All the oligonucleotide sequences used in this study are shown in Table S1 in the supplemental material. Human HP1α, HP1β, HP1γ, and SUV39H1 cDNA fragments were PCR amplified with Pfu DNA polymerase from Human HeLa (S3) QUICK-Clone cDNAs (Clontech) using primers bearing BsiWI and BamHI sites. The amplified DNA fragments were cloned into the EcoRV site of pBluescriptII KS(−) (Stratagene), sequenced, and then digested with BsiWI and BamHI for subcloning.
The phTet-On and phTet-Off plasmids used for transgene activation were humanized codon versions of pTet-On and pTet-Off (Clontech), respectively. The humanized version of the rTetR portion of the phTet-On plasmid was synthesized by sequential PCRs using overlapping oligonucleotides covering the entire rTetR coding region. The humanized version of the normal TetR sequence was generated by the same procedure. The humanized sequences were designed to adapt codon usage for use in mammalian cells and to eliminate potential cryptic splice donor and acceptor sites. The humanized TetR and rTetR sequences and clones are available from W. Reith upon request (Walter.Reith@medecine.unige.ch).
For expression and tethering to the transgenic locus, fusion proteins with rTetR were constructed based on the phTet-On plasmid. One BamHI site between the neomycin resistance gene and the ampicillin resistance gene was removed by digestion, blunt ended, and self-ligated. To insert a FLAG epitope into phTet-On, a BsiWI site was recreated in phTet-On by site-directed mutagenesis at a position corresponding to that in the original plasmid. The resulting plasmid was digested with BsiWI and partly filled in with Klenow DNA polymerase (Takara) in the presence of dGTP and dTTP. A pair of annealed oligonucleotides encoding a FLAG tag was inserted at this site to give phTet-On-FLAG-VP16. phTet-On-FLAG-fused HP1s were created by inserting BsiWI/BamHI-digested HP1 fragments into BsiWI/BamHI-digested phTet-On-FLAG-VP16.
To insert a nuclear localization signal (NLS) into phTet-On-FLAG-VP16, a BsiWI (blunt ended with Klenow)/BamHI fragment containing VP16 AAD was excised from phTet-On-FLAG-VP16 and inserted into PstI (blunt ended with Klenow)/BamHI-digested pECFP-NLS-C1/C3, which was constructed by inserting a pair of annealed oligonucleotides encoding an NLS of the simian virus 40 (SV40) large T antigen into SacI/EcoRI-digested pECFP-C1. The XhoI (blunt ended with Klenow)/BamHI-digested fragment containing NLS-VP16 AAD was then ligated back into BsiWI (blunt ended with Klenow)/BamHI-digested phTet-On-FLAG-VP16, resulting in phTet-On-FLAG-NLS-VP16. Self-ligation of the BsiWI/BamHI (both blunt ended with Klenow)-digested phTet-On-FLAG-NLS-VP16 fragment resulted in a control plasmid, phTet-On-FLAG-NLS-ΔVP16, which encoded a fusion protein without VP16 AAD.
To construct plasmids expressing enhanced cyan fluorescent protein (ECFP)-fused HP1α and SUV39H1 under the control of the SV2 promoter, a BsiWI (blunt ended with Klenow)/BamHI-digested fragment was inserted into EcoRI (blunt ended with Klenow)/BamHI-digested pSV2-ECFP-C1, which was identical to pECFP-C1 (Clontech), except that the original CMV promoter was replaced with the SV2 promoter. The HP1α V22M point mutation was generated by PCR amplification using a mutational primer pair and pBluescriptII KS(−) containing the HP1α cDNA as a template. Following self-ligation of the amplified fragment, the mutated HP1α fragment was released and subcloned into pSV2-ECFP-C1 as described above.
To generate a plasmid expressing a triple-fusion protein consisting of enhanced yellow fluorescent protein (EYFP)-LacR-VP16 AAD, a BsiWI site was introduced by PCR-based mutagenesis just upstream of the stop codon of pEYFP-lacR, which is a CMV promoter-containing plasmid corresponding to pSV2-EYFP-lacR (also known as pSV2-EYFP-lac repressor) (47
). The resulting plasmid was digested with BsiWI and BamHI, and the BsiWI/BamHI-digested VP16 AAD fragment from phTet-On-FLAG-VP16 was inserted to give pEYFP-lacR-VP16.
pSilencer plasmids expressing transgene-directed siRNAs were generated by ligating pairs of annealed oligonucleotides with the BamHI/HindIII-linearized pSilencer 3.1-H1 puro siRNA expression vector (Ambion), according to the manufacturer's instructions. The pSilencer 3.1-H1 puro negative control plasmid (pSilencer-negative; Ambion) encoding an siRNA with limited homology to any known sequences in the human, mouse, and rat genomes was used as a negative control.
The rabbit polyclonal antibodies against human HP1β and HP1γ used for immunofluorescence staining and chromatin immunoprecipitation (ChIP) assays were produced in rabbits immunized with a synthetic peptide, CNEDDDKKDDKN (including amino acids corresponding to 176 to 185 of human HP1β) (unpublished data) and WHSCPEDEAQ (residues 174 to 183 of human HP1γ) (23
), respectively. The rabbit polyclonal antibody against H3K9me3 used in the ChIP assays was obtained from Abcom (ab8898). The secondary antibody used in the immunofluorescence staining was an Alexa Fluor 594-conjugated goat anti-rabbit immunoglobulin G (IgG) (H+L) antibody (A11037; Molecular Probes). The primary antibodies used for Western blot analyses were mouse monoclonal antibody against human HP1α (05-689; Upstate), rabbit polyclonal antibody against Dicer (9
) (a gift from K. Saigo, University of Tokyo, Tokyo, Japan), rabbit polyclonal antibody against human erythrocyte catalase (01-05-030000; Athens Research and Technology), and anti-FLAG M2 monoclonal antibody (F3165; Sigma). The secondary antibodies used in Western blot analyses were horseradish peroxidase-conjugated donkey anti-rabbit IgG (NA934; Amersham Biosciences) and sheep anti-mouse IgG (NA931; Amersham Biosciences) antibodies.
Cell culture and plasmid transfection.
Clone 2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 150 U/ml hygromycin B (Wako) at 37°C with 5% CO2. BHK cells were cultured in a similar manner, but without hygromycin B. The cells were plated 24 h prior to transfections and harvested for biochemical analyses and fluorescence microscopy at the time points indicated in specific experiments. Plasmid transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, using 4 μg of plasmids and 10 μl of reagent per 2 ml of medium in a 35-mm dish.
Immunofluorescence staining and fluorescence microscopy.
Clone 2 cells were cultured on coverslips coated with poly-l-lysine (Sigma). At 24 h after plasmid transfection, the cells were washed with phosphate-buffered saline (PBS) and permeabilized in cold hypotonic buffer (10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol) containing 0.1% Triton X-100 for 10 min on ice. Next, the cells were fixed for 1 min by adding a half-volume of 4% paraformaldehyde (PFA) in PBS, followed by routine fixation in 4% PFA for 1 h. The fixed cells were treated with 0.1% Triton X-100 in PBS for 15 min and blocked with 1% bovine serum albumin in PBS for 1 h. The cells were then incubated in PBS containing 0.1% Tween 20 and antibodies against HP1β (9.8 μg/ml) or HP1γ (52 μg/ml) for 40 min at room temperature with shaking, followed by incubation at 4°C overnight. Next, the cells were refixed in 4% PFA for 1 h and incubated with a secondary antibody (1:500 dilution) for 8 h. Several washes with PBS were performed between the different steps described above. The immunostained cells were mounted on slides with 90% glycerol in PBS and viewed using a Zeiss Axiovert 200 M inverted fluorescence microscope equipped with an ApoTome.
For fluorescence microscopic observation of cells without immunofluorescence staining, cells were cultured on uncoated coverslips, routinely fixed in 4% PFA for 1 h, mounted on slides, and viewed using an Olympus BX-60 or Zeiss Axiovert 200 M inverted fluorescence microscope. Images were taken without the ApoTome. The acquired images were processed using Adobe Photoshop.
ChIP assays were performed using a ChIP assay kit (Upstate) according to the manufacturer's instructions, with some modifications. Briefly, clone 2 cells grown in 35-mm dishes were either mock transfected with 4 μg of pBluescriptII KS(−) or transcriptionally activated by transfection with phTet-Off. At 24 h posttransfection, formaldehyde (37%) was added to the medium to a final concentration of 1% (vol/vol), and the cells were incubated for 10 min at room temperature with gentle shaking. After removal of the medium, the cells were rinsed once with ice-cold PBS containing 20 mM glycine and then twice with ice-cold PBS. Next, the cells were scraped into 1 ml of ice-cold PBS containing protease inhibitors (Roche Applied Science), centrifuged into a pellet, resuspended in 200 μl of sodium dodecyl sulfate (SDS) lysis buffer containing protease inhibitors, and incubated for 10 min on ice. The cell lysates were sonicated with a Bioruptor (Cosmo Bio) for 30 s at the maximum setting 20 times with 1-min intervals, yielding DNA fragments 300 to 500 bp in length. After removal of the cell debris by centrifugation, the supernatants were diluted with 1.8 ml of ChIP dilution buffer containing protease inhibitors and precleared with 80 μl of salmon sperm DNA/protein A agarose slurry for 1 h at 4°C with agitation. Immunoprecipitation was carried out overnight at 4°C with rotation, using antibodies specific for HP1β (5 μg), HP1γ (5 μg), or H3K9me3 (2 μg). The immune complexes were recovered using 60 μl of the salmon sperm DNA/protein A agarose slurry at 4°C for 1 h with rotation and sequentially washed once with low-salt immune complex wash buffer, once with high-salt immune complex wash buffer, once with LiCl immune complex wash buffer, and twice with Tris-EDTA (TE) buffer. The immune complexes were eluted from the agarose beads with 500 μl of freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3), and the cross-links were reversed by incubation with 0.2 M NaCl and 10 μg of RNase A (Wako) at 65°C for 6 h. Following the addition of 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris-HCl (pH 6.5), and 1 μl of 20 μg/μl proteinase K (Takara), the samples were incubated for 1 h at 45°C and then extracted with phenol-chloroform. The DNA obtained was either recovered by ethanol precipitation using glycogen as a carrier (for H3K9me3) (see Fig. ) or purified using a Qiaquick PCR Purification kit (QIAGEN) and finally eluted in 50 μl of TE buffer. A 4-μl aliquot of the recovered DNA was subjected to quantitative real-time PCR using SYBR Green Supermix (Bio-Rad) in an iCycler (Bio-Rad) according to the manufacturer's instructions. The PCR cycling conditions were 3 min at 95°C, followed by 40 cycles of 45 s at 94°C, 30 s at 55°C, and 30 s at 72°C. For amplification of the β-actin promoter, the annealing temperature was set to 64°C.
FIG. 3. The CMVm promoter and transcribed CFP regions of the transgene are associated with HP1β, HP1γ, and H3K9me3. Cells mock transfected with pBluescriptII KS(−) (silent, indicated as OFF) or transcriptionally activated by hTet-Off (indicated (more ...) CpG methylation analysis by bisulfite genomic sequencing.
Bisulfite sequencing of genomic DNA was performed as described previously (15
) with some modifications. Briefly, clone 2 cells grown in 60-mm dishes were either mock transfected with 8 μg of pBluescriptII KS(−) or activated by transfection with phTet-Off. At 24 h posttransfection, genomic DNA was isolated using a GenElute Mammalian Genomic DNA Miniprep kit (Sigma). Next, 12 μg of the genomic DNA was fragmented by digestion with EcoRI and PstI. The reaction mixture was extracted with phenol-chloroform, precipitated with ethanol, and then dissolved in 12 μl of TE buffer. A 3-μl aliquot of the fragmented genomic DNA was denatured by incubation in 0.3 M NaOH in a final volume of 20 μl at 37°C for 15 min. Next, the reaction solution was mixed with 107 μl of 4.04 M sodium bisulfite (Sigma), 7 μl of 10 mM hydroquinone (Sigma), and 6 μl of 6 N NaOH and incubated at 95°C for 1 h in the dark. The bisulfite-treated DNA was subsequently desalted using a Wizard DNA Clean-Up System (Promega) and dissolved in 50 μl of TE buffer. The obtained DNA samples were incubated in 0.3 M NaOH at room temperature for 5 min, neutralized with sodium acetate, precipitated with ethanol in the presence of 20 μg of glycogen as a carrier, and finally dissolved in 20 μl of TE buffer. A 1-μl aliquot of the bisulfite-treated DNA was PCR amplified in a 25-μl reaction mixture containing reaction buffer, 200 μM each deoxynucleoside triphosphate (dNTP), 0.2 U/μl Taq
DNA polymerase (Takara), and 0.4 μM phosphorylated forward and reverse primers. The primers were designed to contain several non-CpG cytosines but exclude CpG dinucleotides, thereby allowing amplification of the DNA only when the cytosines within the primer binding sites were bisulfite converted to uracil. The PCR cycling conditions were 5 min at 94°C; 40 cycles of 30 s at 95°C, 30 s at 52°C, and 1 min at 72°C; and a final extension for 7 min at 72°C. The PCR products were phenol-chloroform extracted, ethanol precipitated, and then blunt ended by Pfu
DNA polymerase treatment in the presence of 200 μM each dNTP for 10 min at 70°C. The 240-bp PCR fragment was subcloned into the EcoRV site of pBluescriptII KS(−) and sequenced.
Northern blot analysis of small RNAs.
Small RNAs were isolated from BHK and clone 2 cells using a mirVana miRNA Isolation kit (Ambion) and subjected to low-stringency Northern blot analysis according to the manufacturer's instructions with some modifications. Briefly, a 7.5-μg portion of the small-RNA fraction was mixed with an equal volume (15 μl) of gel loading buffer II (Ambion), heat denatured, and separated in a 17.5% denaturing polyacrylamide gel containing 7 M urea. The separated RNAs were transferred onto a Hybond N+ nylon membrane (Amersham Biosciences) by electroblotting using a semidry blotting apparatus (ATTO) and then cross-linked by exposure to UV light. Prehybridization was carried out for 2 h at 37°C in a hybridization solution (6× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 5× Denhardt's solution, 0.5% SDS, 0.1 mg/ml denatured and sheared salmon sperm DNA). Hybridization was performed at 37°C in the same solution containing DNA probes, which had been labeled with [α-32P]dCTP (Amersham Biosciences) using a BcaBEST Labeling kit (Takara). After overnight hybridization, the membranes were washed twice in 6× SSC containing 0.2% SDS at 37°C for a total of 30 min. Finally, the blots were exposed to an imaging plate overnight and analyzed using a BAS-2500 BioImaging Analyzer (Fuji Film). Digoxigenin-labeled 22-nucleotide (nt) and 25-nt oligonucleotides (gifts from T. Natsuaki, Utsunomiya University, Utsunomiya, Japan) were used as molecular-size markers. The sequences of the DNA probes corresponded to nucleotides in different regions of the integrated plasmid p3216PCbeta (AB236435) as follows: lac operator (35 to 181), TRE (9391 to 9652), CMVm promoter (13471 to 13616), CFP (13630 to 14360), and nontranscribed vector (15434 to 15689).
Cloning of a hamster Dicer partial cDNA.
Total RNA was isolated from BHK cells using an RNeasy mini kit (QIAGEN) according to the manufacturer's instructions. The first-strand cDNA was synthesized from 1.5 μg of total RNA in a 20-μl reverse transcription reaction mixture containing reaction buffer, 1 mM each dNTP, 2 U/μl ReverTra Ace reverse transcriptase (Toyobo), and 0.25 μM mouse Dicer reverse primer. The mixture was incubated at 42°C for 60 min and then inactivated at 99°C for 5 min. A 5-μl aliquot of the heat-inactivated reverse transcription product was PCR amplified using Pfu DNA polymerase with mouse Dicer forward and reverse primers. The PCR cycling conditions were 1 min at 94°C and 35 cycles of 30 s at 94°C, 30 s at 57°C, and 1.5 min at 72°C, with a final extension for 2 min at 72°C. The 437-bp amplified fragment was subcloned into the EcoRV site of pBluescriptII KS(−) and sequenced.
RNAi-mediated Dicer knockdown.
Hamster Dicer-specific siRNA transfection was carried out with Lipofectamine 2000 according to the manufacturer's instructions, using 5 μg of reagent per 35-mm dish. To achieve complete Dicer knockdown, clone 2 cells were pretreated with three consecutive siRNA (20 pmol) transfections at 48-h intervals, followed by a fourth siRNA (5 pmol) transfection, together with 4 μg of plasmids as indicated for specific experiments. The siRNA transfection efficiency was monitored by cotransfection with 1 μg of pEYFP-SKL (17
), followed by calculation of the percentage of cells showing peroxisomal EYFP signals. The synthetic Dicer siRNA duplex (sense, 5′-GUACUCAAACCUAGAAGUAdTdT-3′, and antisense, 5′-UACUUCUAGGUUUGAGUACdTdT-3′) was purchased from Proligo. A siRNA duplex (sense, 5′-GAUCUGCUCGUACAAGAAUdTdT-3′, and antisense, 5′-AUUCUUGUACGAGCAGAUCdTdT-3′) targeted to the angiotensin-converting enzyme (ACE) gene of the silkworm Bombyx mori
was used as a control (a gift from H. Kawasaki, Utsunomiya University, Utsunomiya, Japan).
Quantitative real-time RT-PCR.
Total RNA was isolated from clone 2 cells using an RNeasy Mini kit (QIAGEN) with on-column DNase I treatment. Quantitative real-time reverse transcription (RT)-PCR analysis was performed in an iCycler (Bio-Rad) using a one-step QuantiTect SYBR Green RT-PCR kit (QIAGEN) according to the manufacturer's instructions. The reverse transcription was carried out at 50°C for 30 min, followed by an initial PCR activation step at 95°C for 15 min. The subsequent PCR conditions were 30 cycles of 15 s at 94°C, 30 s at 55°C, and 30 s at 72°C. The number of cycles required to produce a detectable product above the background level was measured and used to calculate the difference in the starting mRNA level for each sample. The relative mRNA levels of Dicer and CFP were normalized to that of β-actin.
Western blot analysis.
Total cell lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto ClearBlot membrane-P (ATTO) by electroblotting using a semidry blotting apparatus (ATTO) according to the manufacturer's instructions. After blocking with 5% ECL blocking reagent (Amersham Biosciences), the membrane was probed with appropriate primary antibodies specific for Dicer (1:200 dilution), FLAG tag (1:500 dilution), or catalase (1:200 dilution) and subsequently with horseradish peroxidase-conjugated secondary antibodies (donkey anti-rabbit IgG, 1:2,500 dilution; sheep anti-mouse IgG, 1:7,500 dilution). ECL or ECL Plus reagent (Amersham Biosciences) was used for immunological detection of the labeled proteins. Chemiluminescence was detected using an LAS1000UV mini-imager (Fuji Film) and quantified using Image Gauge software (Fuji Film).
Nucleotide sequence accession number.
The GenBank/EMBL/DDBJ accession number of the 437-bp amplified hamster Dicer partial cDNA is AB248867.