The Methyl Donor S-Adenosylmethionine Inhibits Active Demethylation of DNA

CMV-GFP (pEGFP-C1 from Clontech; GenBank^TM accession number U55763) was methylated in vitro by incubating 10 μg of plasmid DNA with 12 units of SssI CpG methyltransferase (New England Biolabs) in the recommended buffer containing 800 μm AdoMet for 3 h at 37 °C. Twelve units of SssI and 0.16 μmol of AdoMet were then added and the reaction was further incubated for 3 additional hours. The methylated plasmid was recovered by phenol/chloroform extraction and ethanol precipitation, and complete methylation was confirmed by observing full protection from HpaII digestion.

Human embryonal kidney HEK 293 cells (ATCC CRL 1573) were plated at a density of 7.5 × 10⁴/well in a 6-well dish and transiently transfected with 80 ng of CMV-GFP (methylated or mock methylated) using the calcium phosphate precipitation method as described previously (). 0.3 μm TSA was added 24 h post-transfection. After an additional 24 h, cells were treated with or without various concentrations of AdoMet or AdoHcy (2–8 mm). Cells were harvested 72 h post-transfection. Each experiment was performed in triplicate, and experiments were performed several times using different cultures of HEK 293 cells.

Whole cell extracts were prepared using radioimmunoprecipitation assay buffer according to the Santa Cruz Biotechnology protocol, and protein concentrations were determined using the Bradford reagent (Bio-Rad). 2.5 μg of protein were resolved on a 12.5% SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride membrane (Amersham Biosciences). After blocking the nonspecific binding with 5% skim milk, GFP protein was detected using rabbit polyclonal IgG (Santa Cruz, sc-8334) at 1:500 dilution, followed by peroxidase-conjugated anti-rabbit IgG (Sigma) at 1:5000, and enhanced chemiluminescence detection kit (Amersham Biosciences). Membranes were stained with 0.2% Ponceau S (Sigma) to determine loading of total protein in each lane. Both the Western blots and Ponceau-stained membranes were quantified using NIH Image 1.62 software, and the GFP signal was normalized to the total protein (which varied only slightly) in each lane.

DNA was extracted from HEK 293 cells using the DNeasy Tissue Kit (Qiagen). DNA was first digested with 50 units of EcoRI, followed by digestion with 20 units of either HpaII or MspI restriction enzymes. Samples were subjected to electrophoresis on a 1.5% agarose gel and then transferred to Hybond-N⁺ membrane (Amersham Biosciences). Blots were probed with a ³²P-labeled CMV-GFP cDNA probe (AvaII-Cfr101 fragment) synthesized using a random priming labeling kit (Roche Diagnostics). Membranes were hybridized at 68 °C for 4–6 h in a buffer containing 0.5 m sodium phosphate, pH 6.8, 1 mm EDTA, 7% SDS, and 0.2 mg/ml herring sperm DNA. Following hybridization, the membranes were washed twice for 10 min in a 5% SDS, 0.04 m sodium phosphate, pH 6.8, 1 mm EDTA solution, and then four times for 10 min in the same solution containing 1% SDS. The demethylation assay measures the fraction of GFP molecules that were demethylated using HpaII restriction enzyme, which cleaves unmethylated CCGG but does not cleave methylated CCGG sequences. The methylated GFP DNA remains intact following HpaII digestion and is identical to the fragment obtained following EcoRI digestion (indicated by M in Fig. 2B), whereas the unmethylated GFP DNA is cleaved by HpaII resulting in a 0.5-kb fragment (indicated by U in Fig. 2B). We scanned each HpaII digested lane and measured the intensity of the total signal hybridizing with the GFP probe in the same HpaII lane (including the unmethylated U and methylated M fragments), this value is equal to 100% of GFP molecules in the lane. We then determined the intensity of the unmethylated signal per HpaII lane, and divided this value (U) by the total signal for GFP (U + M) in the same HpaII lane. To exclude the possibility that the HpaII digestion was skewed by differences in loading, we used the ethidium bromide-stained gels as loading controls for the corresponding Southern blots. We normalized the values obtained upon the calculation [M/(U + M)] × 100 to the amount of DNA in each HpaII lane as determined by quantification of the ethidium bromide-stained gels by NIH Image 1.62. The results of three independent experiments were quantified by densitometry (NIH Image 1.62).

AdoMet was prepared as a 50 mm solution in distilled water by dissolving lyophilized powder (Sigma) in distilled water. AdoHcy was purchased from Sigma and dissolved in distilled water at a 50 mm concentration.

A fragment containing human MBD2/dMTase was excised from pCR2.1-dMTase () with BamHI and XhoI and transferred to the Baculovirus expression transfer vector pBlueBacHis2 C (Invitrogen). PBlueBacHis2 C-MBD2/dMTase and Bac-N-Blue viral DNA were cotransfected into the Sf9 insect cell line, and recombinant viruses were isolated, identified, and amplified according to the manufacturer's protocol (Invitrogen) with no modifications. High titer P3 viral stocks were used for infections. Insect Sf9 cells were cultured in spinner flasks to a density of 2.5 × 10⁶ cells/ml in Grace's insect cell culture medium supplemented (1×) from Invitrogen. For infection, 5 × 10⁶ cells were plated in 10-cm tissue culture plates (Sarstedt) and allowed to settle and attach for 30 min. The culture medium was removed and was replaced with 10 ml of the same medium containing MBD2/dMTase virus at a multiplicity of infection of 10. The cells were cultured with the virus for 5 days at 27 °C and were then harvested by scraping in cold phosphate-buffered saline. Cell pellets from 10 plates were frozen and kept at -70 °C until they were used for enzyme purification. Frozen pellets were thawed in 5 ml of lysis buffer (10 mm Tris-HCl, pH 8.0, 5 mm MgCl₂, 500 mm NaCl, 0.05% Tween 20, 10% glycerol, and 10 mm imidazole) containing 1 μg/ml of the following protease inhibitors: aprotinin, leupeptin, and Pefablock. Protease inhibitors were added to all the solutions used in the purification. The homogenates were subjected to two cycles of freezing and thawing (5 min per step). DNA in the homogenate was sheered by passing through an 18.5-gauge needle 10 times. The extracts were then subjected to 15 cycles of sonication (10 s burst, 10 s gap per cycle at 20% of maximal output). The extracts were centrifuged at 10,000 × g for 35 min. The supernatant was transferred into a fresh tube and was recentrifuged for additional an 25 min at 15,000 × g. The extract was filtered through a 5-micron filter to remove any particulate matter and the buffer was exchanged on a PD-10 buffer exchange column (Amersham Biosciences) with buffer L (10 mm Tris-HCl, pH 8.0, 10 mm MgCl₂) containing 50 mm NaCl. Recombinant MBD2/dMTase was partially purified by Q-Sepharose (Amersham Biosciences) ion exchange chromatography. Q-Sepharose beads (1 ml of swollen beads) were washed extensively and pre-equilibrated with buffer L containing 50 mm NaCl and divided into 3 equal aliquots. The cell extracts were sequentially bound three times to the 3 aliquots of Q-Sepharose beads in batch in 15-ml tubes by shaking gently on a Nutator for 45 min at 4 °C. Following each binding step, the bound beads and unbound supernatant were separated by centrifugation for 2 min at 1000 × g and the supernatant was transferred into a new tube and bound with new pre-equilibrated beads. The bound beads from the three binding steps were joined and resuspended in lysis buffer. The beads were washed in batchs 4 times with 5 ml of buffer L + 50 mm NaCl. For each washing step, the beads were incubated with the wash solution for 15 min and then separated from the wash supernatant by centrifugation for 2 min at 1000 × g. Following washing, the proteins were eluted in batchs (30 min per step) with a stepwise NaCl gradient in buffer L. Each elution step was analyzed for in vitro demethylase activity and for the presence of the recombinant His-tagged MBD2/dMTase by a Western blot analysis using the anti-Xpress antibody from Invitrogen as previously described. MBD2/dMTase peak elution is at the 0.4 m NaCl step. No demethylase activity was observed in the same fractions prepared in a similar manner from uninfected Sf9 cells. For concentration of the 0.4 m NaCl fraction, a Microcon YM-10 concentrator (Millipore) was used at 3300 × g and 4 °C. Spinning time varied according to the volume, and was 25 min for 500 μl.

10 × 10-cm tissue culture plates of HEK 293 cells were used to prepare cell extracts as outlined in the previous section. Q-Sepharose fractionation was performed also as described above and as previously described ().

Methylation of Substrate—Typically, 25 μg of DNA from Micrococcus lysodeikticus (Sigma, Type XI, highly polymerized) were methylated with M.SssI (60 units, New England Biolabs) and AdoMet (3.2 mm, New England Biolabs) in methylation buffer (NEbuffer2, New England Biolabs) in the presence of 50 mm EDTA for 3-4 h at 37 °C. Fresh AdoMet (3.2 mm) and enzyme (40 units) were then added before incubating at 37 °C for an additional 3–4 h. To achieve complete methylation, methylation was repeated after AdoHcy, which is a product of the methylation reaction and inhibits DNA methylation, and was removed using a Microcon 10 concentrator (Millipore) according to the manufacturer's protocol. The degree of methylation was verified by methylation sensitive restriction enzyme analysis (MspI-HpaII digestion) on aliquots of the reaction mixture. The DNA was purified by phenol-chloroform extraction (one part of either phenol or chloroform per three parts reaction mixture). Unincorporated nucleotides were removed by a Nap5 gel filtration chromatography (Amersham Biosciences) column. The Nap5 column was equilibrated with demethylation buffer (10 mm Tris-HCl, 5 mm MgCl₂, pH 7.0). DNA containing fractions were combined, concentrated on a Microcon 10, and subjected to a second Nap5 desalting column. DNA containing fractions were again concentrated as described above.

[α-32P]dGTP Labeling of DNA—We then prepared either methylated or unmethylated DNA that is ³²P-labeled at G, the 3′ neighbor of the methylated C, as previously described () with the following modifications. 5 μg of either methylated or unmethylated DNA in 35 μl of double distilled water were denatured and annealed to a hexanucleotide primer by boiling for 10 min in the presence of 3 μl of random hexanucleotide mixture (0.2 A₂₆₀) (Roche Diagnostics). The primed DNA was then subjected to template-directed extension with the Klenow fragment of DNA polymerase I in the presence of labeled [α-³²P]dGTP, either methyl-dCTP (for methylated DNA) or dCTP for unmethylated DNA, dTTP, and dATP. The 3′ phosphate of all the 5′ neighbors of G including either C or methyl-C is labeled by this procedure. The labeling was performed in polymerase buffer (50 mm NaCl, 6.6 mm Tris-HCl, 6.6 mm MgCl₂, 1 mm dithiothreitol, pH 7.4) with Klenow fragment I (10 units, Roche Diagnostics), methyl-dCTP (1 mm), and dCTP (1 mm), respectively, dATP, dTTP (1 mm each), and [α-³²P]dGTP (50 μCi, PerkinElmer Life Sciences) for 3 h at 37 °C. The reaction mixture was extracted with phenol and chloroform (one part of each per three parts reaction mixture). Trichloroacetic acid precipitation (2 ml of 10% trichloroacetic acid, 20 μg of herring sperm DNA) of an aliquot showed typically 80–95% labeling efficiency (260,000–320,000 cpm/μl, total of 150 μl). The DNA was purified by eluting it twice from a Nap5 column and concentrating on a Microcon 10 as described above with the following variation: distilled water was used for prewashing and elution in the second column. The final concentration was 5 ng/μl and the specific activity was typically 8.0–8.8 × 10⁶ cpm/μg.

A typical reaction mixture (50 μl) consisted of 25 ng of ³²P-prelabeled DNA (prepared as described above) incubated in demethylation buffer (10 mm Tris-HCl, 5 mm MgCl₂, pH 7.0) with either the purified MBD2/dMTase (5 μl, ∼5 ng) or the purified demethylase activity from HEK 293 cells (5 μl) for 24 h at 37 °C in either the absence or presence of AdoMet and AdoHcy, respectively. The DNA was extracted from the enzyme by incubation in 2 volumes of DNA extraction buffer (10 mm Tris-HCl, 0.5 m NaCl, 1% SDS) containing 0.1 unit of proteinase K (Roche Diagnostics) at 50 °C for 2 h. Subsequent phenol-chloroform extraction (one part of either phenol or chloroform per three parts of reaction volume) in the presence of tRNA (50 μg) as a carrier and ethanol precipitation with salt and 95% ethanol resulted in almost quantitative recovery of the input DNA. The DNA pellets were resuspended in distilled water (8 μl) and digested with microccocal nuclease to ³²P-labeled 3′ mononucleotides as described elsewhere (, ). The labeled mononucleotides were separated by thin layer chromatography and visualized by autoradiography on a phosphorimaging plate, and the levels of cytosine (C) and 5-methylcytosine (mC) were quantified by the MCID-M4 software (Imaging Research Inc.). The percent demethylation (C/(C + mC)) was calculated per each sample and then normalized to the value obtained for the demethylase reaction in the absence of inhibitor (0 mm AdoMet/AdoHcy).