The FlaghMBD3a vector was kindly provided by F. Ishikawa (Kyoto University), pGEX-5X-3 mMBD3a and pGEX-5X-3 mMBD3b, antibodies against MBD3 were kindly provided by D. Reinberg (University of Medicine and Dentistry of New Jersey), pCLneo-mHDAC1-Myc and pME18s-Flag-mHDAC2 were kindly provided by C. Seiser (University of Vienna), and pGEX-MTA1/2/3 and pcDNA-Flag-MTA1/2/3 were kindly provided by W. M. Yang (National Cheng Hsing University).
HA mMBD3b was subcloned from pGEX-5X-3 mMBD3b to pcDNA3 by digestion with EcoRV and XhoI enzymes.
Cell lines and transfection.
NB4 cells and U937-PR9 were cultured at 37°C and 5% of CO2 in RPMI medium supplemented with 10% of fetal bovine serum. HEK 293T were cultured at 37°C and 5% of CO2 in Dulbecco modified Eagle medium supplemented with 10% of fetal bovine serum and then transfected by using the calcium phosphate coprecipitation method.
Immunoprecipitation and chromatin immunoprecipitation.
For immunoprecipitations, antibodies were coupled to protein A-Sepharose beads. Cell extracts were prepared in lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride, 0.4 U of aprotinin and leupeptin/ml) and incubated with beads overnight at 4°C. Beads were washed three times with lysis buffer complemented with additional 150 mM NaCl and 0.2% NP-40. Bound proteins were eluted with 2× Laemmli sample buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Input lanes show 10% of the lysates used for precipitation. The extracts were analyzed by Western blotting with antibodies specific for PML (PGM3), RARa (sc551), Mi2 (sc11378), MBD3 (sc9402), and MTA2 (sc28731) (Santa Cruz); Flag (Sigma); and HDAC1, HDAC2, and tubulin (Abcam).
For chromatin immunoprecipitation (ChIP), NB4 or U937-PR9 cells were cross-linked with 1% formaldehyde (Sigma) at 37°C for 10 min. Cells were rinsed twice with ice-cold phosphate-buffered saline and collected. ChIPs were performed and analyzed as described previously (4
) with antibodies specific for either HDAC1 (Abcam), RNA polymerase II (Convence), p300, CBP, acetylated H3, H3K27me3 (Upstate), MTA2, PML, HDAC2, and BRG1 (Santa Cruz). The immunoprecipitated DNA was quantified by real-time quantitative PCR (Roche LightCycler). The sequences of the PCR primers are available upon request.
Bisulfite genomic sequencing and DNA methyltransferase assay.
Bisulfite genomic sequencing was performed as described previously (32
). Immunoprecipitates were assayed for methyltransferase activity in a 100-μl reaction containing a 33-bp hemimethylated oligonucleotide substrate (500 ng), S
H]methionine (2 μl; 77 Ci/mmol), Tris-HCl (50 mM, pH 7.5), EDTA (5 mM), 50% glycerol, dithiothreitol (5 mM), and protease inhibitors. After incubation at 37°C for 1 h, unincorporated nuclides were removed by using Biospin chromatography columns (Bio-Rad), and the incorporation of radioactivity was determined by liquid scintillation counting.
Immunoprecipitation in the presence of ethidium bromide.
A total of 8 × 107 of cells was resuspended in 2 ml of lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% Tween 20) with proteinase inhibitors and then sonicated (Branson sonicator) at 10% output for 15 s. Then, 100 μg of ethidium bromide/ml was added to 1 ml of sonicated material, followed by incubation 30 min at 4°C. After 15 min of centrifugation at 14,000 rpm, the supernatant was collected and used for PML-RAR immunoprecipitation overnight. Immunoprecipitated material was then washed three times with lysis buffer supplemented with 300 mM NaCl, 100 μg of ethidium bromide/ml, and proteinase inhibitors.
RNA interference and retroviral infection.
The target sequence used to silence MBD3 was inserted as a short hairpin into the pRetroSuper (pRS) retroviral vector according to the manufacturer's recommendations (OligoEngine). pLKO.1-Puro lentiviral vectors with the short hairpin target sequence to silence MTA2 and Mi-2 were purchased from Sigma. pRS-Suz12 vector was kindly provided by K. Helin.
Portions (8 μg) of pRS-shMBD3, pRS-shSuz12, and pRS-scramble vectors were transfected with 4 μg of the pVSVG plasmid by using the calcium phosphate precipitation method into HEK-293T/GP2 cells. After 2 and 3 days, supernatants containing the retroviruses were collected. Human U937-PR9 or NB4 leukemic cells were spin infected for 90 min (3,200 rpm) in the presence of protamine sulfate (5 μg/ml) and viruses containing the shMBD3, shSuz12, or shScramble. After 36 h, infected cells were selected with puromycin (2 μg/ml for U937-PR9 or 1 μg/ml for NB4) for at least 72 h.
Next, 8-μg portions of pLKO.1-Puro-MTA2, pLKO.1-Puro-Mi-2, and pLKO.1-Puro-Random vectors were transfected with 4 μg of the pVSVG plasmid and 5 μg of the p8.91 plasmid by using the calcium phosphate precipitation method into HEK-293T/GP2 cells. After 2 and 3 days, supernatants containing lentiviruses were collected. U937-PR9 cells were spin infected for 90 min (3,200 rpm) in the presence of Polybrene (10 μg/ml), and viruses containing shMTA2, shMi-2, or shScramble. After 36 h, infected cells were selected with puromycin (2 μg/ml for U937) for at least 72 h.
For rescue of phenotype experiments, MBD3 from the pcDNA-Flag-MBD3 plasmid was mutated in bp 10 of the MBD3 short hairpin binding site by using a QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's recommendations. Mutated MBD3 (MBD3-rescue) was cloned into the retroviral pMSCV2.2-green fluorescent protein (GFP) plasmid.
Then, 8 μg of pMSCV2.2-MBD3-T369A-GFP and pMSCV2.2-GFP vectors was transfected with 4 μg of the pVSVG plasmid by the calcium phosphate precipitation method into HEK-293T/GP2 cells. After 2 and 3 days, supernatants containing retroviruses were collected. U937-PR9 RNAi-scramble and U937-PR9 RNAi-MBD3 cells were spin infected for 90 min (3,200 rpm) in the presence of protamine sulfate (5 μg/ml) and the virus containing the MBD3-rescue-GFP. After 36 h, the cells were GFP sorted.
The NBT assay was performed using a commercially available nitroblue tetrazolium (NBT; Sigma). A 0.2-ml portion of cell suspension at a density of 2 × 105 cells in RPMI-5% fetal bovine serum was mixed with 0.2 ml of filtered 0.2% NBT solution and 3 μl of TPA 1 μM, followed by incubation for 30 min at 37°C. Subsequently, cytocentrifuge slides were prepared (200 rpm, 4 min). NBT-positive cells were determined by scoring 500 cells under a light microscope. Slides were also stained 1 min with modified Wright-Giemsa stain (Sigma), rinsed in phosphate-buffered saline, stained 10 min in modified Giemsa stain (Sigma) 1:20 with water (Sigma), rinsed with water, and subjected to morphological examination under a light microscopy.
RNA purification and reverse transcription-PCR analysis.
RNA from U937-PR9 MBD3 RNA interference cells (RNAi MBD3) or from U937-PR9 control cells (RNAi control) after no treatment, after RA treatment (1 nM, 36 h), and after RA treatment (1 nM, 12 h) with subsequent Zn induction (100 mM Zn, 24 h) was extracted by using a Qiagen RNeasy minikit, retrotranscripted (avian myeloblastosis virus; Roche), and assayed for the expression of RARβ2 by using quantitative real-time PCR (Roche LightCycler). The sequences of the PCR primers are available upon request.