Promoter-specific Activation and Demethylation by MBD2/Demethylase

MBD2 is the only member of a family of methyl-CpG-binding proteins that has been reported to be both a transcriptional repressor and a DNA demethylase (dMTase). To understand the apparently contradictory function of MBD2/dMTase, we studied the effects of dMTase overexpression on the activity of various in vitro methylated promoters transiently transfected into HEK293 cells. We found that forced expression of a MBD2/dMTase expression vector (His-dMTase) differentially activated two methylated reporters, pSV40-CAT (the SV40 enhancerless promoter adjacent to the chloramphenicol acetyltransferase (CAT) reporter gene) and pGL2T+I4xTBRE (a region of the p21 promoter next to the luciferase reporter gene), in a time- and dose-dependent manner. His-dMTase increased pSV40-CAT expression by 3–10-fold after 96 h, while pGL2T+I4xTBRE expression was increased by 2–3-fold after only 48 h and did not further increase at 96 h. Gene activation was not universal because no effect was seen with the p19-ARF promoter. We then assessed whether activation might be due to demethylation within the promoter region. Using bisulfite mapping, we found that exogenous expression of His-dMTase induced demethylation at 8 of the 10 CpG sites within the SV40 promoter. The observation that His-dMTase increases the demethylase activity in the cells was also confirmed using an in vitro CpG demethylase assay with a mC32pG oligonucleotide substrate and purified Q-Sepharose fractions from HEK293 cells transfected with His-dMTase or empty pcDNA3.1His vector. We propose that a single protein possessing both repressor and demethylase functions has evolved to coordinate a program that requires suppression of some methylated genes and activation of others. MBD2 is the only member of a family of methyl-CpG-binding proteins that has been reported to be both a transcriptional repressor and a DNA demethylase (dMTase). To understand the apparently contradictory function of MBD2/dMTase, we studied the effects of dMTase overexpression on the activity of various in vitro methylated promoters transiently transfected into HEK293 cells. We found that forced expression of a MBD2/dMTase expression vector (His-dMTase) differentially activated two methylated reporters, pSV40-CAT (the SV40 enhancerless promoter adjacent to the chloramphenicol acetyltransferase (CAT) reporter gene) and pGL2T+I4xTBRE (a region of the p21 promoter next to the luciferase reporter gene), in a time- and dose-dependent manner. His-dMTase increased pSV40-CAT expression by 3–10-fold after 96 h, while pGL2T+I4xTBRE expression was increased by 2–3-fold after only 48 h and did not further increase at 96 h. Gene activation was not universal because no effect was seen with the p19-ARF promoter. We then assessed whether activation might be due to demethylation within the promoter region. Using bisulfite mapping, we found that exogenous expression of His-dMTase induced demethylation at 8 of the 10 CpG sites within the SV40 promoter. The observation that His-dMTase increases the demethylase activity in the cells was also confirmed using an in vitro CpG demethylase assay with a mC32pG oligonucleotide substrate and purified Q-Sepharose fractions from HEK293 cells transfected with His-dMTase or empty pcDNA3.1His vector. We propose that a single protein possessing both repressor and demethylase functions has evolved to coordinate a program that requires suppression of some methylated genes and activation of others. methyl-CpG binding domain protein demethylase trichostatin A chloramphenicol acetyltransferase human embryonic kidney methylated nucleosome remodeling and deacetylase The epigenome consists of an additional component that is part of the covalent structure of the genome, a coating of methyl groups. In vertebrates, 80% of cytosine residues within the dinucleotide sequence CpG are modified by methylation in a pattern that is tissue-specific and that is formed during development and maintained in somatic cells (1Razin A. Szyf M. Biochim. Biophys. Acta. 1984; 782: 331-342Crossref PubMed Scopus (258) Google Scholar). It has been well established that the DNA methylation pattern is maintained exclusively by DNA methyltransferase activities, but we have recently proposed that DNA demethylase activities might also participate in the process (2Bhattacharya S.K. Ramchandani S. Cervoni N. Szyf M. Nature. 1999; 397: 579-583Crossref PubMed Scopus (548) Google Scholar, 3Cervoni N. Szyf M. J. Biol. Chem. 2001; 276: 40778-40787Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 4Cervoni N. Detich N. Seo S.B. Chakravarti D. Szyf M. J. Biol. Chem. 2002; 277: 25026-25031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) and that the methylation pattern is a steady state balance of reversible methylation-demethylation reactions (5Szyf M. Trends Pharmacol. Sci. 2001; 22: 350-354Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6Szyf M. Detich N. Prog. Nucleic Acids Res. Mol. Biol. 2001; 69: 47-79Crossref PubMed Google Scholar). We have shown that histone acetylation promotes active demethylation of ectopically methylated genes (3Cervoni N. Szyf M. J. Biol. Chem. 2001; 276: 40778-40787Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) and that inhibitors of histone acetylation inhibit demethylation (4Cervoni N. Detich N. Seo S.B. Chakravarti D. Szyf M. J. Biol. Chem. 2002; 277: 25026-25031Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). It is well documented that the state of activity of a gene, the chromatin structure, and DNA methylation are correlated (7Razin A. EMBO J. 1998; 17: 4905-4908Crossref PubMed Scopus (663) Google Scholar) such that areas of the genome that are methylated are usually less expressed. One molecular mechanism that explains this relationship has recently been elucidated. Repressor complexes are recruited to methylated DNA via the binding of methyl-CpG binding domain proteins (MBDs).1 These complexes contain proteins that have histone deacetylase and chromatin remodeling activities, leading to the formation of a more compact and transcriptionally inactive chromatin (8Nan X., Ng, H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2804) Google Scholar). The earliest discovered MBD, MeCP2, has been found to associate with the Sin3a co-repressor complex (8Nan X., Ng, H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2804) Google Scholar) and can also repress transcription in a histone deacetylase-independent manner (9Yu F. Thiesen J. Stratling W.H. Nucleic Acids Res. 2000; 28: 2201-2206Crossref PubMed Scopus (89) Google Scholar). MBD1, MBD2, and MBD3 were later discovered and were also shown to be involved in transcriptional repression (for review, see Ref. 10Ballestar E. Wolffe A.P. Eur. J. Biochem. 2001; 268: 1-6Crossref PubMed Scopus (281) Google Scholar). In contrast, we have reported that MBD2 is an enzyme (dMTase) capable of actively demethylating DNA (2Bhattacharya S.K. Ramchandani S. Cervoni N. Szyf M. Nature. 1999; 397: 579-583Crossref PubMed Scopus (548) Google Scholar). This activity was shown bothin vitro (2Bhattacharya S.K. Ramchandani S. Cervoni N. Szyf M. Nature. 1999; 397: 579-583Crossref PubMed Scopus (548) Google Scholar) and in vivo (3Cervoni N. Szyf M. J. Biol. Chem. 2001; 276: 40778-40787Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). A demethylase is expected to activate genes by removing the repressive methyl residues. The assignment of a demethylase function to a protein that was independently discovered as a recruiter of repressor complexes has triggered obvious controversy in the field (11Ng H.H. Zhang Y. Hendrich B. Johnson C.A. Turner B.M. Erdjument-Bromage H. Tempst P. Reinberg D. Bird A. Nat. Genet. 1999; 23: 58-61Crossref PubMed Scopus (0) Google Scholar), and several groups have reported that they failed to confirm the demethylase activity of MBD2 (11Ng H.H. Zhang Y. Hendrich B. Johnson C.A. Turner B.M. Erdjument-Bromage H. Tempst P. Reinberg D. Bird A. Nat. Genet. 1999; 23: 58-61Crossref PubMed Scopus (0) Google Scholar, 12Boeke J. Ammerpohl O. Kegel S. Moehren U. Renkawitz R. J. Biol. Chem. 2000; 275: 34963-34967Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 13Wade P.A. Gegonne A. Jones P.L. Ballestar E. Aubry F. Wolffe A.P. Nat. Genet. 1999; 23: 62-66Crossref PubMed Scopus (713) Google Scholar). In this study we tested the hypothesis that MBD2 is a multifunctional protein and that its activity might depend on the context of the promoter with which it interacts. By examining the effects of MBD2/dMTase expression on the activity of various reporter constructs methylated in vitro and transfected into HEK293 cells, we found that MBD2/dMTase differentially activated some but not all promoters in a time- and concentration-dependent manner. Using bisulfite mapping, we found that exogenous expression of MBD2/dMTase induced demethylation within the SV40 promoter, and we also confirmed the demethylase activity of MBD2/dMTase in vitro. These data support our hypothesis that the complex functional role of this protein depends on the promoter context. Enhancerless pSV40-CAT (GenBankTM accession no. X65320), pGL2T+I4xTBRE (14Datto M.B., Yu, Y. Wang X.F. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar), or p19-ARF-LUC (kindly provided by Dr. V. Lobanenkov) were methylated in vitro by incubating 10 μg of plasmid DNA with 12 units of Sss1 CpG methyltransferase (New England Biolabs) in the recommended buffer containing 800 μm S-adenosylmethionine for 3 h at 37 °C. Another 12 units of Sss1 and 0.16 μmol of S-adenosylmethionine were then added, and the reaction was further incubated another 3 h. The methylated plasmid was recovered by phenol/chloroform extraction and ethanol precipitation, and complete methylation was confirmed by observing full protection from HpaII digestion. HEK293 cells were plated at a density of 7 × 104/well in a six-well dish and transiently transfected with 100 ng of reporter plasmid (methylated or mock-methylated) and 1.2 μg of one of the following plasmids: pcDNA3.1HisB vector (Invitrogen), pcDNA-His-dMTase (2Bhattacharya S.K. Ramchandani S. Cervoni N. Szyf M. Nature. 1999; 397: 579-583Crossref PubMed Scopus (548) Google Scholar), which contains a His-tagged human MBD2b/demethylase cDNA as described in the Ref. 2Bhattacharya S.K. Ramchandani S. Cervoni N. Szyf M. Nature. 1999; 397: 579-583Crossref PubMed Scopus (548) Google Scholar, AdTrack-MeCP2 (constructed from GST-MeCP2 kindly provided by Dr. X. Nan; Ref. 15Nan X. Meehan R.R. Bird A. Nucleic Acids Res. 1993; 21: 4886-4892Crossref PubMed Scopus (493) Google Scholar), or pcDNA3.1-Sp1 (16Vallian S. Chin K.V. Chang K.S. Mol. Cell. Biol. 1998; 18: 7147-7156Crossref PubMed Scopus (102) Google Scholar) using the calcium phosphate precipitation method as described previously (17Rouleau J. Tanigawa G. Szyf M. J. Biol. Chem. 1992; 267: 7368-7377Abstract Full Text PDF PubMed Google Scholar). 0.3 μm trichostatin A (TSA) was added 24 h post-transfection, and cells were harvested after 48 or 96 h. Chloramphenicol acetyltransferase (CAT) assays were performed as described previously (17Rouleau J. Tanigawa G. Szyf M. J. Biol. Chem. 1992; 267: 7368-7377Abstract Full Text PDF PubMed Google Scholar), and luciferase activity was assessed using the Promega luciferase assay system. The activity of each extract was measured in triplicate and then normalized to the protein concentration. Fold induction was calculated relative to the activity observed with the HisB vector alone. Experiments were performed several times using different cultures of HEK293 cells and different preparations of plasmids. For dose curve experiments, transfections were performed in triplicate using increasing amounts of HisB or His-dMTase vector (0.05, 0.1, 0.6,1.2, and 3 μg). Cells were then harvested after 96 h for CAT or luciferase assays. Fold induction was calculated relative to the activity observed with the HisB vector alone at each concentration. Bisulfite mapping was performed as described previously (18Clark S.J. Harrison J. Paul C.L. Frommer M. Nucleic Acids Res. 1994; 22: 2990-2997Crossref PubMed Scopus (1627) Google Scholar) with minor modifications. The SV40 promoter sequence was amplified from 5 μg of sodium bisulfite-treated DNA using the following primers: 5′-AAGGGGGATGTGTTGTAAG-3′ (sense) and 5′-CTAAAATACCTCAAAATATTCTT-3′ (antisense). PCR products were then used as templates for subsequent nested PCRs using the primers 5′-GGTTAGTGAATTTTAGATTTGT-3′ (sense) and 5′-TATATCCAATAATTTTTTTCTCC-3′ (antisense). PCR products were subcloned using the TA cloning kit (Invitrogen), and clones were then sequenced using the T7 sequencing kit (Amersham Biosciences). Whole cell extracts were prepared using radioimmune precipitation assay buffer according to the protocol from Santa Cruz Biotechnology. 50 μg of extract were resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. Transfected His-dMTase protein was detected either by anti-MBD2 sheep polyclonal IgG (Upstate Biotechnologies no. 07198) according to the manufacturer's protocol or by anti-Xpress mouse monoclonal IgG antibody (Invitrogen R910-25, which recognizes the Xpress epitope within the pcDNA3.1His vector) at 1:5000 followed by peroxidase-conjugated antimouse IgG (Jackson Immunoresearch) at 1:20,000. An enhanced chemiluminescence detection kit was used for both (Amersham Biosciences). HEK293 cells were transfected with 10 μg of His-dMTase/10-cm plate (×10) or left untransfected, and nuclear extracts were prepared 48 h later as described previously (19Szyf M. Bhattacharya S.K. Methods Mol. Biol. 2002; 200: 163-176PubMed Google Scholar). Approximately 8.5 mg of extract (1.4 ml) was diluted to 50 mm NaCl with 10 ml of buffer L (10 mm Tris-HCl, 10 mm MgCl2, pH 7.8) containing a 1 μg/ml concentration of each of the following protease inhibitors: Pefabloc, aprotinin, and leupeptin. 2 ml of Q-Sepharose beads (Amersham Biosciences) were washed three times with 8 ml of buffer L + 50 mm NaCl and then pre-equilibrated for 30 min in the same buffer. Each extract (HEK or HEK + dMTase) was then subjected to 4 × 40-min bindings, each with 0.25 ml of Q-Sepharose, with rotating at 4 °C. The beads were then pooled (1 ml/sample) and washed 5 × 10 min, each with 4 ml of buffer L + 50 mm NaCl. Batch elution was then performed with 5 × 1 ml of buffer L, each containing the following concentrations of NaCl: 0.2, 0.4, 0.6, 0.8, and 1.0 m for 10 min each. The different fractions, flow through, and washes were then assayed for demethylase activity. Demethylase activity was measured using a methylated (m) C32pG oligonucleotide substrate as described previously (20Szyf M. Bhattacharya S.K. Methods Mol. Biol. 2002; 200: 155-161PubMed Google Scholar) with minor modifications. 1 μl of a mCpG oligonucleotide substrate ([α-P32]dGTP-labeled) was incubated with 30 μl of buffer L and 20 μl of each of the purification samples for 48 h at 37 °C. Samples were then subjected to a phenol/chloroform extraction followed by ethanol precipitation and resuspension in 8 μl of double distilled H20. 1 μl of 10× micrococcal nuclease buffer (250 mm Tris-HCl, 10 mm CaCl2) and 1 μl of micrococcal nuclease were added followed by an overnight incubation at 37 °C. 2 μl of each sample were then resolved by thin layer chromatography and visualized by autoradiography. Since MBD2b was found to act as a DNA demethylase (2Bhattacharya S.K. Ramchandani S. Cervoni N. Szyf M. Nature. 1999; 397: 579-583Crossref PubMed Scopus (548) Google Scholar, 3Cervoni N. Szyf M. J. Biol. Chem. 2001; 276: 40778-40787Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) and since promoter demethylation is associated with gene activation (21Plumb J.A. Strathdee G. Sludden J. Kaye S.B. Brown R. Cancer Res. 2000; 60: 6039-6044PubMed Google Scholar), we first wanted to determine whether expression of MBD2/dMTase to promoter We performed various using several in vitro methylated reporter A of data has established that in vitro methylation can genes genes are ectopically into cells H. R. Y. N. A. Biol. PubMed Google Scholar). both the SV40 and (a of the of the p21 reporter constructs were by in vitro methylation 1 of His-dMTase to activation of the SV40 promoter, but only after 96 h, while the promoter was activated at both 48 and 96 h were with which transcription by histone and which has also been found to DNA demethylation (3Cervoni N. Szyf M. J. Biol. Chem. 2001; 276: 40778-40787Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). was used as a for activation since both the SV40 and promoters are in sites 1 The transcriptional activation seen with MBD2/dMTase is not universal since exogenous MBD2/dMTase was not to activate the p19-ARF promoter after 96 h 1 on different the for methylated promoters by are in HEK293 cells, then this might MBD2 not methylated promoters in HEK293 cells. To this we tested whether another member of the MeCP2, repress methylated The shown in 1 that the SV40 promoter, with (9Yu F. Thiesen J. Stratling W.H. Nucleic Acids Res. 2000; 28: 2201-2206Crossref PubMed Scopus (89) Google Scholar), and the for methylated genes by is active in HEK293 cells. In our that MBD2/dMTase can differentially activate methylated To further that MBD2/dMTase can act as a transcriptional we transfected the methylated SV40 or with different amounts of seen in A and while we observed a dose-dependent increase in activation of both promoters also in to the of transfected His-dMTase as well as in the of to the in A using an antibody MBD2 that the His-dMTase is in HEK293 cells 2 and that the protein with the transcriptional activation These with in that MBD2/dMTase can function as a dose-dependent of gene which is both time- and One for the transcriptional activation by His-dMTase is that it promoter To that the His-dMTase an active demethylase enzyme in our we subjected nuclear extracts from HEK293 cells transfected with His-dMTase to chromatography on Q-Sepharose to the demethylase and the demethylase activity with HEK293 cells. were with a of tested for the of His-dMTase by a using anti-Xpress and also assayed for demethylase activity. The was by the of to within a mC32pG oligonucleotide substrate using thin layer shown in His-dMTase at and m NaCl. These fractions a increase in demethylase of the is to in the m in to the which demethylase activity in the m Since demethylation within a promoter is associated with transcriptional activation (21Plumb J.A. Strathdee G. Sludden J. Kaye S.B. Brown R. Cancer Res. 2000; 60: 6039-6044PubMed Google Scholar), we next wanted to determine whether this was a mechanism by which MBD2/dMTase overexpression to the activation of the SV40 promoter. of methylated with His-dMTase or empty vector as a we used bisulfite mapping to the 10 different CpG sites within the SV40 promoter shown in expression of dMTase increased the of demethylation at 8 of the 10 CpG of the CpG sites or methylated in the clones with the of Since 20 and 30 clones were sequenced from three experiments, it is that this is a These are also with that exogenous expression of MBD2/dMTase to increased demethylation within a promoter (3Cervoni N. Szyf M. J. Biol. Chem. 2001; 276: 40778-40787Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) and that expression of MBD2/dMTase is correlated with demethylation within the promoters of genes M. H. Cancer 2001; PubMed Scopus Google Scholar). We also assessed the CAT activity from the same transfections used for bisulfite mapping and found as in our experiments, dMTase overexpression to transcriptional activation of the SV40 promoter 4 This the hypothesis that the mechanism by which dMTase transcriptional activation is by demethylating the promoter and an chromatin of transcriptional repressor study that MBD2/dMTase can act as a transcriptional with its role as a DNA the activation observed is on several promoter and of are with that MBD2/dMTase is an active demethylase in vitro (2Bhattacharya S.K. Ramchandani S. Cervoni N. Szyf M. Nature. 1999; 397: 579-583Crossref PubMed Scopus (548) Google Scholar) and that of exogenous dMTase can to demethylation in cells with a increase in gene expression (3Cervoni N. Szyf M. J. Biol. Chem. 2001; 276: 40778-40787Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). The activation of methylated promoters by MBD2/dMTase is as is the This the of that the DNA from complete demethylation and activation of it is in that MBD2 as a transcriptional repressor (11Ng H.H. Zhang Y. Hendrich B. Johnson C.A. Turner B.M. Erdjument-Bromage H. Tempst P. Reinberg D. Bird A. Nat. Genet. 1999; 23: 58-61Crossref PubMed Scopus (0) Google Scholar, 12Boeke J. Ammerpohl O. Kegel S. Moehren U. Renkawitz R. J. Biol. Chem. 2000; 275: 34963-34967Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, Zhang Y. 2001; Google Scholar, M. A. A. Y. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), the data not previously data the in are in all the repression by MBD2/dMTase, transcriptional assays were performed from 24 to 48 h Since we not see activation of the SV40 promoter 96 h post-transfection, it is that some of the promoters in be activated by MBD2/dMTase provided that the is The that a is for activation by be by data that demethylation of ectopically methylated DNA in cells is a process (3Cervoni N. Szyf M. J. Biol. Chem. 2001; 276: 40778-40787Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). (11Ng H.H. Zhang Y. Hendrich B. Johnson C.A. Turner B.M. Erdjument-Bromage H. Tempst P. Reinberg D. Bird A. Nat. Genet. 1999; 23: 58-61Crossref PubMed Scopus (0) Google Scholar, M. A. A. Y. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) used the DNA binding domain to to promoters that at the effect of MBD2 on methylated DNA as in this which also for some of the not all promoters are by MBD2, and not all concentrations of transfected MBD2 For J. Ammerpohl O. Kegel S. Moehren U. Renkawitz R. J. Biol. Chem. 2000; 275: 34963-34967Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) that the promoter was by of MBD2 no and in a study by M. A. A. Y. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), concentrations of transfected alone did not repress reporter The data are with our that MBD2 no effect on the p19-ARF promoter 1 and that the effect on transcription we observed is the dose activation with the promoter A and MBD2 has been found to associate with the repressor complex Zhang Y. 2001; Google Scholar), it was not purified as part of this complex Y. G. Reinberg D. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and is that the complex associate with different not only MBD2, on the state of the cell Zhang Y. 2001; Google Scholar). it is that and within promoters MBD2/dMTase act as a transcriptional repressor by the it is that in a different cell and within different promoters MBD2 act independently of as a demethylase and In support of the expression of dMTase is correlated with demethylation within the promoter of and genes M. H. Cancer 2001; PubMed Scopus Google Scholar). In a has that the of MBD2, formed that associated with DNA at the with the activation of the embryonic genome, and also associated with the active J. M. M. D. Ballestar E. B.M. F. 2002; PubMed Scopus Google Scholar). proteins have been found to repressor and such as H. Biochim. Biophys. Acta. 2000; Google Scholar), Gene 2001; PubMed Scopus Google Scholar), and the family of proteins J. J. Cell. 2001; PubMed Scopus Google Scholar), it is that MBD2 is a protein with It is that both repressor and demethylase functions in one protein to coordinate a program of gene expression that requires suppression of some methylated genes and activation of others. be to determine are the involved in the role of MBD2/dMTase in

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