptide N terminus, phosphorylation on tyrosine/serine/threonine, and oxidation on methionine.

       Immunoblotting Analysis—

      293T cells (7 × 107 cells/assay) were left untreated or irradiated with 30 Gy, recovered at 37 °C at the times indicated in each specific experiment, and harvested for nuclei isolation/digestion, immunoprecipitation, and Western blotting. The procedures for nuclei isolation/digestion and immunoprecipitation were essentially the same as described under “Irradiation of Cells, Nuclei Isolation, and Nuclease Digestion” and “Protein Purification” except that buffer volumes in each step were reduced ∼10-fold.

       Mammalian Two-hybrid Analysis—

      Vectors encoding Gal4 DNA-binding domain (BD) and transcription activation domain (AD) were from a mammalian two-hybrid assay kit (BD Biosciences). The Gal4 GFP reporter plasmid was kindly provided by Dr. Toshi Shioda (Massachusetts General Hospital Cancer Center). The coding sequences of H2AX and CaM were inserted into the BD and AD vectors, respectively, and the two constructs were co-transfected into 293T cells with the Gal4 GFP reporter plasmid (2 μg of each plasmid in a 60-mm plate). The negative control was performed by co-transfection of 293T cells with the Gal4 GFP reporter plasmid and the BD and AD constructs in which the BD and AD were fused with two proteins that do not interact. The expression of GFP was analyzed by both flow cytometry and Western blotting.

       Cell Cycle Analysis—

      The exponentially growing cells in DMEM were mock- or γ-ray-irradiated and immediately replated. For the Ca2+ treatments, CaCl2 was added to the culture medium to the indicated concentrations immediately after replating. After allowing cells to recover at 37 °C for the indicated period of time, the cells were harvested, washed, fixed, and stained with propidium iodide, and cellular fluorescence was measured by using a FACSCalibur flow cytometer (BD Biosciences). To detect the phosphorylated histone H3 in mitosis, cells were analyzed as described previously (
      • Xu B.
      • Kim S.
      • Kastan M.B.
      Involvement of Brca1 in S-phase and G2-phase checkpoints after ionizing irradiation.
      ), and 10,000 events were recorded for each measurement.

      RESULTS

       The Dual Tagging Proteomic Strategy for Protein Complex Identification—

      The schematic of our quantitative dual tagging proteomic approach for identifying proteins associated with H2AX is illustrated in Fig. 1A, and representative results are shown in Fig. 1B. The stable cells expressing tag alone and the stable cells expressing the FLAG-H2AX are grown in the “light” and “heavy” medium, respectively. After affinity purification of H2AX using the beads conjugated with anti-FLAG antibody and LC-MS/MS analysis of the bound proteins, all of the Leu-containing peptides appear as pairs in the MS spectrum with one set of peaks from the stable isotope-labeled cells and the other set of peaks from the unlabeled cells. The intensity ratios of the paired peaks reflect the binding profiles of the parent protein to the bait H2AX (Fig. 1).

       The Epitope-tagged H2AX Is Incorporated into Chromatin—

      To determine whether a tag added to the N terminus of H2AX would affect the correct packing and function of H2AX in the nucleosome, we first determined the extraction profiles with increasing concentrations of NaCl of (i) the FLAG-H2AX from the FLAG-H2AX cells and (ii) the endogenous H2AX from the parental 293T cells (Fig. 2A). The results showed that the FLAG-tagged and endogenous H2AX had similar extraction profiles. Both were resistant to NaCl extraction, suggesting that the tagged H2AX has tightly bound within chromatin. Then we digested nuclei with micrococcal nuclease, immunoprecipitated the digested nuclear extract with anti-FLAG beads, and analyzed the DNA fragment size released by the digestion (Fig. 2B). Whereas, as expected, no immunoprecipitates were found for the parental 293T cells, the immunoprecipitates from the FLAG-H2AX cells produced two DNA bands of approximate 150 and 300 bp, which corresponded to the DNA sizes of core particle and two closely adjacent core particles. This result indicates that the tagged H2AX is correctly packed into the core particle. Lastly we mock- and γ-ray-treated (30 Gy) the FLAG-H2AX cells and determined the phosphorylation states of both the tagged and endogenous H2AX using anti-γ-H2AX (Fig. 2C). As for its endogenous counterpart, the tagged H2AX was phosphorylated after IR, suggesting that it responds properly to the induction of DSBs. Furthermore the FLAG-H2AX was expressed at levels similar to those of its endogenous counterpart (Fig. 3), and the cells expressing the FLAG-H2AX showed a growth rate and morphology similar to those of the parental cells, indicating that the expression of the FLAG-tagged H2AX has no effect on the phenotype of the parental cells. Collectively the above results strongly suggest that the FLAG-tagged H2AX, like its endogenous counterpart, is correctly packaged into the nucleosome and functions properly in chromatin.
      Figure thumbnail gr2
      Fig. 2The FLAG-tagged H2AX is incorporated into chromatin.A, FLAG-tagged H2AX shows resistance to NaCl extraction similar to that of its endogenous counterpart. Parental 293T and the FLAG-H2AX cells were lysed in PBS plus 0.1% Triton X-100 and 0–2 m NaCl, and the total cell lysate was fractionated into supernatant and pellet by centrifugation. Proteins from each fraction were resolved by SDS-PAGE and detected by Western blotting (WB). The results showed that the FLAG-tagged and endogenous H2AX had similar extraction profiles, indicating that the FLAG-tagged H2AX has tightly bound within chromatin. B, FLAG-H2AX-containing mono- and dinucleosome core particles harbor the DNA fragments with expected sizes. Nuclei from parental 293T and the FLAG-H2AX cells (1.8 × 107 cells/each) were digested with micrococcal nuclease and immunoprecipitated (IP) with anti-FLAG beads. After extraction/precipitation, the DNA was separated by 2% agarose gel and visualized with ethidium bromide staining. The immunoprecipitates from FLAG-H2AX cells (lane 4) produced two DNA bands of approximately 150 and 300 bp, which corresponded to the expected DNA sizes of core particle and two closely adjacent core particles, indicating that the FLAG-tagged H2AX was correctly packed into the core particle. C, FLAG-tagged H2AX is phosphorylated similarly to its endogenous counterpart upon IR. Parental 293T and the FLAG-H2AX cells were mock- or γ-ray-irradiated and lysed in 2× sample buffer. The proteins were then resolved by SDS-PAGE and detected by Western blotting. The induction of phosphorylation of FLAG-tagged H2AX by irradiation (lane 2) is shown.
      Figure thumbnail gr3
      Fig. 3The FLAG-tagged H2AX is expressed comparably to its endogenous counterpart. Equal amounts of total protein (40 μg) from the parental 293T cells and the cells stably expressing the FLAG-tagged H2AX (FLAG-H2AX cells) were loaded onto a 12% SDS-polyacrylamide gel for Western blot analysis (WB). The bands around 15 and 25 kDa represent the endogenous and FLAG-tagged H2AX, respectively.

       The Assessments of Chromatin Digestion—

      We used nuclease DNase I to digest chromatin to obtain soluble intact H2AX complexes. In such an approach, complete digestion of chromatin is essential for the identification of proteins associated with H2AX directly. For this reason, we tested chromatin digestion conditions extensively. As shown in Fig. 4, when the partially digested chromatin was immunoprecipitated with anti-FLAG beads, Western blotting with anti-H2AX antibody exhibited two bands, one at approximate 14 kDa corresponding to the endogenous H2AX (Fig. 4B, compare lane 1 with lane 3) and another at around 23 kDa corresponding to the FLAG-tagged H2AX (Fig. 4B, compare lane 1 with lane 4). However, when the immunoprecipitation was performed on the completely digested chromatin, only one band around 23 kDa was shown (Fig. 4B, lane 2), corresponding to the FLAG-tagged H2AX (Fig. 4B, compare lane 2 with lane 4). We assume that the endogenous H2AX in the partially digested chromatin was not from the same nucleosome as the tagged H2AX because the two copies of H2A do not interact directly in the same nucleosome (
      • Luger K.
      • Mader A.W.
      • Richmond R.K.
      • Sargent D.F.
      • Richmond T.J.
      Crystal structure of the nucleosome core particle at 2.8 Å resolution.
      ). Further analysis showed that when the completely digested chromatin was used for immunoprecipitation, H2B could be detected (Fig. 4C) but not H3 or H4 (data not shown). Collectively these results strongly suggest that under the complete digestion conditions established in this study, no intact nucleosomes are present in the digested nuclear extract, and the H2AX-H2B dimer is the major stable form of H2AX in solution. Therefore in this study we refer to the H2AX-interacting proteins as those associated with H2AX directly or indirectly through H2B or other closely associated proteins.
      Figure thumbnail gr4
      Fig. 4The assessments of the experimental conditions for chromatin digestion. Nuclei were isolated from cells and digested with DNase I. FLAG-tagged H2AX and its associated proteins were then immunoprecipitated (IP) and visualized with Western blotting (WB). A, partial versus complete digestion of chromatin. After digestion of chromatin with DNase I, the resulting soluble nuclear extract was checked by 2% agarose gel stained with ethidium bromide. The difference between partial digestion (lane 1) and complete digestion (lane 2) with regard to the amount of DNA detected is shown. B, Western blot analyses of the partially and completely digested chromatin. Partially and completely digested chromatin from FLAG-H2AX cells was immunoprecipitated with anti-FLAG beads and Western blotted with the antibodies indicated (note that a weak band around 25 kDa in lane 3 was a contamination). The difference between partially digested chromatin (lane 1), which contained both FLAG-tagged H2AX and endogenous H2AX, and the completely digested chromatin (lane 2), which contained only FLAG-H2AX, is shown. The difference indicates that the partially digested chromatin contained intact polynucleosome core particles, whereas the completely digested chromatin did not (see text for details). C, H2B is complexed with H2AX after the complete digestion of chromatin. After complete digestion, immunoprecipitation with anti-FLAG beads was performed, and the immunoprecipitates were probed with anti-H2B.

       Identification of H2AX-interacting Proteins in the Non-irradiated Control Cells—

      We first characterized the H2AX complex isolated from the cells without IR exposure. Of the 79 proteins identified, 13 proteins were selectively enriched with the anti-FLAG beads by a factor of at least 1.5 (Table I). Reproducibility was assessed primarily according to the method described by Blagoev et al. (
      • Blagoev B.
      • Kratchmarova I.
      • Ong S.E.
      • Nielsen M.
      • Foster L.J.
      • Mann M.
      A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.
      ), that is by (i) determining the variability of ratios for different peptides of the same protein, (ii) determining the variability of ratios for different spectra of the same peptide across the eluted chromatographic profile, (iii) determining the variability of ratios of the same protein detected in different gel fractions (i.e. the same protein appeared in different gel bands; see Supplemental Table I), and (iv) performing independent runs for some of the fractions. The results from the above analyses led to a cutoff value of enrichment-fold of 1.5 for Table I and the first part (30 min section) of Table II and 1.3 for the second part (2.5 h section) of Table II. A smaller cutoff value was set for the second part of Table II (2.5 h section) due to these data being more uniform, which was reflected by smaller standard deviations (compare the standard deviations in the 2.5 h section in Table II with those in Table I and those in the 30 min section in Table II). H2B and H4 were among the histones identified. Because no intact nucleosomes were found in the digested nuclear extract (Fig. 4), the H4 in the H2AX complex probably resulted from free H4. The crystal structure of the nucleosome core particle shows that the histone octamer is composed of two copies of each H2A-H2B dimer and the (H3-H4)2 tetramer formed by H3-H3 “histone fold” interactions (
      • Luger K.
      • Mader A.W.
      • Richmond R.K.
      • Sargent D.F.
      • Richmond T.J.
      Crystal structure of the nucleosome core particle at 2.8 Å resolution.
      ). Each H2A-H2B dimer interacts with the tetramer through the H2B-H4 histone fold. Consistent with the direct interactions of H2A and H2B in the structure, the enrichment ratio for H2B was clearly different from the ratio for H4 (Table I) whose interaction is mediated through H2B (
      • Luger K.
      • Mader A.W.
      • Richmond R.K.
      • Sargent D.F.
      • Richmond T.J.
      Crystal structure of the nucleosome core particle at 2.8 Å resolution.
      ).
      Table IProteins identified to associate with H2AX from the cells without being irradiated
      NCBI accession no.Protein name
      Only the proteins with two or more peptides matched are listed.
      Enrichment-fold
      Fold of enrichment was calculated as the ratio of Leu-d3-labeled peptide to unlabeled peptide.
      S.D.
      Standard deviation was determined from multiple peptides.
      No. of peptides
      Histones
      gi|4504253Histone H2AX (bait)99.49.294
      gi|28173554Histone H2B2.260.392
      gi|4504301Histone H41.600.334
      Ca2+ binding/signaling
      gi|49037474Calmodulin7.21.727
      gi|4757900Calreticulin precursor1.760.236
      gi|825671Nucleophosmin/B231.540.093
      gi|6470150BiP protein1.720.2116
      Transcription elongation
      gi|4507241Structure-specific recognition protein 12.20.326
      gi|6005757Chromatin-specific transcription elongation factor1.790.264
      Others
      gi|5031755Heterogeneous nuclear ribonucleoprotein R1.610.034
      gi|337457Ribonucleoprotein La1.580.183
      gi|7705433HSPC0211.830.383
      gi|3183544Polyadenylate-binding protein 11.640.2913
      a Only the proteins with two or more peptides matched are listed.
      b Fold of enrichment was calculated as the ratio of Leu-d3-labeled peptide to unlabeled peptide.
      c Standard deviation was determined from multiple peptides.
      Table IIProteins identified to associate with H2AX from the cells irradiated with γ-ray and allowed to recover at 37 °C for 30 min and 2.5 h
      NCBI accession no.Protein name
      Only the proteins with two or more peptides matched are listed.
      Enrichment-fold
      Fold of enrichment was calculated as the ratio of Leu-d3-labeled peptide to unlabeled peptide.
      S.D.
      Standard deviation was determined from multiple peptides except for proteins H2B and H2BE from the cells recovered for 2.5 h whose enrichments were estimated from a single Leu-containing peptide. NA, not applicable.
      No. of peptides
      30 min
      Histones
      gi|4504253Histone H2AX (bait)97.429.64
      gi|28173554Histone H2B2.180.203
      Ca2+ binding/signaling
      gi|4757900Calreticulin precursor1.500.078
      gi|825671Nucleophosmin/B231.690.232
      gi|6470150BiP protein1.510.2011
      DNA repair
      gi|178152Poly(ADP-ribose) polymerase-11.600.122
      gi|20336294DEAH box polypeptide 30 isoform 11.510.236
      2.5 h
      Histones
      gi|4504253Histone H2AX (bait)102.217.44
      gi|28173554Histone H2B1.57NA3
      gi|4504263Histone H2B, member E1.36NA4
      gi|184086Histone H2B.11.370.014
      gi|4504301Histone H41.300.043
      Ca2+ binding/signaling
      gi|4757900Calreticulin precursor1.310.042
      gi|825671Nucleophosmin/B231.340.114
      Others
      gi|2135011DNA-binding protein A1.320.093
      gi|38683855FLJ20758 protein1.310.095
      gi|87528DnaK-type molecular chaperone HSPA5 precursor1.330.083
      a Only the proteins with two or more peptides matched are listed.
      b Fold of enrichment was calculated as the ratio of Leu-d3-labeled peptide to unlabeled peptide.
      c Standard deviation was determined from multiple peptides except for proteins H2B and H2BE from the cells recovered for 2.5 h whose enrichments were estimated from a single Leu-containing peptide. NA, not applicable.
      CaM is a Ca2+-dependent regulatory protein, and its target enzymes are among important regulators such as protein kinases and phosphatases. Calreticulin is also a Ca2+-binding protein. Although calreticulin is mainly located in the endoplasmic reticulum, it is also found in the nucleus and can bind to histones (
      • Nigam S.K.
      • Goldberg A.L.
      • Ho S.
      • Rohde M.F.
      • Bush K.T.
      • Sherman M.
      A set of endoplasmic reticulum proteins possessing properties of molecular chaperones includes Ca2+-binding proteins and members of the thioredoxin superfamily.
      ). BiP is a chaperone protein localized in the endoplasmic reticulum, and its function is highly regulated by Ca2+. It has been reported that BiP binds to denatured histones in vitro (
      • Nigam S.K.
      • Goldberg A.L.
      • Ho S.
      • Rohde M.F.
      • Bush K.T.
      • Sherman M.
      A set of endoplasmic reticulum proteins possessing properties of molecular chaperones includes Ca2+-binding proteins and members of the thioredoxin superfamily.
      ).
      The structure-specific recognition protein 1 and the chromatin-specific transcription elongation factor are the two components of the transcription elongation factor FACT. The two proteins form a stable heterodimer, which is required for transcription elongation when chromatin is used as a template (
      • Orphanides G.
      • Wu W.H.
      • Lane W.S.
      • Hampsey M.
      • Reinberg D.
      The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins.
      ). In vitro biochemical studies with purified proteins have shown that FACT interacts with the H2A-H2B dimer of the nucleosome and forms a FACT-H2A-H2B complex. It facilitates transcription elongation by displacing the H2A-H2B dimer from the nucleosome (
      • Belotserkovskaya R.
      • Oh S.
      • Bondarenko V.A.
      • Orphanides G.
      • Studitsky V.M.
      • Reinberg D.
      FACT facilitates transcription-dependent nucleosome alteration.
      ). In this study, both subunits of the FACT were identified, suggesting that FACT may play a crucial role in DNA damage repair or transcription in vivo.
      Nucleophosmin/B23 is a nuclear phosphoprotein and is more abundant in cancer cells than in normal resting cells. UV radiation triggers an immediate up-regulation of nucleophosmin/B23 expression in mammalian cells, suggesting that nucleophosmin/B23 may be involved in the acute response of cells to environmental stress (
      • Wu M.H.
      • Yung B.Y.
      UV stimulation of nucleophosmin/B23 expression is an immediate-early gene response induced by damaged DNA.
      ). It has been reported that nucleophosmin/B23 binds to histone proteins in HeLa cells (
      • Okuwaki M.
      • Iwamatsu A.
      • Tsujimoto M.
      • Nagata K.
      Identification of nucleophosmin/B23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus DNA complexed with viral basic core proteins.
      ).

       Identification of H2AX-interacting Proteins in the Irradiated Cells—

      Previous reports have shown that ∼30 min after the exposure of cells to IR, H2AX was strongly phosphorylated and formed IR-induced foci around the DSB site (
      • Paull T.T.
      • Rogakou E.P.
      • Yamazaki V.
      • Kirchgessner C.U.
      • Gellert M.
      • Bonner W.M.
      A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.
      ). We analyzed the H2AX protein complex to identify the factors associating with H2AX in this dynamic phase. Thirty minutes after IR, four proteins that were found to interact with H2AX from the non-irradiated cells, H2B, calreticulin precursor, nucleophosmin/B23, and BiP (Table I), were again identified to complex with H2AX (Table II). The repeated identification of these four proteins on one hand suggests that these proteins form stable complexes with H2AX and on the other hand confirms the reproducibility of our experimental approach. In addition to the four proteins, two proteins involved in DNA damage and repair, PARP-1 and the DEAH box polypeptide 30 isoform 1, were identified (Table II). Interestingly CaM and FACT, which were shown to be associated with H2AX in the non-irradiated cells (Table I), had dissociated from H2AX after IR. PARP-1 is the enzyme that uses NAD+ as a substrate to catalyze the transfer of ADP-ribose to a variety of nuclear protein acceptors. The most poly(ADP-ribosyl)ated proteins in vivo are PARP-1 itself and histones H1 and H2B (
      • Huletsky A.
      • de Murcia G.
      • Muller S.
      • Hengartner M.
      • Menard L.
      • Lamarre D.
      • Poirier G.G.
      The effect of poly (ADP-ribosyl)ation on native and H1-depleted chromatin. A role of poly (ADP-ribosyl)ation on core nucleosome structure.
      ). It has been shown that PARP-1 plays important roles in DNA damage repair and in transcription (
      • Satoh M.S.
      • Lindahl T.
      Role of poly (ADP-ribose) formation in DNA repair.
      ,
      • Tulin A.
      • Spradling A.
      Chromatin loosening by poly (ADP)-ribose polymerase (PARP) at Drosophila puff loci.
      ). Although histones are the preferred targets in vivo and in vitro (
      • Huletsky A.
      • de Murcia G.
      • Muller S.
      • Hengartner M.
      • Menard L.
      • Lamarre D.
      • Poirier G.G.
      The effect of poly (ADP-ribosyl)ation on native and H1-depleted chromatin. A role of poly (ADP-ribosyl)ation on core nucleosome structure.
      ), the stable histone-PARP-1 complex has not been reported. DEAH box polypeptide 30 isoform 1 is a member of the DEAH helicase family, which plays important roles in basal transcription, DNA repair, and chromosome transmission (
      • Hoeijmakers J.H.
      • Egly J.M.
      • Vermeulen W.
      TFIIH: a key component in multiple DNA transactions.
      ). A member of the DEAH helicase family has been shown to directly interact with the highly conserved C-terminal BRCT repeat of the tumor suppressor BRCA1, and the disruption of this interaction leads to defects in DNA repair and progression of breast and ovarian cancer (
      • Cantor S.B.
      • Bell D.W.
      • Ganesan S.
      • Kass E.M.
      • Drapkin R.
      • Grossman S.
      • Wahrer D.C.
      • Sgroi D.C.
      • Lane W.S.
      • Haber D.A.
      • Livingston D.M.
      BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function.
      ). BRCA1 was reported to co-localize with H2AX at DSB sites (
      • Paull T.T.
      • Rogakou E.P.
      • Yamazaki V.
      • Kirchgessner C.U.
      • Gellert M.
      • Bonner W.M.
      A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.
      ,
      • Stewart G.S.
      • Wang B.
      • Bignell C.R.
      • Taylor A.M.
      • Elledge S.J.
      MDC1 is a mediator of the mammalian DNA damage checkpoint.
      ). However, to our knowledge no physical interaction between BRCA1 and H2AX has been observed. The identified association between H2AX and DEAH box polypeptide 30 isoform 1 suggests that the DNA damage-induced co-localization of BRCA1 and H2AX may be mediated by members of the DEAH helicase family.
      The next step was to profile the H2AX complex when cells were irradiated and allowed to recover at 37 °C for a longer period of time, 2.5 h. As shown in Table II, histone H2B, calreticulin precursor, and nucleophosmin/B23 were reproducibly identified. At this time point, DNA repair proteins, PARP-1 and the DEAH box polypeptide 30 isoform 1, were found to be dissociated from H2AX. One interesting phenomenon was that two histone H2B isoforms, gi|4504263 and gi|184086, were identified to join the H2AX complex. The two H2B isoforms (gi|4504263 and gi|184086) are distinguished from the bulk H2B (gi|28173554) by peptide EIQTAVRLLLPGELAK. The two H2B isoforms (gi|4504263 and gi|184086) are distinguished from one another by two unique peptides, KESYSVYVYK for gi|4504263 and KESYSIYVYK for gi|184086 (Supplemental Table I).

       Immunochemical and Mammalian Two-hybrid Analyses Validate the Proteomic Data—

      In agreement with the proteomic data (Tables I and II), Western blotting showed that the two H2AX partners, nucleophosmin/B23 and calreticulin, were co-precipitated with H2AX under all three conditions (Fig. 5A). However, there were some inconsistencies between the proteomic and the Western data. The Western data showed that when cells were irradiated and allowed to recover for 2.5 h more nucleophosmin/B23 and less calreticulin were associated with H2AX than those under the other two conditions (Fig. 5A, compare lane 6 with lanes 2 and 4). Proteomic data showed that the two proteins had similar isotopic enrichment ratios under three different treatment conditions (Tables I and II). A possible explanation is that for the mass spectrometric analysis we had mixed the labeled and unlabeled protein lysates together before affinity purification, and some in vitro binding or exchange might have occurred between the unlabeled proteins and the labeled FLAG-H2AX protein complexes. There were two possible mechanisms: (i) the components of the H2AX complex, which were Leu-d3-labeled, might exchange with the unlabeled counterparts in the mixed protein lysate or (ii) the labeled H2AX protein complex might bind additional unlabeled components in addition to its existing ones (i.e. increasing the copy numbers of the components). Both reactions would lead to lower labeled-to-unlabeled ratios (i.e. the “Enrichment-fold” in Tables I and II) in the mass spectrometric analysis, which have been observed in this experiment (Tables I and II) and another report that used similar experimental strategy (
      • Blagoev B.
      • Kratchmarova I.
      • Ong S.E.
      • Nielsen M.
      • Foster L.J.
      • Mann M.
      A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.
      ). The fact that the proteins that specifically associate with bait protein have significantly higher labeled-to-unlabeled ratios than those for nonspecific bound proteins, which were observed in this study and the other report (
      • Blagoev B.
      • Kratchmarova I.
      • Ong S.E.
      • Nielsen M.
      • Foster L.J.
      • Mann M.
      A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.
      ), suggests that the unlabeled proteins cannot reach a copy number similar to the existing labeled components of the protein complex under defined experimental conditions. In short, the reason for the inconsistency between proteomic data and Western data is that the isotopic ratios in the mass spectrometric analysis may not reflect the stoichiometry profile of the protein complex as Western blotting indicates. We also tested for PARP-1, which the proteomic data had shown to be associated with H2AX when the cells were irradiated and allowed to recover for 30 min but not under the other two conditions (Tables I and II). The immunoprecipitation and Western blot analysis results (Fig. 5A) were consistent with the proteomic data. The H2AX-H2B interaction in the non-irradiated control cells has been demonstrated in Fig. 4C. To ensure that similar amounts of target protein H2AX were loaded for each treatment, the Western blot filter was also probed with anti-H2AX. As expected, the tagged H2AX was present in similar amounts for all three treatments (Fig. 5A, compare lanes 2, 4, and 6).
      Figure thumbnail gr5
      Fig. 5Immunochemical and mammalian two-hybrid analyses of the association of nucleophosmin/B23, calreticulin, PARP-1, and CaM with H2AX.A, immunochemical analyses. The mock- or γ-ray-irradiated cells were allowed to recover for 30 min as well as for 2.5 h. The nuclei were then completely digested with DNase I, and the resulting soluble proteins were immunoprecipitated (IP) with anti-FLAG beads. The immunoprecipitated proteins from untreated or irradiated cells were analyzed with Western blotting (WB). The co-immunoprecipitation of nucleophosmin/B23 and calreticulin with H2AX under three conditions and PARP-1 with H2AX under the second condition (i.e. cells were irradiated and allowed to recover for 30 min) is shown. B and C, mammalian two-hybrid analyses of the interaction between H2AX and CaM. B, the expression of reporter GFP was detected by flow cytometry: panel 1, non-transfected cells; panel 2, negative control; panel 3, testing for the interaction between H2AX and CaM. The percentage of fluorescent cells is indicated in each panel. C, the expression of GFP was detected by Western blotting. An equal amount of total protein (40 μg) was loaded for each lane. In both B and C, the induction of GFP expression by the H2AX-CaM interaction is indicated (panel 3 in B and lane 3 in C).
      We analyzed the interaction between H2AX and CaM with a mammalian two-hybrid system with the expression of the reporter GFP detected by flow cytometry and Western blotting (Fig. 5, B and C). There was no GFP expression in the non-transfected 293T cells (Fig. 5, B, lane 1, and C, lane 1). Co-transfection of 293T cells with the Gal4 GFP reporter plasmid and the constructs encoding fusion proteins Gal4 BD-H2AX and AD-CaM resulted in the expression of GFP in 13.6% of the total cells (Fig. 5, B, lane 3, and C, lane 3) relative to the negative control of 3.9% (Fig. 5, B, lane 2, and C, lane 2). These results suggest that H2AX interacts with CaM in mammalian cells in situ in agreement with our proteomic data (Table I).

       Cell Cycle Is Arrested at the G2 Phase after IR, and the Elevated Extracellular Ca2+ Partially Relieves the Arrest—

      After observing the dynamic alterations of the components of the H2AX complex upon IR, we investigated how chromatin structure and cell cycle checkpoint responded to IR. The results from both 4′,6-diamidino-2-phenylindole staining of the cellular DNA and the salt extraction analysis of nuclei showed that chromatin was condensed after cells were exposed to IR (Supplemental Fig. 1); this has also been observed in HeLa cells (
      • Nur-E-Kamal A.
      • Gross S.R.
      • Pan Z.
      • Balklava Z.
      • Ma J.
      • Liu L.F.
      Nuclear translocation of cytochrome c during apoptosis.
      ). We then examined the effects of IR on cell growth and division. Whereas the non-irradiated cells continued to grow from about 50% confluency to almost full confluency in 24 h, the irradiated cells stopped dividing and appeared larger than the non-irradiated cells, suggesting that the IR had induced cell cycle arrest (Supplemental Fig. 2). We then analyzed the cell cycle by flow cytometry. As shown in Fig. 6A, panel a, the non-irradiated cells exhibited a similar profile of heterogeneous log phase distribution in the 12-h period. When irradiated with 8 Gy of γ-ray, cells gradually accumulated in the G2/M phase (Fig. 6A, compare panel b with panel a), indicating IR-induced G2/M arrest. Because multiple Ca2+-binding/signaling proteins were identified to be involved in the dynamics of the H2AX complex during DNA repair (Tables I and II), we asked whether Ca2+ binding/signaling was involved in the IR-induced G2/M arrest. To test this speculation, we examined the effects of Ca2+ on cell cycle progression of the irradiated cells. As shown in Fig. 6A, panel c, when CaCl2 was added to the culture medium of the irradiated cells immediately after IR, to increase the Ca2+ concentration from 1.8 mm (the original Ca2+ concentration in culture medium) to 15 mm, the IR-induced accumulation of cells in the G2/M phase was significantly reduced (Fig. 6A, compare panel c with panel b). For control purposes, we also tested the effects of elevated Ca2+ concentration (15 mm) on non-irradiated cells. As shown in Fig. 6A, panel d, no obvious effects of Ca2+ on the cell phase distribution of the untreated cells were observed (Fig. 6A, compare panel d with panel a).
      Figure thumbnail gr6
      Fig. 6Ca2+ partially relieves the IR-induced G2 arrest.A, Ca2+ partially relieves the IR-induced G2 arrest. The cells in the exponential growth phase in DMEM were mock- or γ-ray-irradiated and immediately replated. For the Ca2+ treatments, CaCl2 was added to the culture medium to a final concentration of 15 mm immediately after replating. After allowing cells to recover for the indicated period of time, the cells were harvested for analysis by flow cytometry. Panel a, non-irradiated cells in normal medium (1.8 mm Ca2+); panel b, the irradiated cells in normal medium (1.8 mm Ca2+); panel c, the irradiated cells in the medium with elevated Ca2+ (15 mm); panel d, non-irradiated cells in the medium with elevated Ca2+ (15 mm). The effects of elevated extracellular Ca2+ concentration on restoring IR-induced G2/M arrest are indicated (compare panel c with panel b). B, Ca2+ enhances the G2-to-M transition of the irradiated cells. The cells were grown, irradiated, Ca2+-treated as in A, and allowed to recover for 30 min. The cells were then harvested and costained with propidium iodide and antibody to phospho-histone H3 (Ser-10) as described (
      • Xu B.
      • Kim S.
      • Kastan M.B.
      Involvement of Brca1 in S-phase and G2-phase checkpoints after ionizing irradiation.
      ). Panel a, non-irradiated cells in normal medium (1.8 mm Ca2+); panel b, the irradiated cells in normal medium (1.8 mm Ca2+); panel c, the irradiated cells in the medium with elevated Ca2+ (15 mm); panel d, non-irradiated cells in the medium with elevated Ca2+ (15 mm). It is shown that the IR-induced reduction in G2-to M transition (compare panel b with panel a) was restored by the elevated extracellular Ca2+ concentration (compare panel c with panel b).
      To further examine the effects of Ca2+ on cell cycle progression, we evaluated the transition of cells from the G2 to M phase (Fig. 6B). In Fig. 6B, propidium iodide was used to monitor the cellular DNA content (y axis), and the antibody to histone H3, which is phosphorylated exclusively during mitosis, was used to identify the mitotic cells from the G2 cells (x axis). Compared with the non-irradiated cells, 30 min after IR the transition from the G2 to M phase was reduced significantly in the irradiated cells (Fig. 6B, compare panel b with panel a). When CaCl2 was added to the culture medium of the irradiated cells, the IR-induced reduction in the G2-to-M transition was restored (Fig. 6B, compare panel c with panel b). We did not observe any significant effects of elevated extracellular Ca2+ concentration on the G2-to-M transition of the non-irradiated cells (Fig. 6B, compare panel d with panel a). Collectively these results suggest that Ca2+ is involved in the G2-to-M transition in mammalian cells, and IR-induced G2 arrest may involve Ca2+ binding/signaling.

       Ca2+ Promotes the Proliferation of the Irradiated Cells—

      We also examined the effects of Ca2+ on cell growth and division. The exponentially growing cells were irradiated (8 or 30 Gy), and equal numbers of cells were plated on 60-mm plates. CaCl2 was added to the culture medium to the indicated concentrations, and the cells were allowed to grow at 37 °C. After 2 days, viable cells were counted using trypan blue exclusion assay (Sigma). Fig. 7 shows that in contrast to the somewhat inhibitory effects of elevated extracellular Ca2+ concentration (over ∼5 mm) on non-irradiated cell numbers, increasing extracellular Ca2+ concentrations resulted in increased viable cell numbers for the irradiated cells, and the responses were dose-dependent.
      Figure thumbnail gr7
      Fig. 7Ca2+ promotes the division of the irradiated cells. The cells in the exponential growth phase were mock- or γ-ray-irradiated (8 or 30 Gy), and immediately equal numbers of cells were replated in 60-mm plates. CaCl2 was added to the culture medium to the indicated concentrations, and the cells were grown at 37 °C for 2 days. Viable cells were counted using trypan blue exclusion assay. Note that increasing extracellular Ca2+ concentration of the irradiated cells resulted in increased viable cell numbers.

      DISCUSSION

      By combining DNA nuclease digestion of chromatin, which releases intact protein complexes from the insoluble chromatin, and a quantitative dual tagging proteomic strategy, which identifies real time specific protein-protein interactions, we observed dynamic changes in the H2AX protein complex in response to the induction of DSBs. One noteworthy observation is that multiple Ca2+-binding/signaling proteins were found in the H2AX protein complexes (Table I). This result led us to examine whether Ca2+/CaM is involved in the IR-induced G2 arrest and cell proliferation. The results demonstrate that the elevated extracellular Ca2+ partially relieves the IR-induced G2 arrest (Fig. 6A), enhances the IR-affected G2-to-M transition (Fig. 6B), and promotes cell division of the irradiated cells (Fig. 7). It has been established that entry into mitosis requires the activation of the mitotic cyclin-dependent kinase Cdc2 through the dephosphorylation of phosphorylated Thr-14 and Tyr-15 by the mitosis-promoting phosphatase Cdc25c (
      • Rhind N.
      • Furnari B.
      • Russell P.
      Cdc2 tyrosine phosphorylation is required for the DNA damage checkpoint in fission yeast.
      ). In response to DNA damage, the checkpoint kinases Chk1 and Chk2 inactivate Cdc25c by phosphorylating Ser-216, leading to the G2 arrest (
      • Peng C.Y.
      • Graves P.R.
      • Thoma R.S.
      • Wu Z.
      • Shaw A.S.
      • Piwnica-Worms H.
      Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216.
      ). The results observed in this study (Fig. 6) suggest that Ca2+-dependent factors may also be involved in the IR-induced G2 checkpoint in addition to Chk1 and Chk2. CaM-dependent protein kinase II is a CaM-dependent protein kinase and can phosphorylate Cdc25c on Ser-216 in vitro (
      • Hutchins J.R.
      • Dikovskaya D.
      • Clarke P.R.
      Regulation of Cdc2/cyclin B activation in Xenopus egg extracts via inhibitory phosphorylation of Cdc25C phosphatase by Ca2+/calmodium-dependent kinase II.
      ,
      • Patel R.
      • Holt M.
      • Philipova R.
      • Moss S.
      • Schulman H.
      • Hidaka H.
      • Whitaker M.
      Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells.
      ). It was proposed that CaM-dependent protein kinase II controls the G2 checkpoint through the phosphorylation of Cdc25c on Ser-216 (
      • Hutchins J.R.
      • Dikovskaya D.
      • Clarke P.R.
      Regulation of Cdc2/cyclin B activation in Xenopus egg extracts via inhibitory phosphorylation of Cdc25C phosphatase by Ca2+/calmodium-dependent kinase II.
      ,
      • Patel R.
      • Holt M.
      • Philipova R.
      • Moss S.
      • Schulman H.
      • Hidaka H.
      • Whitaker M.
      Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells.
      ). However, the results in this study argue against this hypothesis because if CaM-dependent protein kinase II plays a major role in controlling the G2 checkpoint, it is difficult to explain the observation that the elevated extracellular Ca2+ promotes the G2-to-M transition in the irradiated cells (Fig. 6). Therefore, a mechanism other than CaM-dependent protein kinase II must be responsible for the Ca2+/CaM-dependent regulation of the cell cycle progression in mammalian cells.
      CaM is the primary intracellular Ca2+ receptor and is universally required for cell proliferation in eukaryotes (
      • Kahl C.R.
      • Means A.R.
      Regulation of cell cycle progression by calcium/calmodulin-dependent pathways.
      ). In this study, we found that CaM associated with H2AX in the non-irradiated cells but dissociated in the irradiated cells. In vitro biochemical studies have shown that CaM interacts with histones and forms a CaM-histone complex (
      • Iwasa Y.
      • Iwasa T.
      • Higashi K.
      • Matsui K.
      • Miyamoto E.
      Modulation by phosphorylation of interaction between calmodulin and histones.
      ,
      • Wolff D.J.
      • Ross J.M.
      • Thompson P.N.
      • Brostrom M.A.
      • Brostrom C.O.
      Interaction of calmodulin with histones. Alteration of histone dephosphorylation.
      ). The interaction between CaM and histones is both Ca2+- and charge density-dependent, and the removal of Ca2+ and phosphorylation of histones cause dissociation of the complex (
      • Iwasa Y.
      • Iwasa T.
      • Higashi K.
      • Matsui K.
      • Miyamoto E.
      Modulation by phosphorylation of interaction between calmodulin and histones.
      ). It has also been shown that the binding of CaM to histones dramatically promotes the dephosphorylation of histones by phosphatase (
      • Wolff D.J.
      • Ross J.M.
      • Thompson P.N.
      • Brostrom M.A.
      • Brostrom C.O.
      Interaction of calmodulin with histones. Alteration of histone dephosphorylation.
      ). The results in Fig. 6 suggest that IR induced the decreased level of intracellular Ca2+ or CaM. Consistent with this finding, it was reported that IR resulted in dramatic reductions in CaM expression in multiple cell lines (
      • Watson C.A.
      • Chang-Liu C.M.
      • Woloschak G.E.
      Modulation of calmodulin by UV and X-rays in primary human endothelial cell cultures.
      ). Perhaps the IR-induced rapid phosphorylation of H2AX (
      • Rogakou E.P.
      • Pilch D.R.
      • Orr A.H.
      • Ivanova V.S.
      • Bonner W.M.
      DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.
      ,
      • Paull T.T.
      • Rogakou E.P.
      • Yamazaki V.
      • Kirchgessner C.U.
      • Gellert M.
      • Bonner W.M.
      A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage.
      ) and the IR-repressed expression of CaM (
      • Watson C.A.
      • Chang-Liu C.M.
      • Woloschak G.E.
      Modulation of calmodulin by UV and X-rays in primary human endothelial cell cultures.
      ) contributed to the dissociation of the CaM-H2AX complex in vivo (Tables I and II). Because H2AX is a structural component of chromatin, it is possible that changes in H2AX complex structure and H2AX phosphorylation after IR result in chromatin condensation (Supplemental Fig. 1) (
      • Nur-E-Kamal A.
      • Gross S.R.
      • Pan Z.
      • Balklava Z.
      • Ma J.
      • Liu L.F.
      Nuclear translocation of cytochrome c during apoptosis.
      ). On the other hand, it was reported that H2AX−/− cells exposed to low doses of irradiation exhibited mild G2 arrest (
      • Celeste A.
      • Petersen S.
      • Romanienko P.J.
      • Fernandez-Capetillo O.
      • Chen H.T.
      • Sedelnikova O.A.
      • Reina-San-Martin B.
      • Coppola V.
      • Meffre E.
      • Difilippantonio M.J.
      • Redon C.
      • Pilch D.R.
      • Olaru A.
      • Eckhaus M.
      • Camerini-Otero R.D.
      • Tessarollo L.
      • Livak F.
      • Manova K.
      • Bonner W.M.
      • Nussenzweig M.C.
      • Nussenzweig A.
      Genomic instability in mice lacking histone H2AX.
      ,
      • Fernandez-Capetillo O.
      • Chen H.T.
      • Celeste A.
      • Ward I.
      • Romanienko P.J.
      • Morales J.C.
      • Naka K.
      • Xia Z.
      • Camerini-Otero R.D.
      • Motoyama N.
      • Carpenter P.B.
      • Bonner W.M.
      • Chen J.
      • Nussenzweig A.
      DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1.
      ). Furthermore most of the factors in the checkpoint signaling pathway were reported to be localized at the DNA damage sites in chromatin (
      • Zou L.
      • Cortez D.
      • Elledge S.J.
      Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin.
      ). Several lines of recent evidence support the hypothesis that chromatin structure plays crucial roles in DNA repair and checkpoint signaling (
      • Fernandez-Capetillo O.
      • Nussenzweig A.
      Linking histone deacetylation with the repair of DNA breaks.
      ,
      • Iizuka M.
      • Smith M.M.
      Functional consequences of histone modifications.
      ,
      • Jazayeri A.
      • McAinsh A.D.
      • Jackson S.P.
      Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair.
      ,
      • Morrison A.J.
      • Highland J.
      • Krogan N.J.
      • Arbel-Eden A.
      • Greenblatt J.F.
      • Haber J.E.
      • Shen X.
      INO80 and γ-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair.
      ,
      • Nakamura T.M.
      • Du L.L.
      • Redon C.
      • Russell P.
      Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast.
      ). We propose that in mammalian cells the repression of CaM expression and the phosphorylation of H2AX are important cellular responses to the induction of DSBs. These changes, along with other histone modifications and chromatin remodeling, may trigger the reconfiguration of chromatin structure and hence regulate cell cycle progression and DNA repair. According to this model, the elevated extracellular Ca2+ is required to compensate the IR-repressed level of intracellular CaM for the maintenance of chromatin structure (
      • Kahl C.R.
      • Means A.R.
      Regulation of cell cycle progression by calcium/calmodulin-dependent pathways.
      ).
      Multiple repair/signaling proteins are recruited to DSB sites (
      • Celeste A.
      • Fernandez-Capetillo O.
      • Kruhlak M.J.
      • Pilch D.R.
      • Staudt D.W.
      • Lee A.
      • Bonner R.F.
      • Bonner W.M.
      • Nussenzweig A.
      Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks.
      ). Among those factors, NBS1, 53BP1, and MDC1 have been shown to physically interact with γ-H2AX (
      • Stewart G.S.
      • Wang B.
      • Bignell C.R.
      • Taylor A.M.
      • Elledge S.J.
      MDC1 is a mediator of the mammalian DNA damage checkpoint.
      ,
      • Kobayashi J.
      • Tauchi H.
      • Sakamoto S.
      • Nakamura A.
      • Morishima K.
      • Matsuura S.
      • Kobayashi T.
      • Tamai K.
      • Tanimoto K.
      • Komatsu K.
      NBS1 localizes to γ-H2AX foci through interaction with the FHA/BRCT domain.
      ,
      • Ward I.M.
      • Minn K.
      • Jorda K.G.
      • Chen J.
      Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX.
      ). In yeast, an ATP-dependent chromatin-remodeling complex, INO80, was shown to interact with γ-H2AX (
      • Morrison A.J.
      • Highland J.
      • Krogan N.J.
      • Arbel-Eden A.
      • Greenblatt J.F.
      • Haber J.E.
      • Shen X.
      INO80 and γ-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair.
      ). The absence of those proteins in the list of proteins we identified here suggests that the use of more cells for complex purification and employment of higher sensitivity mass spectrometry techniques for protein detection may lead to identification of additional H2AX-interacting partners. It should be pointed out that previous relevant studies usually used many more cells than used in this study. For example, 4.25 × 1010 cells were used to purify a histone deacetylase-dependent corepressor complex (
      • Fleischer T.C.
      • Yun U.J.
      • Ayer D.E.
      Identification and characterization of three new components of the mSin3A corepressor complex.
      ) in comparison with 8 × 108 cells used in the present study.
      In the present study, to obtain soluble intact H2AX protein complexes, we used nuclease DNase I to cleave chromatin every 10 bp of DNA under mild conditions. To identify those proteins associated with intact nucleosomes, micrococcal nuclease can be used for the digestion. For the identification of the protein-protein interactions, many relevant studies used overexpression of the proteins of interest, which may affect the cell phenotypes. The tandem affinity purification approach was developed to isolate complexes in high purity, but it requires multiple steps of affinity purification to reduce contaminating proteins (
      • Rigaut G.
      • Shevchenko A.
      • Rutz B.
      • Wilm M.
      • Mann M.
      • Seraphin B.
      A generic protein purification method for protein complex characterization and proteome exploration.
      ,

      Deleted in proof

      ,

      Deleted in proof

      ). The repetitive washings often result in loss of weak or transient protein interactions of biological relevance. In our dual tagging approach for complex component profiling, a stable cell line is first established that expresses a tagged bait protein whose expression level is comparable to that of the endogenous counterpart. The technology then integrates the capabilities of natural complex formation, epitope affinity isolation, and in-spectra quantitative measurements to distinguish systematically those interacting proteins from a large nonspecific binding background. Sensitive and unambiguous identification of genuine components relies on the isotope ratio in MS spectra, not on the purity of the complexes through multistep purifications. This approach should be useful in other areas such as identification of transcription mediators/factors.
      In summary, using DNA nuclease digestion of chromatin and a quantitative dual tagging proteomic system we identified several proteins that have been shown to interact with members of the histone family, including H2B, CaM, two components of FACT, BiP, calreticulin, and nucleophosmin/B23. We also identified several novel binding partners of H2AX under the defined conditions. That is, we showed that DNA repair proteins PARP-1 and DEAH box polypeptide 30 isoform 1 interacted with H2AX when cells were exposed to γ-ray and allowed to recover for a relatively short period of time (e.g. 30 min). More importantly, we demonstrated that the H2AX protein complex undergoes dynamic changes upon induction of DNA damage and during DNA repair. Finally we characterized the biological functions for part of the identified proteins, i.e. the Ca2+-binding/signaling proteins. The results demonstrated that Ca2+/CaM played important roles in regulating IR-induced cell cycle arrest. Our data support the hypothesis that chromatin structure may play crucial roles in DNA repair and checkpoint signaling (
      • Fernandez-Capetillo O.
      • Nussenzweig A.
      Linking histone deacetylation with the repair of DNA breaks.
      ,
      • Iizuka M.
      • Smith M.M.
      Functional consequences of histone modifications.
      ,
      • Jazayeri A.
      • McAinsh A.D.
      • Jackson S.P.
      Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair.
      ,
      • Morrison A.J.
      • Highland J.
      • Krogan N.J.
      • Arbel-Eden A.
      • Greenblatt J.F.
      • Haber J.E.
      • Shen X.
      INO80 and γ-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair.
      ,
      • Nakamura T.M.
      • Du L.L.
      • Redon C.
      • Russell P.
      Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast.
      ).

      Acknowledgments

      We thank Claire K. Sanders for help with using the fluorescence microscope and Michael Harris for reading the manuscript.

      Supplementary Material

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      1. Deleted in proof

      2. Deleted in proof