Cell culture and reagents.
The human renal cancer cell line 786-O (ATCC no. CRL-1932) and the human embryonic kidney cell line HEK293 were maintained in Dulbecco modified Eagle medium with 10% fetal bovine serum (HyClone Laboratories, Logan, UT). 786-O is a human primary clear-cell carcinoma line with an epithelial morphology. Expression wild-type vectors as well as mutant plasmids of CDP/Cut were kind gifts from Alain Nepveu, McGill University, Quebec, Canada. The pxm/Cux/Mut CDP 6-mutant plasmid contains Ser402, Thr415, Ser789, Thr804, Ser972, and Ser987 and also Ser987 alone (pxm/Cux/CR#-HD/S987A), residues which were replaced with codons for Ala (4
). Note that the Ser987 mutant should not be recognized by anti-CDP antibody (Santa Cruz Biotechnology, Inc.) as this is a much smaller version of the wild-type CDP as described earlier (4
FIH-1 reporter constructs and deletion of the FIH-1 promoter.
A 6,950-bp FIH-1 promoter construct was cloned into pGL3-basic vector to create an approximately full-length FIH-1 promoter. Details were described in our previous paper (6
). Based on this construct, a series of deletion mutants (see Fig. ) of the FIH-1 promoter were generated by direct PCR amplification from genomic DNA prepared from HEK293 cells by using the DNeasy kit (QIAGEN Inc., Valencia, CA). The length was counted with the transcription start site as 1, and upstream of the ATG codon was a minus count. PCR primers were designed containing an MluI site in the 5′ forward primers and an XhoI site in the 3′ reverse primers. The 5′ forward primer used for the −2.7 kb construct was 5′-CG AGC CGT CTA CAT GGT GTG TTA TAT TGA TC-3′, and the reverse primer is 5′-CCG CTC GAG CAT CTC TTT CAC CAT TC-3′. The 480-bp forward primer is 5′-CG ACG CGT CAT TTC TTC AGC AGT-3′, and its reverse primer is identical to the 2.7-kb reverse primer. The −220-bp forward primer is identical to that of the 480-bp construct, and its reverse primer is 5′-CCG CTC GAG CTC CGC CGC CAG GGA C-3′. The −180-bp forward primer is 5′-CAG CGC GTC TAC CAT TAG GAG GCG CAC-3′, the −114-bp forward primer is 5′-CGA CGC GTC GCA GCT CAT CTC TAT G-3′, and the −92-bp forward primer is 5′-CGA CGC GTT GGG TGT GTG GTT T-3′. The reverse primer for the 180-bp, 114-bp, and 92-bp constructs is identical to the 220-bp construct. PCR products were generated using DNA polymerase (Eppendorf-Brinkmann Instruments, Inc., Westburg, NY). PCR conditions were as follows: 98°C for 30 s and 94°C for 5 min, with 40 cycles of 94°C for 30 s, 65°C for 45 s, and 72 °C for 4 min, followed by 72°C for 10 min. PCR products were digested with MluI and XhoI restriction enzymes and ligated into the pGL3-basic vector. All the constructs generated with PCR were sequenced to monitor for any PCR-associated nucleotide incorporation errors. The automated sequencing was performed by the Mayo Clinic Molecular Biology Core facility.
FIG. 1. FIH protein level was increased in the presence of PKC ζ DN. The 786-O RCC cells were transfected with 2 μg of expression vector plasmids carrying a dominant-negative mutant (DN) of PKC ζ or HA-FIH or empty vector in 60-mm tissue (more ...) Determination of FIH-1 potential transcriptional start site(s).
Briefly, the total RNA abstracted from 786-O cells was treated with calf intestinal phosphatase to remove the 5′ phosphates. This eliminates truncated mRNA and non-mRNA, leaving only full-length, capped mRNA. Intact full-length mRNA was then dephosphorylated with tobacco acid pyrophosphatase to remove the 5′ cap and leave a 5′ phosphate at the 5′ end of the mRNA. T4 RNA ligase was used to ligate the GeneRacer RNA oligonucleotide (sequence, 5′-CGA CUG GAG CAC GAG GAC ACU GAC AUG GAC UGA AGG AGU AGA AA-3′) to the 5′ end of the mRNA. This was followed with reverse transcription of the ligated mRNA by using SuperScript III reverse transcriptase and the GeneRacer random primers to create RACE (rapid amplification of cDNA ends)-ready first-strand cDNA with known priming sites at the 5′ end. We amplified the first-strand cDNA using a reverse FIH-1 gene-specific primer (5′-CGGT CAGC ACCA CAGG CTCC TCAT TC-3′) and the GeneRacer 5′ primer (5′-CGA CTG GAG CAC GAG GAC ACT GA-3′) at 94°C for 15 min, with 40 cycles of 94°C for 30 seconds, 60°C for 45 seconds, and 72°C for 45 seconds, followed by 72°C for 10 min. HotStar Taq DNA polymerase (QIAGEN Inc., Valencia, CA) was used for PCR. The PCR product was analyzed on a 2% agarose gel, and three bands were found, ranging from 150 bp to 500 bp. Three microliters of these PCR products was ligated into the pCR4-TOPO vector, and 36 clones were selected and sent for sequencing. Through BLAST, sequencing results were found to match the PubMed sequence.
Transfection and whole-cell extract preparation.
786-O cells and 293 cells (5 × 105) were seeded in a 60-mm dish 1 day before transfection. Transfections were carried out with the Effectene transfection kit (QIAGEN Inc., Valencia, CA). Briefly, 2 μg of the PKC ζ dominant-negative plasmid or the CDP dominant-negative plasmid was resuspended in DNA condensation buffer (150 μl) and 16 μl of Enhancer was added prior to incubation of the samples at room temperature for 5 min. Effectene (40 μl) was then added, and the whole mixture was incubated for another 10 min. Dulbecco modified Eagle medium with 10% fetal bovine serum (350 μl) was then added to the DNA mixtures. Forty-eight hours after transfection, cells were washed three times with phosphate-buffered saline and whole-cell extracts were prepared in accordance with the protocol listed below.
RNA interference for PKC ζ and CDP.
PKC ζ (catalog no. M-003526-02; Dharmacon, Chicago, IL), nontarget control (sc-36869), and the CDP siRNA pool (catalog no. sc-35051) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. Transfections were performed according to the instructions of the Transfect I kit (Dharmacon, Chicago, IL). Briefly, for mixture A, 95 μl of serum-free medium was mixed with 5 μl of transfect reagent I for 5 min at room temperature (RT); for mixture B, 10 μl of siRNA pool (10 μM) was mixed with 90 μl of serum-free medium for 5 min at RT. Mixture A was then added to mixture B and incubated for another 20 min at RT. The medium was then replaced with 800 μl of complete medium, added dropwise to the dish. For whole-cell lysate preparation and Western blot analysis, the cells were lysed 72 h after siRNA transfection. For the luciferase assay, 24 h after the siRNA transfection, samples were then transfected with 0.5 μg of FIH-1 promoter constructs using the Effectene transfection kit (described above) and incubated for an additional 48 h. The cells were then lysed with 1× lysis buffer for the luciferase assay.
Preparation of whole-cell extracts.
786-O cells and 293 cells were washed twice with 10 ml of cold phosphate-buffered saline, lysed with ice-cold RIPA lysis buffer (50 mM Tris [pH 7.5], 1% NP-40, 150 mM NaCl, 1 mM Na3VO4, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, leupeptin [10 μg/ml], 0.5% aprotinin, 2 mM pepstatin A), incubated on ice for 30 min, and centrifuged at 14,000 rpm at 4°C for 10 min.
Western blot analysis.
The whole-cell extracts were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the immunodetection antibodies used were FIH-1 (catalog no. BC-100-428A; Novus Biologicals, Littleton, CO), CDP (catalog no. sc-13024; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), phospho-mitogen-activated protein (phospho-MAP) kinase (catalog no. 07-467; Millipore, Billerica, MA), phosphoserine (catalog no. AB1603; Chemicon, Temecula, CA), and PKC ζ (Santa Cruz Biotechnology). Primary incubations were followed with an appropriate secondary antibody and detected with the ECL enhancer reagent from Amersham Biosciences Corp. (Piscataway, NJ). For immunoprecipitation assays with the phosphoserine antibody, anti-PKC ζ antibody was used for Western blotting.
Luciferase reporter assays.
786-O cells and 293 cells (5 × 104) were seeded in a 12-well plate 1 day before transfection. All transfections were carried out using the Effectene reagent as described above. A total of 1 μg of FIH-1 promoter construct and 0.1 μg of pRL-TK Renilla luciferase vector (Promega, Pittsburgh, PA) were used for each transfection. The pRL-TK Renilla luciferase activity was used as the control for transfection efficiency. In some of the experiments, we equilibrated the luciferase activities by protein concentrations of the cell extracts with respect to the control vectors. Each transfection experiment was performed in triplicate and repeated for a minimum of three times. For cotransfection experiments, cells received 0.3 μg of different deletion constructs of pGL3-FIH-1, 0.1 μg of pRL-TK Renilla luciferase vector, 1 μg of the indicated expression plasmids (PKC ζ DN) or siRNA of CDP/Cut, and carrier DNA. Firefly luciferase and Renilla luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega). Approximately 48 h after transfection, cells were washed twice with 1× phosphate-buffered saline and harvested with 200 μl of passive lysis buffer. Cell lysates were cleared by centrifugation, and 5 μl of lysate was added to 100 μl of firefly luciferase substrate. Light units were measured in a LB960 microplate luminometer. Renilla luciferase activities were measured in the same well after addition of 100 μl of the Stop and Glo reagent. The average was calculated among three wells; the ratio between the firefly luciferase and Renilla luciferase was calculated, and the relative change (n-fold) was obtained after comparing the results to the control sample. Data were expressed as the means ± standard deviations of triplicate values.
Double-stranded oligonucleotides generated from the single-stranded oligonucleotide list in Table were used as electrophoretic mobility shift assay (EMSA) probes. Sequence W is the wild-type FIH-1 promoter region between 114 bp and 92 bp; sequence M is the mutant sequence. The upper-strand (sense) oligonucleotide (30 ng) was 5′ end labeled using polynucleotide kinase with [γ-32P]ATP (Amersham Pharmacia Biotech). After the labeling reaction, a twofold excess of the lower-strand (antisense) oligonucleotide was annealed to the upper strand. Double-stranded DNA probes were then purified from the reaction mixture using a Bio-Gel P-100 column (Bio-Rad). Whole-cell extracts were isolated from 786-O cells and 293T cells. DNA-protein binding was performed in 0.5× Dignam buffer D (20 mM HEPES, pH 7.9, 100 mM KCl, 20% glycerol, 0.2 mM EDTA) supplemented with 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 10 mM MgCl2, and 100 μg/ml poly(dI-dC). Binding reactions were initiated with a 30,000-cpm addition of DNA probe in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) to 5 to 10 μl of whole-cell extracts. Electrophoresis was performed in 4% acrylamide gels, and the gels were vacuum dried and exposed to film for 16 to 48 h. Competition experiments were carried out in the same way as described above except that increasing amounts of double-stranded wild-type oligonucleotide (200-fold) were mixed with 30,000 cpm of M-1 probe (see Table ) and then added to the binding reaction mixture.
Oligonucleotides used for gel shift assay
The chromatin immunoprecipitation (ChIP) assay was performed using the ChIP assay kit from Upstate Biotechnology, Inc. (Lake Placid, NY). Briefly, 7 × 106 to 10 × 106 cells were used for each assay. Protein-DNA cross-linking was carried out by the addition of 1% formaldehyde directly to the cell cultures, followed by incubation at 37°C for 10 min. After the cells were thoroughly washed with ice-cold phosphate-buffered saline, the cells were scraped off and harvested. Cells were lysed with 200 μl of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.1) supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A). Sonication was then performed on ice using a sonicator (Lab-Line Ultra Tip; Lab-Line Instrument. Inc.) preset for 10-s pulses with 10-s intervals. Ten repeated sonication cycles (as previously standardized by us) were applied to achieve chromatin fragmentation in the 200- to 1,000-bp range. Fragmented chromatin was diluted 10-fold in ChIP dilution buffer (0.01% SDS, 1% Triton X-100, 2 mM EDTA, 16.7 mM Tris, pH 8.1, and 150 mM NaCl). Diluted chromatin fragments were precleared by incubation with protein A agarose beads under constant rotation for 2 h at 4°C. For immunoprecipitations, an antibody specific for CDP-1 or the respective immunoglobulin G (IgG) control was used for an overnight incubation with constant rotation at 4°C. The protein-DNA-antibody complex was pulled down by protein A agarose-salmon sperm DNA beads. After thorough and sequential washings with low-salt, high-salt, and LiCl-containing buffers, the resulting immune complex was eluted with 1% SDS and 0.1 M NaHCO3. Formaldehyde cross-links were reversed by adding 5 M NaCl and heating at 65°C for 4 h. DNA fragments were then recovered by ethanol precipitation following proteinase K digestion and phenol-chloroform extraction.
PCR was performed with FIH promoter-specific primers amplifying the CDP binding region (forward, 5′-GTT CTT TAT GTC TCT GGA CCG-3′, and reverse, 5′-CCA CAC ACC CAA CGG AA AC-3′). The 200-bp amplicon was analyzed with 2% agarose gel electrophoresis.