) and anti-Nemo-like kinase (anti-NLK) were generated as polyclonal antisera in rabbits. Additional antibodies were anti-p300/CBP (05-267), anti-p300 (05-257), anti-PCAF (07-141), anti-acetyl-histone H3 (Lys14) (06-760), anti-phospho-histone H3 (Ser10) (06-570), anti-histone H3 (05-499), and anti-α-actinin (05-384) (Upstate, Lake Placid, NY); anti-CBP (sc-369), anti-KLF13 (sc-9605), anti-Brg-1 (sc-10768), and anti-RNA polymerase II (sc-9001x) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-acetyl-histone H4 (Lys 8) (ab1760-100) (Abcam, Cambridge, MA); anti-V5 (A7345) (Sigma-Aldrich, St. Louis, MO); and anti-RANTES (Pierce, Rockford, Ill.).
Plasmids used included the following: pGL3-RP-luc, constructed by inserting bp −195 to +54 of the RANTES
promoter into pGL3 (Promega, Madison, WI); pREP4-RP-luc, constructed by inserting bp −195 to +54 of the RANTES
promoter into the pREP4 plasmid (a gift from Keji Zhou at National Institutes of Health, Bethesda, Md.); pREP4-ΔA-RP-luc, constructed by subcloning the insert from the previously described mutant of the A site of the RANTES
) into the XhoI-HindIII sites of pREP4-luc; pBJ5-Brg-1 plasmid (a gift from Jerry Crabtree, Stanford University); pcDNA3.1(+) (Invitrogen, Carlsbad, CA); pcDNA-KLF13, described previously (49
); and pcDNA/V5/His-NLK, constructed by inserting the full-length cDNA of NLK into pcDNA 3.1/V5/His (Invitrogen).
T-lymphocyte isolation and cell culture.
Human peripheral blood T lymphocytes were prepared from LeukoPacs (Stanford Blood Bank, Stanford, CA) by negative selection (RosetteSep) according to the manufacturer's protocol (StemCell Technologies, Vancouver, BC, Canada). Purified T lymphocytes or Jurkat cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1 mM glutamine, and 100 U/ml penicillin-streptomycin and stimulated with 5 μg/ml phytohemagglutinin (PHA) for up to 7 days at 37°C, 5% CO2. HeLa cells and SW13 cells (ATCC) were maintained in Dulbecco's modified eagle medium containing 10% bovine calf serum, 1 mM glutamine, and 100 U/ml penicillin-streptomycin at 37°C, 5% CO2.
RANTES protein secreted into the cell culture supernatant was quantified by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (Pierce, Rockford, IL).
Real-time quantitative PCR.
Total RNA was prepared using the RNeasy kit (QIAGEN, Valencia, CA). cDNA was made using Superscript II with random primers (Invitrogen). Primers for human RANTES (forward primer, 5′-AGTCGTCTTTGTCACCCGAAA-3′; reverse primer, 5′-AGCTCATCTCCAAAGAGTTGATGTAC-3′) were purchased from Elim Biopharmaceuticals, Inc. (Hayward, CA). Primers for β-glucuronidase (Applied Biosystems, Foster City, CA) were used as an internal control. Primers for gamma interferon (IFNG; Applied Biosystems) were used as a negative control in the small interfering RNA (siRNA) experiment. PCR was performed in triplicate using SYBR Green or TaqMan Universal PCR master mix (Applied Biosystems) with a GeneAmp 7900 sequence detection system (Applied Biosystems) for 40 cycles of PCR under the following conditions: 2 min at 50°C and 10 min at 95°C for 1 cycle, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The expression of the RANTES gene was represented as the fold increase (2−ΔΔCt), where ΔΔCt = [ΔCt(stimulated)] − [ΔCt(unstimulated)] and ΔCt = [Ct(sample)] − [Ct(Gus)].
Nuclear run-on analysis.
The RANTES transcription rate was measured by nuclear run-on analysis. Resting and PHA-activated T lymphocytes (2 × 107 cells) were harvested, and nuclei were isolated in hypotonic buffer (10 mM Tris, pH 7.6, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40). Isolated nuclei were incubated with 25 mM HEPES, pH 7.5, 2.5 mM MgCl2, 2.5 mM dithiothreitol (DTT), 75 mM KCl, 5% glycerol, 2.8 mM ATP, 2.8 mM GTP, 2.8 mM CTP, 0.003 mM UTP, and 500 μCi [γ-32P]UTP (New England Nuclear, Wilmington, DE) for 30 min at 37°C. After in vitro transcription, total RNA was isolated and hybridized using a slot blot to RANTES and actin cDNA and to pUC18 plasmid DNA that had been immobilized on nylon membranes using ExpressHyb (Clontech). After washing, the membranes were exposed to X-ray film (Kodak Biomax MS).
Knock-down of KLF13 by KLF13-specific siRNA.
Double-stranded siRNA oligonucleotides directed against KLF13 mRNA (5′-CCUCAGGUGUCAAAGUAAAdTdT-3′) and nonsilencing siRNA (5′-UUCUCCGAACGUGUCACGUdTdT-3′) were purchased from QIAGEN. A total of 5 × 106 T lymphocytes were nucleofected with 1.5 μg KLF13 or nonsilencing siRNA using the Amaxa nucleofector system (Cologne, Germany) according to the manufacturer's protocol (program U-14). Cells were plated in six-well plates in RPMI plus 10% fetal bovine serum and incubated at 37°C, 5% CO2. After 5 h, PHA (5 μg/ml) was added and cells were incubated for an additional 48 h and harvested for use in Western blotting assays, real-time quantitative PCR, and ELISAs.
In vivo footprinting and LM-PCR analysis.
Dimethyl sulfate (DMS) was added to cell cultures (5 μl of DMS/ml of culture medium) and incubated for 2 min at room temperature. After washing cells with phosphate-buffered saline, 2 ml of stop buffer (20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 20 mM EDTA, 1% sodium dodecyl sulfate, 600 μg/ml proteinase K) was added to each tube and incubated at 37°C for 3 h. Genomic DNA was purified and analyzed by ligation-mediated PCR (LM-PCR) as described elsewhere (40
) using the following primer set: primer 1 (located at bp −332), 5′-TAACTGCCACTCCTTGTTGTCC-3′, for the first-strand reaction; primer 2 (located at bp −311), 5′-CCCAAGAAAGCGGCTTCCTGCTCTC-3′, for 15 cycles of PCR amplification; primer 3 (located at bp −294), 5′-CTGAGGAGGACCCCTTCCCTGGAAGGTA-3′, for the labeling reaction. The products were analyzed on 8% acrylamide-urea gels. After electrophoresis, the gel was dried and exposed to film overnight at −80°C using intensifying screens.
ChIP and re-ChIP assay.
A chromatin immunoprecipitation (ChIP) assay was performed using the ChIP-IT kit (Active Motif, Carlsbad, CA), following the manufacturer's instructions using 2 × 107 human T lymphocytes per condition and specific antibody. For the chromatin reimmunoprecipitation (re-ChIP) assays, the chromatin complexes were eluted from the first ChIP with 10 mM DTT at 37°C for 30 min and diluted 20 times with ChIP dilution buffer (1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, and 150 mM NaCl) and reimmunoprecipitated with a second antibody. Input DNA and DNA immunoprecipitated with either specific antibodies or immunoglobulin G (IgG) were PCR amplified using primers flanking the proximal RANTES promoter and the TATA box from bp −209 to +100 (−209 primer, 5′-CACCATTGGTGCTTGGTCAAAGAGG-3′; +100 primer, 5′-GCAGTAGCAATGAGGATGACAGCGA-3′). Reaction mixtures were cycled with an initial melt step at 94°C for 5 min and then 24 to 30 cycles of 94°C for 45 seconds, 56°C for 30 seconds, and 72°C for 60 seconds, followed by 72°C for 10 min. Products were analyzed by electrophoresis on a 2% gel. As a negative control, primers corresponding to a genomic region distal to the RANTES promoter from −3789 to −3459 were used (5′ primer, −3789, 5′-GCAGATTACGAGGTCAGGAG-3′; 3′ primer, −3459, 5′-TTATGCTTTTCAACAGTCT-3′).
Nuclear extract preparation and Western blotting.
Nuclear extracts were prepared according to the manufacturer's protocol (Transfactor extraction kit; Clontech, Mountain View, CA). Western blots with nuclear extracts or immunoprecipitates were detected using ECL reagent (Amersham Pharmacia Biotech, Piscataway, NJ).
In vivo DSP cross-linking-mediated coimmunoprecipitation assay.
In vivo cross-linking and immunoprecipitation were performed as described previously (28
) with the following modifications: 5-day-activated T lymphocytes were cross-linked by the addition of 0.6 mg dithio-bis(succinimidylpropionate) [DSP; Pierce]/ml for 15 min at room temperature. For IP, 1 mg of nuclear extract was mixed with 2 or 5 μg of antibody and rotated overnight at 4°C. Protein A/G beads were added and incubated for an additional 4 h at 4°C with rotation. The beads were then pelleted and washed five times with 1 ml of IP wash buffer (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1 mM EDTA, 0.2% Triton X-100). The beads were incubated in 40 μl of 2× sodium dodecyl sulfate (SDS) loading buffer (0.1 M Tris, pH 6.8, 4% SDS, 10% glycerol, 0.2 M DTT) for 10 min at 95°C. After electrophoresis, samples were analyzed by Western blotting.
In vitro kinase assay.
HeLa cells were transiently transfected with either pcDNA3.1/V5/His vector or pcDNA/V5/His-NLK using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Cells were lysed 48 h after transfection, and 500 μg of lysate was subjected to immunoprecipitation with anti-V5 antibody (Sigma). To determine whether NLK phosphorylates histone H3 protein, aliquots of immunoprecipitant were incubated at 30°C with 10 mM HEPES (pH 7.4), 5 mM MgCl2, 5 mM DTT, 1 μCi [γ-32P]ATP, and 1 μg of recombinant histone H3 (Upstate) for 15 min in a final volume of 25 μl. The reaction was stopped by addition of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, and the samples were subjected to SDS-PAGE. The γ-32P-labeled histone H3 substrate was visualized by autoradiography. To identify the specific phosphorylation sites of NLK on histone H3, 1 μg of synthetic H3 peptide and 0.2 mM unlabeled ATP were used instead of recombinant histone H3 and [γ-32P]ATP in the kinase reaction mixture. Histone H3 peptides (United Biochemical Research, Inc., Seattle, WA) that included residues 1 to 27 (ARTKQTARKSTGGKAPRKQLASKAARK) or residues 1 to 35 (ARTKQTARKSTGGKAPRKQLASKAARKSAPATGGV) were used to determine serine 10 and serine 28 phosphorylation, respectively. Samples were separated by 20% SDS-PAGE and subjected to Western blotting with anti-phospho-histone H3 (serine 10 or serine 28) antibody following the in vitro kinase reaction.
Histone acetyltransferase (HAT) activity was assayed in nuclear extracts using a HAT assay kit (Upstate).
Transient transfection and luciferase assay.
Transient transfection was carried out using Superfect (QIAGEN) transfection reagent according to the manufacturer's protocols. Luciferase was measured with the Dual Luciferase assay kit (Promega) following the manufacturer's instructions. Luciferase activity was measured over 30 s in an EG&G Lumat LB 9507 luminometer.