Stable Transfectants and Cell Culture
The CMV-neo vector plasmids were constructed as reported (Huang et al., 1996
; Zhang et al., 2001a
). Mouse epidermal JB6 promotion-sensitive Cl41 and stable transfectants, CMV-neo mass (Cl41), dominant negative mutant (DNM) JNK1 (DNM-JNK1), p38 (DNM-p38β) and ERK2 (DNM-ERK2) were established as described (Zhang et al., 2001a
). Cells were cultured in Eagle’s minimum essential medium (MEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and 25 μg/ml gentamicin in a 37° C 5% CO2
incubator. Before each experiment, transfectants were selected with G418 and verified with their respective antibodies. JNK-wt and JNK2−/−
MEFs, ATM-wt and ATM−/−
MEFs, immortalized H2AX-wt and H2AX−/−
MEFs (passage 16-21, derived from C57/Bl6 mice) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented as above.
Treatment of Cells with UV
Cells were seeded in 10- or 15-cm dishes and cultured as above until 80% confluence. JB6 cells were treated for 1 h prior to UV with kinase inhibitors PD98059, SB202190, SP600125 or wortmannin (Calbiochem, La Jolla, CA). H2AX-wt and H2AX−/− MEFs were treated with Z-VAD or SP 600125 (Calbiochem, La Jolla, CA) 1 h before UV. To create JNK1/2 deficient cells, JNK1 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) was transfected into JNK2−/− cells to knock down JNK1 expression; JNK2−/− cells transfected with control siRNA served as controls. See Supplemental Experimental Procedures for description of UV sources.
Total Cellular Protein or Histone Extraction and Western Analysis
Cellular proteins were extracted after UV exposure by disrupting cells in lysis buffer (50 mM Tris-HCI, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM Na3
, 1 mM NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM PMSF). For histone extraction after UV exposure, cells were lysed in NETN buffer (150 mM NaCl, 1mM EDTA, 20 mM Tris, pH 8, 0.5% NP-40) and centrifuged 5 min. Histones were extracted from the pellets with 0.1 M HCl (Ward and Chen, 2001
). The protein samples were resolved by SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked at room temperature for 1 h with 5% nonfat milk in Tris-buffered saline containing Tween 20 (TBST). Primary antibodies to detect phosphorylated H2AX at Ser139 (γH2AX), H2A, H2AX (Upstate Biotechnology, Inc., Lake Placid, NY MA), caspase-3, phosphorylated JNK, total JNK, phosphorylated ERK1/2, total ERK1/2, phosphorylated p70 S6K (Thr421/424), total p70 S6K, phosphorylated ATF2 (Thr69/71), total ATF2, phosphorylated c-Jun (Ser63), total c-Jun, phosphorylated ATM (Ser1981), total ATM, phosphorylated ATR (Ser428), total ATR, β-actin (Cell Signaling Biotechnology, Inc., Beverly, MA) were incubated with membranes at 4 °C overnight. Membranes were incubated with the appropriate secondary antibody in 5% nonfat milk/TBST for 3h at 4 °C. Proteins were detected by enhanced chemiluminescence (ECL) (Amersham, Biosciences Corp., Piscataway, NJ).
Construction of Expression Vectors
Ser139 in H2AX was mutated to Ala (139A) to study phosphorylation (QuikChange II Site-Directed Mutagenesis Kit, Stratagene, La Lolla, CA). Mutated H2AX or H2AX-wt was inserted into pcDNA4 (Invitrogen, Carlsbad, CA) for transfection in HEK293 cells and H2AX−/− MEFs and pGEX-5X-1 vectors (Amersham Biosciences Corp) to generate GST-H2AX-wt and GST-H2AX-139A fusion proteins. H2AX was also introduced into the Bam HI/Xba I site of the pACT vector to generate pBIND-H2AX for the mammalian two-hybrid assay.
Immunoprecipitation and Chromatin Immunoprecipitation (ChIP)
For immunoprecipitation, JB6 cells were treated or not treated with UVA (80 kJ/m2) and then disrupted in lysis buffer as above but including 25 U/ml of benzonase (Novagen). Cell lysates were incubated with a JNK or pJNK antibody (Upstate Biotechnology, Inc.) at 4 °C overnight and protein A/G-Sepharose beads (Santa Cruz Biotechnology) for an additional 4 h. Duplicate blots were made from the same set of experiments. One blot was probed with an H2AX antibody (Upstate Biotechnology, Inc.) to detect H2AX in the JNK-H2AX immunocomplex and the other blot was probed with a JNK antibody (Upstate Biotechnology, Inc.). Proteins were revealed using ECL reagents (Amersham Biosciences Corp.). To investigate whether H2AX is phosphorylated, pcDNA3.1/JNK2 and pcDNA4/H2AX or pcDNA4/H2AX-139A plasmids were transfected into HEK 293 cells or H2AX−/− MEFs. After UVA, these cells were disrupted as above. H2AX or H2AX-139A was precipitated by anti-his (Santa Cruz Biotechnology) and phosphorylation of H2AX at Ser139 (γH2AX) was detected. The cell lysate was also used to detect the expression of JNK2 with anti-V5 (Invitrogen, Carlsbad, CA).
For chromatin immunoprecipitation (ChIP) experiments, JB6 cells were exposed to UVA (80 kJ/m2) and then processed according to the instructions from the manufacturer (Upstate Biotechnology, Inc.; see Supplemental Experimental Procedures). Immunoprecipitated histones and histone-bound protein complexes were analyzed by immunoblotting with JNK, pJNK, H2AX, or γH2AX antibodies.
To study co-localization of endogenous pJNK and H2AX or pJNK and γH2AX in vivo, two groups of JB6 cells were fixed in 30 % paraformaldehyde and permeabilized in 0.5% Triton X-100 30 min after UVA (80 kJ/m2). For group 1, fixed cells were incubated with pJNK mouse monoclonal (Upstate Biotechnology, Inc.) and H2AX rabbit polyclonal antibodies (Cell Signaling Biotechnology, Inc.), followed by incubation with green-fluorescent Alexa Fluor 488 dye-labeled anti-mouse and red-fluorescent Alexa Fluor 568 dye-labeled anti-rabbit IgG (Invitrogen, Carlsbad, CA), respectively. For group 2, the fixed cells were incubated with pJNK rabbit polyclonal and γH2AX mouse monoclonal antibodies (Upstate Biotechnology, Inc.), followed by incubation with green-fluorescent Alexa Fluor 488 dye-labeled anti-rabbit and red-fluorescent Alexa Fluor 568 dye-labeled anti-mouse IgG (Invitrogen, Carlsbad, CA). All samples were viewed with a fluorescence microscope (Leica, Bensheim, Germany).
In Vitro Kinase Assay
To detect H2AX phosphorylation, the H2AX protein (Upstate Biotechnology, Inc.) was mixed together with active ERK1, JNK1, JNK2 or p38α proteins (Upstate Biotechnology, Inc.), 0.2 mM ATP and 1x kinase buffer and incubated at 30 °C for 15 min. The reactive products were divided into two parts. One part was separated by 15% SDS-PAGE for western blot analysis and the other was separated by 15% SDS-PAGE for Coommassie blue staining. In addition, H2AX phosphorylation was also measured using immunoprecipitated JNK and a kinase assay according to the method recommended by the manufacturer (Cell Signaling Biotechnology, Inc.; see Supplemental Experimental Procedures). For the in vitro
kinase assay to detect γ-32
p incorporation, GST-c-Jun (Choi et al., 2005
) and H2AX (Upstate Biotechnology, Inc.) proteins or GST-H2AX or GST-H2AX-139A proteins were mixed together with active JNK1 and JNK2 (Upstate Biotechnology, Inc.), 1 μCi of [γ-32p] ATP and 1x kinase buffer and incubated at 30 °C for 15 min. The reactive products were separated by SDS-PAGE for autoradiography and Coommassie blue staining. To further investigate whether Ser139 in H2AX is targeted by JNK directly, we designed five peptides (16A, 18A, 19A, 130S and 139S) for peptide mapping analysis. 16A, 18A and 19A comprised 25 N-terminal residues (aa 10–24) in H2AX. In 16A, 18A or 19A, Ser16, Ser18 or Ser19, respectively, was mutated to Ala to block the respective phosphorylation. 130S contained amino acids 123–137 harboring Ser130 and 139S contained amino acids 128–142 in H2AX. In 139S, Ser130 was mutated to Ala to block possible phosphorylation of Ser130. Then the synthesized peptides (Invitrogen) were mixed with active JNK1 (Upstate Biotechnology, Inc.), 1 μCi of [γ-32p] ATP and 1x kinase buffer, incubated at 30 °C for 15 min and analyzed by SDS-PAGE and autoradiography.
Mammalian Two-Hybrid Assay
To identify the binding domain in vivo
, we used the mammalian two-hybrid assay as described previously (Choi et al., 2005
Determination of Sub-G1 by Flow Cytometry
Determination of sub-G1 fractions was determined according to published protocols (see http://www.cancer.ucsd.edu/Research/Shared/flow/Protocols
). Briefly, after UVA exposure, cells were fixed and then stained with propidium iodide (PI). The DNA content was determined by flow cytometry and the sub-G1 portion was considered to be the apoptotic cell population.
Cell Survival Assays
To measure the sensitivity of cells to UVA, H2AX-wt and H2AX−/− MEFs were plated at 10,000 cells/well on 96-well plates and incubated for 24 h prior to UV. Following UVA, cell viability was determined in triplicate at various time points or UVA dose using the MTS assay according to the instructions from the manufacturer (CellTiter 96® AQueous One Solution Cell Proliferation assay kit, Promega, Madison, WI). The results were compiled using the Multiskan MS plate reader (Labsystems, Helsinki, Finland).
DNA Fragmentation or In Vitro DNA Degradation Assay
H2AX-wt and H2AX −/− MEFs were treated with UVA (40 kJ/m2) and parallel groups of H2AX-wt cells were treated with SP600125 or Z-VAD for 1 h before UVA radiation. The cells were disrupted by adding DNA STAT-60 (Tel-Test, Inc., Friendswood, TX) at various times and cells were centrifuged at 12,000xg for 15 min at 4 °C. The supernatant fraction containing fragmented DNA was mixed with 0.5 ml of isopropanol at room temperature for 10 min and centrifuged at 12,000xg for 10 min at 4 °C. The DNA pellet was washed with 75% ethanol and resuspended in Tris-HCI (pH 8.0) with 100 μg/ml RNAse for 2 h. DNA fragments were separated by 1.8% agarose gel electrophoresis, stained with ethidium bromide and photographed under UV light. To investigate whether H2AX expression in H2AX−/− MEFs restores UVA-induced apoptosis, pcDNA4/H2AX-wt or pcDNA4/H2AX-139A was individually transfected into H2AX−/− MEFs following the manufacturer’s recommended protocol (Lipofectamine Plus Reagents, Invitrogen). The transfected pcDNA4 empty vector was used as a control. Cells were treated with UVA (40 kJ/m2) 48 h after transfection and used for the DNA fragmentation test.
To investigate in vitro
DNA degradation, we used a cell-free system. Six hours after UVA exposure (40 kJ/m2
), H2AX-wt or H2AX−/−
MEFs were harvested and the respective cytosolic fractions (S-100 fractions) were prepared as described previously (Liu et al., 1996
). DNA cleavage was assayed by incubating the S-100 fractions (80 μg) and naked DNA (pcDNA3) (8 μg) (Invitrogen) at 37 °C for 2 h in a final volume adjusted to 60 μl with buffer A (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2
, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, and 0.1 PMSF). To investigate whether H2AX phosphorylation affects DNA cleavage, H2AX (2 μg) was first phosphorylated by active JNK2 (Upstate Biotechnology, Inc.) at 30 °C for 30 min. Then the kinase reaction products were mixed with the S-100 fractions or CAD and pcDNA3 (Invitrogen) and incubated for 2 h at 37 °C. The products were loaded onto a 1.8% agarose gel containing 5 μl ethidium bromide and photographed under UV light.
In Vitro Protein Binding Assay
The expression plasmid (pET-15b-DFF40His) was transformed into BL21 (DE3) Escherichia coli. The expression of DFF40 was induced by isopropyl-β-D-thiogalactoside (IPTG), and the recombinant DFF40 protein was purified using Ni-NTA-agarose beads (QIAGEN, Hilden, Germany). Then 2 μg of the H2AX protein (Upstate Biotechnology, Inc.) was incubated with Ni-NTA-agarose-DFF40 or Ni-NTA-agarose at 4°C for 2 h. The Ni-NTA-agarose-DFF40-H2AX complex sample was boiled and separated by SDS–PAGE for western blot with antibodies against total H2AX (Upstate Biotechnology, Inc.) or DFF40 (Chemicon, Temecula, CA) and Coommassie blue staining.