Demethylase Activity Is Directed by Histone Acetylation

Mammalian genomes are compartmentalized into dense inactive chromatin that is hypermethylated and active open chromatin that is hypomethylated. It is generally accepted that this bimodal pattern of methylation is established during development and is then faithfully inherited through subsequent cell divisions by a maintenance DNA methyltransferase (DNMT1). The pattern of methylation is believed to direct local histone acetylation states. In contrast to this well accepted consensus, we show here using a transient transfection model that an active demethylase is involved in shaping patterns of methylation in somatic cells. Demethylase activity is directed by the state of histone acetylation, and therefore, the resulting methylation pattern is determined by local histone acetylation states contrary to the accepted model. Our data support a new model suggesting that the pattern of methylation is maintained by a dynamic balance of methylation and demethylation activities and the local state of histone acetylation. This provides a simple mechanism for explaining why active genes are not methylated. Mammalian genomes are compartmentalized into dense inactive chromatin that is hypermethylated and active open chromatin that is hypomethylated. It is generally accepted that this bimodal pattern of methylation is established during development and is then faithfully inherited through subsequent cell divisions by a maintenance DNA methyltransferase (DNMT1). The pattern of methylation is believed to direct local histone acetylation states. In contrast to this well accepted consensus, we show here using a transient transfection model that an active demethylase is involved in shaping patterns of methylation in somatic cells. Demethylase activity is directed by the state of histone acetylation, and therefore, the resulting methylation pattern is determined by local histone acetylation states contrary to the accepted model. Our data support a new model suggesting that the pattern of methylation is maintained by a dynamic balance of methylation and demethylation activities and the local state of histone acetylation. This provides a simple mechanism for explaining why active genes are not methylated. DNA methyltransferase green fluorescence protein enhanced GFP trichostatin A DNA demethylase chloramphenicol acetyl transferase micrococcal nuclease cytomegalovirus polymerase chain reaction base pair(s) kilobase(s) chromatin immunoprecipitation A hallmark of mammalian genomes is the compartmentalization of the genome into dense inactive chromatin that is hypermethylated and active, open chromatin that is hypomethylated (1Razin A. Cedar H. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2725-2728Crossref PubMed Scopus (155) Google Scholar). However, the mechanism responsible for establishing this tight relationship remains unclear. The accepted model is that a sequence of methylation and demethylation events fashion the methylation pattern during development, but it is then faithfully inherited by a semiconservative DNA methyltransferase, DNMT11(2Razin A. Riggs A.D. Science. 1980; 210: 604-610Crossref PubMed Scopus (1509) Google Scholar). Methylation of newly synthesized DNA is exclusively determined by the state of methylation of the parental strand. The pattern of methylation is therefore believed to be fixed in somatic cells. Based on the assumption that DNA conserves its pattern of methylation in somatic cells, numerous experiments used transiently transfected methylated DNA to study the effects of DNA methylation on gene expression. In many of these studies the state of methylation of these ectopically methylated genes following transfection was not determined assuming that the pattern of methylation of the transfected gene did not change. Methylated DNA is associated with methyl CpG binding proteins, such as MeCP2, which reside in a complex with histone deacetylase activity (3Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2240) Google Scholar). The current model is therefore that the pattern of methylation dictates the state of histone acetylation and chromatin configuration (4Eden S. Hashimshony T. Keshet I. Cedar H. Thorne A.W. Nature. 1998; 394: 842Crossref PubMed Scopus (239) Google Scholar). This attractive model explains the compartmentalization of the genome and its inheritance in somatic cells, but it can not explain how genes are demethylated upon their activation. An alternative and opposite interpretation of the tight correlation between histone acetylation is that active chromatin causes demethylation of associated sequences. Such a model can explain why active genes are not methylated and how their unmethylated state is maintained through cell division. This hypothesis that active chromatin can cause demethylation is supported by previous data. Treatment of mammalian cells with general histone deacetylase inhibitors can cause global demethylation of human Epstein-Barr virus producer cell lines' genomes (5Szyf M. Eliasson L. Mann V. Klein G. Razin A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8090-8094Crossref PubMed Scopus (86) Google Scholar). Similarly inNeurospora the deacetylase inhibitor TSA causes selective demethylation (6Selker E.U. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9430-9435Crossref PubMed Scopus (187) Google Scholar). Recent data support the claim that inhibition of histone deacetylation can cause selective demethylation of some genes such as the IgfII receptor (7Hu J.F. Pham J. Dey I. Li T. Vu T.H. Hoffman A.R. Endocrinology. 2000; 141: 4428-4435Crossref PubMed Scopus (55) Google Scholar) but not certain tumor suppressor genes (8Cameron E.E. Bachman K.E. Myohanen S. Herman J.G. Baylin S.B. Nat. Genet. 1999; 21: 103-107Crossref PubMed Scopus (1678) Google Scholar). Whereas this data shows that activation of genes by histone deacetylase inhibitors can lead toward loss of methylation, the mechanism is unclear. This loss of methylation might either be caused by inhibition of the maintenance DNA methyltransferase during replication, by site-specific proteins, by triggering site-specific repair activity, or by active site-specific or general demethylation. In this report we use a transient transfection approach to directly measure demethylation activity in human cells (HEK 293) and show that the state of methylation of DNA is not fixed in somatic cells but is dynamically modulated to correlate with the state of gene activity. This is accomplished by active demethylase activity that is directed by histone acetylation. These data provide a simple mechanism for explaining how active genes are demethylated and maintained in their unmethylated state. HEK 293 cells were plated at a density of 8 × 104/well in a six-well tissue culture dish and transiently transfected with 80 ng of plasmid DNA using the calcium phosphate precipitation method as described previously (9Rouleau J. Tanigawa G. Szyf M. J. Biol. Chem. 1992; 267: 7368-7377Abstract Full Text PDF PubMed Google Scholar). Transfections were repeated a minimum of three times using different cultures of HEK 293 cells. CAT assays were performed in triplicate as described previously (9Rouleau J. Tanigawa G. Szyf M. J. Biol. Chem. 1992; 267: 7368-7377Abstract Full Text PDF PubMed Google Scholar). HEK 293 cells were maintained as a monolayer in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% calf serum (Colorado Serum Co). To serum starve the cells, confluent HEK 293 cells were cultured in a medium containing 0.5% fetal calf serum for 72-h post-transfection. To determine the percentage of cells at different stages of the cell cycle, cells were stained with propidium iodide and the DNA content was measured by flow cytometry. pMetCAT+,SV40CAT, pCMV-GFP, and GFP plasmids were methylated in vitro by incubating 10 µg of plasmid DNA with 20 units of SssI CpG DNA methyltransferase (10Nur I. Szyf M. Razin A. Glaser G. Rottem S. Razin S. J Bacteriol. 1985; 164: 19-24Crossref PubMed Google Scholar) (New England BioLabs Inc.) in a buffer recommended by the manufacturer containing 160 µmS-adenosylmethionine, at 37 °C for 2 h. After repeating this procedure three times, full protection from HpaII digestion was observed. Bisulfite mapping was performed as described previously with minor modifications (11Clark S.J. Harrison J. Paul C.L. Frommer M. Nucleic Acids Res. 1994; 22: 2990-2997Crossref PubMed Scopus (1622) Google Scholar). 5 µg of sodium bisulfite-treated DNA samples was subjected to PCR amplification using the first set of primers described below. PCR products were used as templates for subsequent PCR reactions utilizing nested primers. The PCR products of the second reaction were then subcloned using the Invitrogen TA cloning kit (we followed the manufacturer's protocol), and the clones were sequenced using the T7 sequencing kit (Amersham Pharmacia Biotech) (we followed the manufacturer's protocol, procedure C). The primers used for the bacterial DNA chloramphenicol acetyltransferase (CAT) genomic region (GenBank 228 accession numberU65077) were: CAT5′1, 5′-ttgtttaatgtatttataattacat-3′; CAT5′(nested), 5′-taaagaaaaataagtataagtttta-3′; CAT3′1, 5′-ctcacccaaaaattaactaaaa-3′; CAT3′(nested), 5′-tttaaaaaaataaaccaaattttca-3′. The primers used for the enhanced green fluorescence protein (pEGFP-1) (CLONTECH) (GenBankTM accession numberU55761) were: GFP5′1, 5′-gttattatggtgagtaaggg-3′; GFP5′(nested), 5′-ggggtggtgtttattttgg-3′; GFP3′1, 5′-tataactattataattatactcca-3′; GFP3′ (nested), 5′-cttataccccaaaatattacc-3′. CHIP assays (12Crane-Robinson C. Myers F.A. Hebbes T.R. Clayton A.L. Thorne A.W. Methods Enzymol. 1999; 304: 533-547Crossref PubMed Scopus (40) Google Scholar) were performed by following the Upstate Biotechnology Chromatin Immunoprecipitation (CHIP) assay kit protocol (catalog no. 17-295). HEK 293 cells were transfected with 80 ng of in vitro methylatedpMetCAT+, SV40CAT, and pCMV-GFPplasmids, using the calcium phosphate method (see above). A final concentration of 0.3 µm TSA was added or not added to fresh medium 24 h was added to the culture at a final concentration of and at 37 °C for 10 and chromatin was using an histone as recommended by the of the was to the of DNA in different samples DNA from the and samples was from to and was subjected to PCR amplification using the following primers for the CAT and GFP CAT CAT GFP GFP nuclease (Amersham Pharmacia Biotech) were performed on M. G. G. A. M. Full Text PDF PubMed Scopus Google Scholar) for 20 at 20 °C in and The reaction was and DNA was using the cell were using and on a for and for GFP After to and the binding with GFP protein was using at followed by at protein was using at followed by at and enhanced kit (Amersham Pharmacia the methylation pattern of genes in somatic cells is as by the semiconservative model of methylation, an ectopically methylated gene methylated in somatic cells. the methylation pattern and its correlation with gene activity are a dynamic balance of methylation and then the cell ectopically methylated DNA and fashion its pattern of methylation in with its state of activity. therefore first the hypothesis that somatic cells can an ectopically methylated gene and it it from a but not it is from an inactive or to the state of methylation of an gene sequence to the that in demethylation are a of a of the demethylated the bacterial acetyl CAT gene the of 2 of the DNA region was previously to be active in cells (9Rouleau J. Tanigawa G. Szyf M. J. Biol. Chem. 1992; 267: 7368-7377Abstract Full Text PDF PubMed Google Scholar) but is inactive in HEK 293 cells In the CAT which is by the is active in HEK 293 cells were methylated in vitro with SssI and transfected into HEK 293 cells using the calcium phosphate protocol (9Rouleau J. Tanigawa G. Szyf M. J. Biol. Chem. 1992; 267: 7368-7377Abstract Full Text PDF PubMed Google Scholar). After DNA from these cells was and the methylation state of in a region the CAT gene was determined by of demethylated the CAT region the of demethylation was the CAT region from region This that can be demethylated in HEK 293 cells on their state of expression. determined this demethylation is to gene and it exclusively a gene is from the we performed the assay using an in GFP fluorescence which is the of a different from mapping of a region the GFP from that the GFP demethylated to a This that HEK 293 cells a demethylation activity that ectopically methylated from or the in the region of of GFP are demethylated in some plasmid suggesting that demethylation is and not are responsible for and binding of histone acetylation and and of a complex 1998; PubMed Scopus Google Scholar). To determine demethylation histone acetylation, we the histone deacetylase trichostatin A the demethylation of ectopically methylated and Bisulfite mapping of the in with 0.3 TSA for demethylation with demethylation in the of TSA A of in vitro plasmid transfected into HEK cells or with TSA is in The in the of demethylation of gene as determined by The of demethylation was determined by the of the HpaII by The and the of demethylation from three A in demethylation of transfected plasmid in the of These that ectopically methylated DNA from active demethylation in HEK 293 cells. TSA a in the state of of as shows HEK cells as with with the the an of of cells GFP with of in the of demethylation from be a of the of with in the or the of a complex that might be by TSA inhibition of histone deacetylation 1998; PubMed Scopus Google Scholar). To determine histone acetylation can demethylation of the of a we a demethylated in HEK cells. in TSA demethylation of a GFP sequence in a as by the digestion of the GFP sequence with the the sequence it is not The of from experiments was using and in A in demethylation of transfected plasmid in the of This data the hypothesis that histone acetylation and not the of a region the demethylation of a sequence of DNA in HEK cells. cells were with of TSA and subjected to to the between demethylation and inhibition of histone acetylation. HpaII from with µm to with 0.3 µm that demethylation with TSA The of demethylation in the of TSA with HpaII from at h to h. This of the transfected plasmid the transfection these and that demethylation of methylated plasmid h post-transfection. the used to demethylation the of cells with TSA for we determined cell is not a in the methylation In vitro HEK cells were with either Dulbecco's modified Eagle's medium containing either 10% fetal calf serum in the or of or 0.5% fetal calf serum and h post-transfection. cells were stained with propidium iodide and subjected to cell to determine the cell cell that were either fetal calf or with TSA the in cell cycle, from The state of methylation of transfected GFP plasmid the different was by sodium mapping to cells with which demethylation in transfected in cells. the in the cell of and cells were this is with the that cell caused by TSA are not responsible for the demethylation. To that transiently transfected DNA is associated with either or and that TSA can the state of acetylation of to the GFP and CAT we performed a chromatin immunoprecipitation assay (CHIP) using The in that is an of the CAT gene sequence with reside from the to from the region TSA the of CAT associated with and TSA not acetylation of associated with which is with previous studies that that not are following TSA (8Cameron E.E. Bachman K.E. Myohanen S. Herman J.G. Baylin S.B. Nat. Genet. 1999; 21: 103-107Crossref PubMed Scopus (1678) Google Scholar). the of with some to with the plasmid is not demethylated TSA not To provide a direct of DNA associated with chromatin is plasmid DNA from histone or not with was subjected to mapping clones in contrast to DNA The of demethylation was plasmid DNA from in this DNA was from cells with TSA DNA not be to cells, which were not with from cells transfected with plasmid in the or of This is with previous studies that methylated DNA is associated with as a of of histone by methylated DNA binding such as A. Nature. 1998; PubMed Scopus Google Scholar). The to be by methylation as by the that it is by directed The that DNA that is associated with is demethylated in the of TSA the hypothesis that demethylation following TSA acetylation of the associated with the gene and is not a of a general in demethylase activity. This can explain why TSA not in demethylation of genes (8Cameron E.E. Bachman K.E. Myohanen S. Herman J.G. 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Nature. 1999; PubMed Scopus Google Scholar) into HEK 293 cells. the state of methylation of the transfected GFP gene by mapping in The of demethylation the of the GFP gene is with demethylation is with shows a of transfected GFP in the or of The in the of demethylation of gene as determined by The of demethylation was determined by the of the HpaII by The and the of demethylation from three These experiments that demethylation of a sequence is on the of demethylase in to the state of histone acetylation. Our data from report that that as a of a methylated gene and that it did not cause demethylation J. S. U. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). A simple to this is that the of with methylated genes on the as well as the of Our data that of not which is not associated with TSA we previously that DNA in vitro S. N. Szyf M. Nature. 1999; PubMed Scopus Google we determined demethylase DNA in the of a sequence in the in of the demethylation of and the as by the in the of the plasmid that is The of from experiments was using and in A using the the of in transfected cells but not in the of added in transfected cells. This shows that the of demethylase in demethylation of a gene in different and that demethylase not some can its activity as To determine the demethylation is in of the plasmid and to that not the activity of the as for H. D. A. Nat. Genet. 1999; PubMed Scopus Google cell from HEK cells transfected plasmid and or were subjected to using an directed toward GFP were in and was determined by HEK cells with GFP and protein GFP In HEK cells which were either with GFP plasmid or GFP were h by use of a with a fluorescence The shows the from Whereas of the percentage of cells it is that of by is by TSA with These data that demethylation is not to the by The DNA methylation pattern is a of the gene in It is therefore why it is to how DNA methylation patterns are and how their with chromatin The data in this report support a simple but attractive model that explains the correlation between active genes and Our data that the state of demethylation is determined by chromatin The state of methylation of a sequence is determined by a dynamic balance of the of demethylase activity in the cell and the state of acetylation of show that an sequence of which is ectopically is demethylated in HEK 293 cells it is associated with acetylation is either by the that histone acetyl to and or by a inhibitor of histone deacetylation and DNA that is to demethylated direct that DNA associated with is The DNA associated with is in cells that were not with the hypothesis that active demethylation is a of the with and not of TSA demethylase activity by of a demethylase demethylation and gene the data in this a model that the in demethylation is the between the demethylase and The of such an is enhanced by either histone acetylation or an in the of It is to that demethylase is from the DNA by histone as to be the with D. Wolffe A.P. Full Text PDF PubMed Scopus Google Scholar). Bisulfite which is followed by PCR to at the demethylation events in a DNA A that from this is with a active demethylation of ectopically methylated plasmids is and not of are demethylated in some plasmid and in 2 This pattern of demethylation is with demethylation but is with a mechanism as previously on an in vitro N. S. Szyf M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). and demethylation of CpG is with the that from the mapping of the state of methylation of in the CpG in the first of the tumor suppressor L. A. D. Res. Google F.A. P.A. 2000; PubMed Scopus Google Scholar) and the tumor suppressor S. Szyf M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) are not methylated in but the first is methylated in some that not L. A. D. Res. Google Scholar). histone acetylation is involved in demethylation of CpG of a region as by then histone acetylation not be to the region show that the genes we are associated with This is in with previous data that histone acetylation is not to the region but of the L. A. Hebbes T.R. M. Biol. 1998; PubMed Scopus Google which can explain the demethylation of a from a and Recent data that of histone acetylation can at from M. Biol. 21: PubMed Scopus Google Scholar). directed in the toward site-specific demethylation associated with and activation of Razin and Riggs A. Riggs A.D. Science. 1980; 210: 604-610Crossref PubMed Scopus (1509) Google Scholar) that demethylation in a site-specific and by binding of to during in development that it from methylation during This model support C.L. Biol. 2000; PubMed Scopus Google Scholar). However, data that demethylation is not on the of DNA binding S. P.L. Wolffe A.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). experiments and active demethylation in transient transfection assays Keshet I. J. Cedar H. Full Text PDF PubMed Scopus Google suggesting the of and demethylation A. Keshet I. Razin A. Cedar H. Full Text Full Text PDF PubMed Scopus Google Scholar). The data here are not in with these previous It is that different are responsible for the site-specific demethylation that the activation of genes during development and the demethylation An alternative hypothesis is that or to the demethylation activity described in this report are responsible for site-specific demethylation. demethylation might be by a histone acetylation, site-specific and cell demethylation might be directed by a of histone acetyl by certain This model is with a for or for and demethylation Keshet I. J. Cedar H. Full Text PDF PubMed Scopus Google Scholar). demethylation that is at the cell of the in of the and N. A. Cedar H. A. 1998; PubMed Scopus Google Scholar). It is to that the that with these demethylation by and histone acetylation. The of the demethylase activity by transient transfection assay is unclear. of the is the of demethylase in somatic it responsible for repair of methylation or it a dynamic in the DNA methylation pattern and its correlation with chromatin It is that the demethylase activity here is an that is in some cells. the assay described in this study some of the of the demethylation reaction in cells. These might explain a of of demethylation events in as well as on of the of mammalian the correlation of gene chromatin and the DNA methylation The of demethylase that of ectopically methylated DNA in a somatic cell to of the DNA methylation pattern in somatic cells and the relationship between DNA methylation and histone acetylation. The DNA methylation pattern at a might be a of methylation and the of the reaction is determined by the local state of histone acetylation. Recent data on how DNA methylation histone data that it is that histone acetylation DNA for

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