Reagents and antibodies
Chemical reagents, including Tris, NaCl, and SDS for molecular biology and buffer preparation, were purchased from Sigma-Aldrich (St. Louis, MO, USA). EGF, PD 98059, SB 202190, and the Cot kinase inhibitor (4-(3-chlor-4-fluorophenyl-amino)-6-(pyridin-3-yl-methylamino)-3-cyano-1, 7-naphthylridine) were purchased from Calbiochem-Novabiochem (San Diego, CA, USA). Restriction enzymes and some modifying enzymes were obtained from New England BioLabs, Inc. (Beverly, MA, USA). Cell culture medium and other supplements were purchased from Invitrogen (Carlsbad, CA, USA). The DNA ligation kit (version 2.0) was from TAKARA Bio Inc. (Otsu, Shiga, Japan). [γ-32P]ATP and [35S]methionine were purchased from Amersham Biosciences (Piscataway, NJ, USA). Antibodies for immunoblotting and immunocytochemial analysis were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), or Upstate Biotechnology, Inc. (Charlottesville, VA, USA). NE-PER, nuclear, and cytoplasmic extraction reagent were purchased from Pierce (Rockford, IL, USA). The Check-Mate mammalian two-hybrid system, including expression vectors and the reporter luciferase vector, was obtained from Promega Corp. (Madison, WI, USA). The NE-PER Nuclear and Cytoplasmic Extraction Reagents for fractionation of cells were purchased from Pierce (Rockford, IL, USA).
Cell culture conditions and transfection
Human embryonic kidney 293 (HEK293), human cervix adenocarcinoma (HeLa), and mouse embryo fibroblast (NIH3T3) cells were purchased from American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) or 10% bovine calf serum (BCS). DNA transfection of cells was performed using Fugene 6 (Roche, Palo Alto, CA, USA).
Construction of mammalian expression and small interfering RNA vectors
For the mammalian two-hybrid (M2H) system, the cDNAs of 60 human kinases were amplified by polymerase chain reaction (PCR), and each was introduced into the pBIND two-hybrid system vector. The cDNA encoding histone H3.3 (a generous gift from Aichi Cancer Center Research Institute, Nagoya, Japan) was recombined into the Bam
I site of the pACT vector. The point mutation of histone H3 at Ser-10 (S10A), Ser-28 (S28A), or Ser-10/Ser-28 (S10/28A) was generated by using the Quick Change II site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and introduced into the pcDNA3.1/V5-His vector (pV5-H3-S10A, -S28A, -S10/28A), respectively. The pRK-myc-Cot plasmid was provided by Warner C. Greene (University of California, San Francisco, CA, USA) and subcloned into Bam
RI site of pcDNA4-hisMaxA. To construct the siRNA-Cot, pSilencer 3.0-H1 (Ambion, Austin, TX, USA) was digested with Xba
I and BbsI
. The annealed synthetic primers were then introduced following the recommended protocols (Ambion, Austin, TX, USA): 1
) sense siRNAs: GATCCACTGA TCCCAGTAGA TCAAT-TCAAG AGATTGATCT ACTGGGATCA GTTTTTTTGG AAA; 2)
antisense siRNAs: AGCTTTTCCA AAAAAACTGA TCCCAGTAGA TCAATCTCTT GAATTGATCT ACTGGGATCA GTG. The human c-fos
promoter was a gift from Akihiko Yoshimura (Kyushu University, Fukuoka, Japan) and the AP-1 luciferase reporter plasmid (−73/+63 collagenase-luciferase) was constructed as reported (38
Isolation of histone proteins
To isolate histone proteins, cells (2×107–5×107) were homogenized in 1 ml of nuclear preparation buffer (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 1.5 mM MgCl2, 0.65% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) in the presence of protein phosphatase inhibitors (10 mM NaF, 1 mM sodium orthovanadate, and 25 mM β-glycerophosphate). Nuclei were recovered by centrifugation at 1500 g for 10 min. All centrifugations were carried out at 4°C. Nuclei were resuspended in 0.3 ml of resuspension buffer (10 mM Tris-HCl pH 7.6, 3 mM MgCl2, 10 mM NaCl, 1 mM PMSF, and protein phosphatase inhibitors). Nuclei were extracted with 0.4 N H2SO4 to isolate total histones. The samples were precipitated with trichloroacetic acid (TCA), then resuspended in double distilled H2O.
The proteins were resolved by sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. The membranes were blocked and hybridized with the appropriate primary antibody overnight at 4°C. Histone H3 and phospho-histone H3 (Ser-10) were detected with the respective specific antibodies. Protein bands were visualized by the chemiluminescence detection kit (ECL, Amersham Biosciences) after hybridization with the horseradish peroxidase-conjugated secondary antibody from rabbit or mouse.
In vitro binding assay and GST protein expression
For expression of the Xpress epitope-tagged Cot, DYRK3, deletion mutant Cot, the appropriate plasmids (pcDNA4/Xpress-Cot, deletion mutants Cot) were in vitro translated with l-[35S]methionine using the TNTQuick coupled transcription/translation system (Promega). For the glutathione-S-transferase (GST) pulldown assay, 5 μg GST fusion proteins were collected on glutathione-Sepharose beads (Amersham Biosciences) and incubated for 4 h at 4°C with [35S]-labeled Cot. The bound proteins were denatured in sample buffer and separated by 10–20% SDS-PAGE, and expression was detected by autoradiography (KODAK, New Haven, CT, USA).
Phosphorylation assay for histone H3 in vitro
Phosphorylation of histone H3 by Cot in vitro
was carried out as described previously (39
). In brief, the myc-Cot protein was immunoprecipitated from transiently transfected HEK293 cells, combined with 1 μg of bacterial expressed histone H3 in 50 μl kinase buffer (20 mM Tris pH 7.4, 20 mM β-glycerophosphate, 0.1 mM Na3
, 10 mM MgCl2
, 50 mM NaCl, 1 mM dithiothreitol, 50 mM ATP, 1 mM NaF, and 10 μCi [γ-32
P]) for 1 h at 30°C. The samples were separated by 15% SDS-PAGE and gels were dried. The [γ-32
P] ATP-labeled histone H3 was visualized by autoradiography or by using a phospho-specific antibody against histone H3 (Ser-10).
Mammalian two-hybrid assay
The DNAs, pACT-histone H3, pBIND-kinases, and pG5-luciferase were combined in the same molar ratio and the total amount of DNA was not more than 100 ng/well. The transfection was performed using the Fugene 6 reagent as described by the manufacturer’s recommended protocols. The cells were disrupted by the addition of 200 μl of cell lysis buffer directly into each well of the 48-well plate, then aliquots of 100 μl were added to individual wells of a 96-well luminescence plate. Luminescence activity was measured automatically by computer program (MTX Lab, INC, Vienna, VA, USA). The relative luciferase activity was calculated and normalized based on the pG5-luciferase basal control. To assess transfection efficiency and protein amount, the Renilla luciferase activity assay or Lowry protein assay was used.
Reporter gene assays
The reporter gene assay for firefly luciferase activity was performed using lysates from transfected cells. In addition, the reporter gene vector phRL-SV40 (Promega) was co-transfected into each cell line and the Renilla luciferase activity generated by this vector was used to normalize the results for transfection efficiency. Cell lysates were prepared by first washing the transfected HEK293 cells (grown in 60 mm diameter dishes) once in phosphate-buffered saline (PBS) at 37°C. After removing the PBS completely, 500 μl of passive lysis buffer (PLB, Promega Dual Lucif-erase Reporter Assay System) was added, then cells were incubated for 1 h with gentle shaking. The supernatant fraction was used to measure firefly and Renilla luciferase activities. Cell lysates (20 μl each) were mixed with 100 μl of luciferase assay II reagent and firefly luciferase light emission was measured by a Luminoskan Ascent plate reader (Thermo Electron Corp., Helsinki, Finland). Subsequently, 100 μl of Renilla luciferase substrate (Promega) was added in order to normalize the firefly luciferase data. The results are expressed as relative c-fos or AP-1 activity (fold) and are presented as luciferase activity relative to the c-fos- or AP-1-only transfected control cells.
For translocation of endogenous Cot, the cells were fixed in 4% paraformaldehyde and Cot was detected with a monoclonal Cot antibody and a Texas Red-conjugated secondary antibody. Phosphorylation of histone H3 (Ser-10) was detected with monoclonal anti-phospho-histone H3-FITC (Ser-10). Nuclei were stained with 4, 6-diamidino-2-phenylindole. Cells were incubated for 24 h, starved in serum-free media for an additional 24 h, then were or were not irradiated with UVB (4 kJ/m2) and harvested after 30 min additional incubation. Samples were analyzed using a fluorescence microscope system (Leica) and the Image-Pro software program v.4.
Chromatin immunoprecipitation assay
Nuclear factors associated with chromatin in HEK293 cells were cross-linked to DNA by using formaldehyde (1%). Cells were harvested, and cross-linked chromatin was sheared by sonication. DNA fragments were < 1 kb and averaged 450 bp as verified by agarose gel electrophoresis. Immunoprecipitation was performed with 100 μg (DNA content) of chromatin extracts diluted in ChIP dilution buffer (1.1% Triton X-100, 0.01% SDS, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, and 167 mM NaCl). Samples were precleared with Salmon Sperm DNA/Protein A agarose beads (Upstate) for 30 min, then incubated overnight (16 h) with 4 μg of anti-histone H3, anti-phospho-histone H3 (Ser-10), or anti-myc, respectively, at 4°C. DNA present in the immunoprecipitated chromatin was isolated after reversed cross-link and proteinase K digestion, then the specific region of the c-fos promoter was confirmed by PCR amplification using the following primer sets: 5′-CCCGACCTCGGGAACAAGGG-3′ (− 491 forward) and 5′-ATGAGGGGTTTCGGGGATGG-3′ (−233 reverse).
Anchorage-independent cell transformation assay (soft agar assay)
EGF-induced cell transformation was investigated in mock, psi-H3, or pV5-H3 stably transfected cells. In brief, cells (8×103/ml) were exposed to EGF (0.1–10 ng/ml) in 1 ml of 0.3% basal medium Eagle (BME) agar containing 10% FBS. The cultures were maintained at 37°C in a 5% CO2 incubator for 10 days, and the cell colonies were scored using a microscope and the Image-Pro PLUS computer software program (Media Cybernetics, Silver Spring, MD, USA).
Transformation of NIH3T3 cells was performed following standard protocols (40
). Cells were plated in 100 mm dishes at a density of 1 × 104
cells and, after incubation for 3 wk, were transiently transfected with 0.1 μg of the H-RasG12V
, 2.5 μg pV5, 2.5 μg pRK-myc-Cot, and/or 2.5 μg pV5-H3 plasmid. Cells were kept in MEM with 5% BCS and media were changed every 3 days for a period of 3 wk. Foci were enumerated by staining the monolayer with methanol for fixation and 0.4% crystal violet for visualization. Data shown represent data obtained from three independent plates for each transfection.