Feedback regulation of DNA methyltransferase gene expression by methylation

Eur. J. Biochem. 264, 191±199 (1999) q FEBS 1999

Feedback regulation of DNA methyltransferase gene expression bymethylation

Andrew Slack*, Nadia Cervoni*, Marc Pinard and Moshe Szyf

Department of Pharmacology, McGill University, Montreal, PQ, Canada

This paper tests the hypothesis that expression of the DNA methyltransferase, dnmt1, gene is regulated by a

methylation-sensitive DNA element. Methylation of DNA is an attractive system for feedback regulation of DNA

methyltransferase as the final product of the reaction, methylated DNA, can regulate gene expression in cis. We

show that an AP-1-dependent regulatory element of dnmt1 is heavily methylated in most somatic tissues and in

the mouse embryonal cell line, P19, and completely unmethylated in a mouse adrenal carcinoma cell line, Y1.

dnmt1 is highly over expressed in Y1 relative to P19 cell lines. Global inhibition of DNA methylation in P19

cells by 5-azadeoxycytidine results in demethylation of the AP-1 regulatory region and induction of dnmt1

expression in P19cells, but not Y1 cells. We propose that this regulatory region of dnmt1 acts as a sensor of the

DNA methylation capacity of the cell. These results provide an explanation for the documented coexistence of

global hypomethylation and high levels of DNA methyltransferase activity in many cancer cells and for the

carcinogenic effect of hypomethylating diets.

Keywords: 5-azadeoxycytidine; DNA metylation; cytosine-5-DNA methyltransferase (dnmt1); feedback

regulation; transfection.

Mammalian DNA is modified by methylation of the majority ofthe cytosine moieties residing in CpG dinucleotide sequences[1]. A long list of data supports the hypothesis that themethylation pattern plays an important role in the regulation ofgene expression programs [2,3] and possibly other functions ofthe genome [4]. Recent data has provided a molecularmechanism for the inhibition of gene expression by DNAmethylation by linking the binding of methylated CpGs in thegenome by the methylated DNA binding protein MeCP2 withrecruitment of histone deacetylase [5,6]. One of the mostimportant, or possibly the only guardian of the methylationpattern is the DNA methyltransferase enzyme which catalyzesthe transfer of a methyl residue from S-adenosyl-methionine toa hemimethylated CpG sequence [1,7]. The main DNAmethyltransferase activity in vertebrates is encoded by thednmt1 gene [8,9]. A homozygous knock out of the dnmt1 geneis embryonal lethal [10]. Partial inhibition of DNA methyl-transferase activity by a pharmacological inhibitor, 5-azacyti-dine (5-azaCdR) [11], expression of an antisense RNA to dnmt1mRNA [12], or treatment with dnmt1 antisense oligonucleo-tides [13] results in changes in methylation of certain sequencesand alterations in gene expression profiles [12,13]. It stands toreason therefore that expression of dnmt1 is tightly regulated,both in somatic cells in homeostasis as well as at junctionpoints when programmed changes in the identity of the celloccur, such as in differentiation. Several modes of transcrip-tional and post-transcriptional regulation of dnmt1 have beenpreviously suggested [14]. Transcription of dnmt1 is regulatedby the Ras signalling pathway in cancer cells [15,16] as well as

in activated T cells [17], and post-transcriptional regulation ofDNA methylase has been proposed to be responsible for theregulation of dnmt1 mRNA levels during the growth state ofcells [18]. Induction of dnmt1 activity by oncogenic signalingpathways has been proposed to play a causal role inoncogenesis [4,14]. One of the most common mechanisms ofregulation in biological systems is feedback regulation ofexpression of a rate-limiting enzyme by the end product of aphysiological pathway. For example, expression of steroido-genic enzymes is regulated in response to the levels ofglucocorticoids [19]. It is tempting to suggest that theexpression of dnmt1 is similarly regulated by the methylationcapacity of a cell. Complex paradigms are sometimes invokedto allow for negative feedback regulation of expression of arate-limiting enzyme by its product [19]. Previously publisheddata supports the hypothesis that cells respond to a pharmaco-logical inhibition of methylation by induction of DNAmethyltransferase activity. De novo methylation has beendemonstrated in T cells following 5-azaCdR treatment [20].Similarly, it has been recently shown that 5-azaCdR treatmentof T cells results in induction of dnmt1 mRNA and DNAmethylase activity levels [17]. Subjecting rats in vivo to amethionine-deficient diet which triggers DNA hypomethylationalso results in induction of DNA methyltransferase activity andcarcinogenesis [21].

Methylation is an attractive system for feedback regulationbecause the final product of the reaction, methylated DNA, candirectly regulate gene expression. A simple model is that dnmt1bears a DNA element regulating its expression that is controlledby its state of methylation. In this paper we tested thehypothesis that a methylation-sensitive regulatory elementexists in the dnmt1 gene using 5-azaCdR-treated embryonalcarcinoma P19 cells as a model system. We have previouslydescribed a regulatory element in the mouse dnmt1 residingupstream of the third somatic exon of the gene and proximaltranscription initiation sites. This element directs promoter

Correspondence to M. Szyf, Department of Pharmacology, McGill

University, 3655 Drummond Street, Montreal PQ, Canada H3G 1Y6.

Fax: + 1 514 398 6690, Tel.: + 1 514 398 7107.

Abbreviations: 5-azaCdR, 5-azadeoxycytidine.

* Note: these authors contributed equally to the manuscript.

(Received 26 April 1999, accepted 1 June 1999)

192 A. Slack et al. (Eur. J. Biochem. 264) q FEBS 1999

activity in a reporter chloramphenicol acetyltransferase assay,bears consensus AP-1 sites and is activated by the Ras±Junproto-oncogenic signaling pathway [16,22]. In this report wetested the hypothesis that a hypomethylation challenge inducedby a pharmacological inhibitor of DNA methyltransferasewould induce dnmt1 expression and that this induction ismediated by a methylation-sensitive element.

M A T E R I A L S A N D M E T H O D S

Cell culture, 5-azaCdR treatment and transient transfectionassays

Y1 cells (American Tissue Culture Collection; CCL-79) [23]were maintained as monolayers in F-10 medium, which wassupplemented with 7.25% heat-inactivated fetal equine serumand 2.5% heat-inactivated fetal bovine serum (Immunocorp,Montreal). P19 cells (CRL-1825) [24] were maintained as amonolayer in Dulbecco's modified Eagle's medium supple-mented with 10% heat-inactivated fetal bovine serum. For5-azaCdR treatment, 1 � 105 cells were plated in growthmedium. Twenty-four hours after plating, the medium wasreplaced with fresh medium containing various concentrations(0, 0.1 and 1 m) of 5-azaCdR (Sigma). The medium wasremoved and replaced with fresh medium containing 5-azaCdRevery 12 h for a period of 72 h. For transient transfectionassays, P19 (and Y1) cells were plated at a density of 8 � 104

per well in a six-well tissue culture dish. A 1-mL DNA/calciumphosphate precipitate was prepared according to standardprotocols [22,25]. Two hundred microliters of calciumphosphate precipitate containing 5 g´mL21 of plasmid DNAwere added to each well containing 1.5 mL of the appropriatemedium as previously described [22]. Transfections wereperformed in triplicate. The medium was changed after 24 hand cells were harvested by scraping 48 h post-transfection,followed by lysis in 100 mL of 100 mm Tris pH 8 by a fivefoldfreeze/thaw cycle. Fifty microliters of this extract wereincubated for 3 h at 37 8C with 0.5 Ci of [3H]-labelled acetylcoenzyme A (Dupont) and acetylated chloramphenical acetyltransferase was extracted in ethyl acetate followed by liquidscintillation counting as previously described [22].

Preparation of RNA and DNA

Tissues were homogenized in standard DNA extraction buffer(1% SDS, 5 mm EDTA, 150 mm NaCl) followed by pro-teinase K digestion, phenol/chloroform extractions and ethanol

precipitations [26]. Cell lines DNA was prepared from pelletednuclei [18] according to standard protocols [26]. RNA wasprepared from cell lines and tissues using RNAzol (TelTest).

DNA methylation analysis.

Ten grams of DNA were subjected to digestion with HindIII(10 U´mg21) for 6 h followed by phenol extraction and ethanolprecipitation. The HindIII-digested DNA was digested witheither 25 U of MspI which is insensitive to CC*GG methylationor HpaII (Boehringer Mannheim) which is sensitive tomethylation, for 8 h at 37 8C. The DNA was electrophoresedand transferred to Hybond N+ membrane (Amersham) andsubsequently hybridized to a 1.7-kb BamHI fragment encodinggenomic sequences upstream to the proximal promoter ofthe dnmt1 gene (see Fig. 2 for physical map) [16,22].Radionucleotides (3000 mCi´mmol21) were purchased fromAmersham and the probes were labelled by the randompriming method according to the manufacturer's instructions(Boehringer Mannheim).

Bisulfite mapping

Bisulfite mapping was performed as previously described withsmall modifications [12]. Sodium bisulfite, free weight = 104,was used. A 3.6-m solution of sodium bisulfite (ACS grade,Sigma) (pH 5) was prepared fresh each time and a 20-mm stockof solution of hydroquinone was prepared and stored at 220 8C.Five micrograms of DNA (digested with EcoRI), wereincubated for 15 min at 37 8C with 54 mL of double distilledH2O and 6 mL of 3 m NaOH. Following this incubation,431 mL of a 3.6-m sodium bisulfite/1 mm hydroquinonesolution was added. One hundred microliters of mineral oilwere added to overlay the solution and the tube was heated at55 8C for 12 h. The bisulfite reaction was recovered frombeneath the mineral oil and desalted using the Promega WizardPrep (following manufacturer's protocol). Six microliters of3 m NaOH were added to the desalted solution and the tube wasincubated for 15 min at 37 8C. Following ethanol precipitation(in the presence of 0.3 m ammonium acetate) the DNA wasresuspended in 100 mL double distilled H2O. Approximately50 ng of DNA were used in each of the PCR amplifications.PCR products were used as templates for subsequent PCRreactions utilizing nested primers. The PCR products of thesecond reaction were then subcloned using the Invitrogen TAcloning Kit (following the manufacturer's protocol) and theclones were sequenced using the T7 Sequencing Kit (Pharmacia)(following the manufacturer's protocol, procedure C). The primersused for the dnmt1 genomic region (GenBank accession M84387)were: MET5 01 5 0-GGATTTTGGTTTATAGTATTGT-3 0 MET 5 0(nested) 5 0-GGAATTTTAGGTTTTTATATGTT-3. MET3 015 0-CTCTTCATAAACTAAATATTATAA-3 0 and MET3 0(nested) 5 0-TCCAAAACTCAACATAAAAAAAT-3 0.

RNase protection assays

RNA was prepared from exponentially growing cells usingstandard protocols [22]. RNAse protection assays wereperformed as described [22] using a 0.7-kb HindIII/BamHIfragment as a riboprobe. This probe is a genomic fragmentbearing exons 3 and 4 of the murine dnmt1 probe. It protectstwo major bands of 112 and 100 nucleotides corresponding tothese exons as well as a number of minor alternatively splicedand alternate initiations as previously described [22]. Tonormalize the signal obtained for dnmt1 with the amount of

Fig. 1. Physical map of the murine AP-1 dependent dnmt1 regulatory

region. The first three exons of the murine dnmt1 locus are indicated as

filled boxes with exon numbers below. Sex-specific exons are shown as

open boxes with exon numbers below [34]. CpG sites in the regulatory

region upstream of exon 2 and 3 are indicated as vertical lines. Intronic

regions are shown as solid lines between exons. Transcription initiation

sites are indicated as arrows above the exons. The AP-1 recognition sites

are indicated. The HpaII site located nearest the second exon is denoted by

an asterisk

q FEBS 1999 Feedback regulation of dnmt1 (Eur. J. Biochem. 264) 193

total RNA present in each sample, and to verify equal loading,the RNA was simultaneously hybridized with a [32P]-labelledriboprobe complementary to 18S RNA and subjected to RNasedigestion and protection (Ambion) [22].

Electrophoretic mobility shift assays

Nuclear extracts were prepared from untreated and 5-azaCdRtreated P19 and Y1 cell lines as described previously. Fordetection of AP-1 binding activity, an oligonucleotide homo-logous to the regulatory region of dnmt1 containing the AP-1consensus sequence and AP-1 consensus oligonucleotide [16]were labelled by polynucleotide kinase and purified by NAPcolumns (Pharmacia) for use as a probe. For detection of thedifferent complexes bound to methylated and nonmethylatedDNA, the dnmt1 regulatory region probe fragments wereamplified by PCR using primers homologous to the genomicmurine dnmt1 sequence. The resulting products were thenend-labelled by polynucleotide kinase and twice methylatedusing MSssI (NEB) or mock methylated. The primers used forDNA methylase probe B were 5 0 5 0-GAATCCCAGGCCTC-CACA-3 0 and 3 0 5 0-GAGGAGCTGTCAGTCAGGG-3 0. Thoseused for probe A were 5 0 5 0-AATGCAGCATGACTCATGCT-3 0and 3 0 5 0-GAGGAGCTGTCAGTCAGGG-3 0. For each assay,10 mg extract was added to 1.0 � 105 c.p.m. labelled probe in4% glycerol, 1 mm MgCl2, 0.5 mm EDTA, 0.5 mm dithio-threitol, 50 mm NaCl, 10 mm TrisCl (pH 7.5) and 0.1 mg´mL21

E. coli DNA and incubated 30 min at room temperature. Thereaction products were electrophoresed on a 5% nondenaturingacrylamide gel at 4 8C. Gels were then dried and autoradio-graphed. These experiments were performed with minormodifications essentially as described in [16].

R E S U LT S

An AP-1 regulatory element is located <1.7 kb 5 0 to theproximal promoter of dnmt1 [16,22] and is nested in a CpG-rich

area as indicated in the map presented in Fig. 1. There are 29CpG dinucleotides including a HpaII site in close proximity tothe AP-1 regulatory sites. To test the hypothesis that methyl-ation of these CpG sites might serve as a sensor of the state ofmethylation of the genome, we first determined whether thesesites are differentially methylated in the genomes of differentcell lines and tissues. The map of this area is presented inFig. 1.

The state of methylation of this region could be monitored bydetermining the sensitivity of two CCGG sites located in theCG-rich region and 2.2 kb upstream to cleavage by HpaII (seeFig. 2 for map). We have analyzed the state of methylation ofthis site in somatic tissues that are not dividing and do notexpress high levels of DNA methylase [18,27]. We have alsoanalyzed the state of methylation of this site in Y1 cells, anadrenocortical cell line that expresses high levels of Ras, AP-1and DNA methylase activity [15] as well as an embryonalcarcinoma line P19 that expresses very low levels of AP-1activity [16,25]. Genomic DNA prepared from the differenttissues was first cut with HindIII and then subjected to digestionwith either MspI (which cleaves the sequence CCGG irrespectiveof the state of methylation of the internal cytosine) or HpaII(which is sensitive to methylation at the same site). Thedigested DNA was subjected to Southern blot analysis using a1.7-kb BamHI/HindIII fragment bearing the genomic region ofdnmt1 as indicated in the physical map in Fig. 2.

The data presented in Fig. 2 suggest that the HpaII sitelocated upstream to the AP-1 regulatory region is heavilymethylated in all tissues (as indicated by the relative abundanceof the partially methylated 2.3-kb and fully methylated 4.5 -kbHpaII fragments). This HpaII site is completely methylated inP19 cells (as indicated by the absence of the nonmethylated1.7-kb HpaII fragment and the presence of the fully methylated4.5-kb fragment). In contrast, the HpaII site is completelyunmethylated in Y1 cells (as indicated by the presence of thenonmethylated 1.7-kb HpaII fragment and the absence of the

Fig. 2. Methylation of the regulatory region of

dnmt1 in different murine tissues and cell lines.

Ten micrograms of genomic DNA prepared from

Y1, P19 and mouse somatic tissues (as indicated

in the figure) were digested with HindIII which

was followed with either MspI or HpaII digestion

(as indicated in the figure). The digested DNA

was Southern blotted and hybridized to a 1.7-kb

genomic fragment of the dnmt1 [22]. The

autoradiograms are presented in the upper panel.

The physical map of the dnmt1 5 0 region is

presented in the lower panel. The position of the

probe is indicated above the dnmt1 map and the

expected fragments if the HpaII sites are fully or

partially hypomethylated, are indicated below the

map. The first three exons are indicated as boxes

with exon numbers below.


methylated 4.5-kb and 2.3-kb fragments). Similarly, all tissuesare differentially methylated at the 5 0 CCGG site located 2.3 kbupstream (see physical map in Fig. 2) as indicated by thedifferent abundances of the fully methylated 4.5-kb HpaIIfragment in many tissues. Some differences exist betweensomatic tissues. The abundance of the unmethylated 1.7-kbHpaII fragment relative to the fully methylated HindIIIfragment is higher in brain tissues than in spleen or testis.However in all tissues, the partial methylation is consistent withthese tissues containing at least two different populations ofcells bearing either methylated or nonmethylated HpaII sites atthese positions. Although this methylation could be potentiallycorrelated with expression of dnmt1, the compound pattern ofmethylation in somatic cells makes it hard to correlate it to aspecific role in regulating dnmt1 gene expression.

Y1 cells and P19 cells show, in contrast to somatic tissues,two clear diametrically opposed states of methylation of this

region. The methylation of this region corresponds to itsfunctional role in regulating dnmt1 activity. Y1 cells expresshigh levels of the AP-1 transcriptional activators [15], whichinteract with this region, whereas nondifferentiated P19 cellsexpress low levels of AP-1 [16,25] activity which render thisregion relatively inactive in these cells (Fig. 4).

We tested the hypothesis that the AP-1 regulatory region ofdnmt1 regulates the expression of dnmt1 in response to a globalhypomethylation challenge. If this region is indeed responsiblefor the induction of dnmt1 following a global hypomethylationchallenge the following conditions have to be fulfilled. First, incells where this region is methylated in the basal state, a globalhypomethylation should result in demethylation of this regionand a parallel induction of dnmt1 expression. Second, thereshould be no induction of dnmt1 following 5-azaCdR treatmentin cells where this region is not methylated in the basal state.Third, in vitro methylation of this region should be shown to

Fig. 4. 5-azaCdR causes a dose-dependent

induction of dnmt1 mRNA in P19, but not the

Y1 cells. Ten micrograms of total RNA was used

for RNAse protection assay analysis of the level

of dnmt1 mRNA. A riboprobe homologous to the

third and fourth exon of the DNA methylase [22]

was hybridized to RNA from P19 and Y1 cell

lines treated with 0, 0.1, 0.3 and 1.0 mm

5-azaCdR. The bands protected by an 18S

riboprobe are shown below. tRNA control lanes

are shown at the right. Quantification of the

results by densitometric analysis is shown in the

lower panel. The results were standardized by the

intensity of the hybridization to the 18S

ribosomal RNA probe in each lane. (To maintain

a linear film exposure, the Y1 dnmt1

autoradiograph shown was exposed for a shorter

period of time than the corresponding P19

autoradiograph.)

Fig. 3. 5-azaCdR causes demethylation of the 5 0 regulatory region of the DNA methylase locus in the P19 cell line. Representative bisulfite sequencing

films are shown for 5-azaCdR treated and untreated P19 DNA and untreated Y1 DNA. The total percentages and numbers of demethylated cytosines for all

sequenced clones are shown in the table. Seven to 14 clones were sequenced per condition. The DNA methylase physical map is shown below with all of the

CpG sites in this area indicated by vertical lines. The region mapped by bisulfite mapping with HpaII and AP-1 sites indicated is magnified above the map.

CpG sites are shown as circles, filled if methylated, open if nonmethylated. The arrow indicates site 101 which is hypersensitive to demethylation by

5-azaCdR in P19 cells.


inhibit its interaction with AP-1 and transcriptional activation.Y1 and P19 cells offer an excellent model system to test thishypothesis as this region is heavily methylated in P19 cells andcompletely unmethylated in Y1 cells, as determined by thebisulfite mapping shown in Fig. 3.

We first addressed the question of whether global demethy-lation by a demethylating agent alters the pattern of methyla-tion of this region in P19 cells? A dose-dependent andsequence-specific demethylation was observed by bisulfitemapping of the methylation state of this region in P19 cells thatwere treated with 5-azaCdR (0.1±0.5 mm). When the averagestate of hypomethylation of all CpG sites in this region in allsequenced clones is compared, the total percentage ofdemethylated cytosines in all sequenced clones increasesfrom 32% in untreated P19 cells to 46% in 0.1 mm treatedcells, and 52% in 0.5 mm treated cells. Whereas there is ageneral decrease in methylation of this region, as indicated bythe presence of clones that are unmethylated in all CpG sitesfollowing 5-azaCdR treatment (Fig. 3), one site (101) isespecially sensitive to demethylation. This data suggests, aspreviously proposed, that even under conditions of generalinhibition of methylation some sites are especially sensitive todemethylation [18]. It is unclear whether this site is also criticalfor activation of dnmt1 or whether other sites in this region playa similar role.

We then determined whether 5-azaCdR treatment alters thelevels of expression of dnmt1 in Y1 and P19 cells. 5-azaCdRcauses global demethylation in Y1 cells [15] but clearly doesnot alter the state of methylation of this AP-1 region which isfully unmethylated in untreated cells. The basal level ofexpression of dnmt1 is much higher in the Y1 cell line relativeto the P19 cell line. 5-azaCdR treatment does not alter the levelof expression of dnmt1 in Y1 cells. However, in P19 cells,5-azaCdR treatment induces an eightfold induction of dnmt1mRNA as determined by densitometric analysis (Scanalytics)(Fig. 4). The expression level of dnmt1 in the P19 cells treatedwith even the highest dose of 5-azaCdR does not equal that of

the Y1 cell line, possibly due to the constitutively low levels ofAP-1 activity in the P19 cell line [16,25]. In summary, ourresults demonstrate that global hypomethylation by a pharmaco-logical agent causes demethylation of the dnmt1 AP-1regulatory region in P19 cells and induction of dnmt1expression. No induction is observed in Y1 cells where thisregion is nonmethylated in untreated cells. These results areconsistent with the hypothesis that the methylation status of thedescribed region is important for transcriptional activation ofdnmt1.

It has been previously reported that 5-azaCdR can induceHa-ras and c-jun mRNA levels in human T cells [17]. Toexclude the possibility that induction of dnmt1 in P19 cells by5-azaCdR is an indirect effect of induction of AP-1 activity, weassayed AP-1 binding activity in 5-azaCdR-treated P19 cells(Fig. 5). Nuclear extracts were prepared from untreated and5-azaCdR-treated P19 cells and incubated in vitro with eitheran excess of [32P]-labelled double stranded oligonucleotidebearing the 5 0 AP-1 sequences from the dnmt1 regulatoryregion or a consensus AP-1 oligonucleotide [16]. As shown inFig. 5, there is no increase in AP-1 binding activity in nuclearextracts following 5-azaCdR treatment. This data stronglysuggests that the transcriptional upregulation of dnmt1 is not aconsequence of an induction of AP-1 activity and subsequenteffects on promoter activity.

Three mechanisms have been previously proposed to explainthe inhibition of gene expression by methylation. First,methylation can attract the binding of methylated DNA bindingproteins such as MeCP2 which in turn results in recruiting of ahistone deacetylase and precipitation of an inactive chromatin[5,6]. This mechanism can explain an on/off switch of geneexpression, but not the modulation of activity of an expressedgene. It is therefore highly unlikely in our case because dnmt1is actively transcribed in P19 cells, moreover there is little or noexpression of MeCP1 or MeCP2 in embryonal cells [28].Second, methylation has been shown to directly inhibit bindingof transcription factors [29]. This is highly unlikely in our casebecause the AP-1 consensus recognition sequences and theAP-1 sites in dnmt1 do not include a CG dinucleotide [16]. Oneplausible mechanism for inhibition of the AP-1 regulatoryregion by methylation is that it alters the interaction of otherDNA binding proteins with this region, which in turn impedesAP-1 binding. A similar mechanism has been suggested toexplain the inhibition of interferon-g expression upon methy-lation [30]. If this is so, then the identity of factors bound to thisregulatory region should change depending upon its methy-lation status.

To test the hypothesis that the binding of factors bound to theregulatory region change depending upon its methylationstatus, we have tested the ability of either mock or in vitromethylated DNA fragments bearing the two AP-1 sites in thednmt1 region to generate DNA-protein complexes with proteinsincluded in P19 nuclear extracts. Two fragments were used (Aand B in Fig. 6). Fragment A includes the two AP-1 recognitionelements and two CG dinucleotides residing between theseelements. These CG sites are unmethylated in P19 cells.Fragment B also contains a cluster of CG dinucleotidesupstream to the 5 0 AP-1 site that are methylated in P19 cells.Methylation of fragment A does not alter its ability to formDNA±protein complexes in this region and identical complexesare formed under both conditions (Fig. 6B, complexes 2 and 3).This is consistent with the fact that the two CG sites included inthis site are not methylated in vivo in P19 cells (Fig. 3) andwith the hypothesis that methylation per se does not alter theinteraction of AP-1 complexes with their recognition sequence.

Fig. 5. The observed induction of dnmt1 is not due to a nonspecific

effect of 5-azaCdR on AP-1 activity. Ten micrograms of P19 nuclear

extracts treated with 0, 0.1, 0.3 and 1.0 mm 5-azaCdR were incubated with

an oligonucleotide probe homologous to the AP-1 site contained in the

dnmt1 5 0 regulatory region and an AP-1 consensus oligonucleotide probe.

The relative positions of the AP-1-shifted complexes and the free (unbound)

probe are indicated.


However, when additional methylated CGs are included in thefragment, a clear difference is observed between the methylatedand nonmethylated fragments. Whereas nonmethylated probe Bforms complexes 2 and 3 identified with the short fragment(probe A), the methylated probe B forms different additionalDNA±protein complexes 1 and 4 (Fig. 6). This experiment isconsistent with the hypothesis that methylation of a cluster ofCG sites upstream to the AP-1 recognition sequences attract adifferent set of DNA-binding factors, which in turn inhibit theformation of the cognate DNA±protein complexes.

The dnmt1 regulatory region bearing the proximal dnmt1promoter can direct chloramphenical acetyltransferase activityin transient transfection assays [16,22]. To test the effect ofmethylation of the dnmt1 regulatory region on its trans-criptional activation capacity, we determined whether in vitromethylation of all CGs in the 2.3-kb genomic fragment similarto the observed situation in P19 cells (Figs 2 and 3) with MSssImethylase [31] would inhibit the expression of a reporterbacterial chloramphenicol acetyltransferase in P19 cells. Fivegrams of the methylated or mock methylated pMetCAT were

Fig. 6. Methylation status of the 5 0 dnmt1 regulatory region determines the identity of bound factors from P19 cell extracts. Ten micrograms of P19

nuclear extract were incubated with methylated or mock-methylated (indicated as + and ±, respectively) DNA fragments homologous to the regulatory

region of dnmt1. The bound complexes are indicated as complexes 1±4. Probe A (as indicated in the physical map below) spans the region containing the

AP-1 sites and the two CpG sites between. Probe B spans the entire regulatory region including an additional five upstream CpG sites. Reactions with

methylated and nonmethylated probes were performed without extracts as negative controls (indicated above the lanes as X).


transiently transfected into P19 cells in triplicate. Methylationof the reporter construct pMetCAT by MSssI results in29 ^ 7% of control (mock-methylated) chloramphenicolacetyltransferase activity directed by this region in P19 cells(data not shown). Methylation of the single HpaII site does notinhibit the promoter as has been observed with other promoters(data not shown). Whereas our experiment is consistent withthe hypothesis that methylation of the dnmt1 regulatory regioninhibits its transcriptional activity, it does not exclude, however,the possibility that the methylation of the vector sequencesresults also in silencing of the promoter activity. It should alsobe pointed out that transient expression assays do not reproducethe full range of transcriptional control seen in the endogenouslocus as has been previously shown by Buschhausen et al. [32]and Kass et al. [33]. This work has shown that time-dependentassembly of repressive nucleoprotein complexes is required toblock transcription of methylated genes.

D I S C U S S I O N

The important role that DNA methylation plays in regulatinggenome functions necessitates tight regulation of the levels ofDNA methyltransferase with altering physiological and non-physiological conditions. Whereas transcriptional and post-transcriptional regulatory controls over dnmt1 are in place tocoordinate the level of dnmt1 in response to changes in growthor differentiation [4,5], some mechanisms must maintain thesteady-state level of DNA methylation under homeostaticconditions. How does the cell sense the state of DNAmethylation and respond to a sudden challenge such as globalhypomethylation? Because methylation of regulatory regions isa ubiquitous mechanism of regulation of gene expression, anattractive hypothesis is that the dnmt1 gene is regulated bymethylation. In this paper we show that dnmt1 has evolved adistinct way of responding to a demethylation challenge (for amodel, see Fig. 7). An AP-1 regulatory element, which isflanked by a CG-rich sequence upstream to the proximalpromoter, is heavily methylated in many tissues in vivo and inP19 cells (Fig. 2) resulting in low basal transcriptional activity

of the dnmt1 gene. We have shown that this region isdemethylated when the cell is challenged by a DNAmethyltransferase inhibitor (Fig. 3) and that this is followedby induction of dnmt1 mRNA (Fig. 4). Consistent with thehypothesis that demethylation of this region is critical for thisinduction, we show that a demethylation challenge of a cell thatbears a nonmethylated dnmt1 regulatory region, Y1 cells, doesnot result in an increase in dnmt1 mRNA (Fig. 4). We proposethat methylation of CG sites results in DNA±binding interac-tions that impede the binding of AP-1 to this region (Fig. 6) andsilences its transcriptional activation capacity (for a model, seeFig. 7). The nature of the proteins involved remains to bedetermined in future experiments.

The model proposed above is consistent with two previouslywell documented, but paradoxical, phenomena. Firstly, phar-macologic inhibition of DNA methyltransferase has beenshown to increase DNA methyltransferase activity and thetranscriptional rate of dnmt1 mRNA [17]. Secondly, rodentstreated with methionine-deficient diets exhibit both globalhypomethylation of their genome as well as a higher level ofDNA methyltransferase activity [21]. It was previously believedthat the hypomethylation of DNA plays a causal role in thetransformation process. An alternative explanation that issuggested by our data and the model proposed here is thatthe global hypomethylation induced by a methionine-deficientdiet results in a feedback induction of dnmt1. Increased levelsof dnmt1 per se have been previously suggested to play a causalrole in cellular transformation. Future experiments will beneeded to test this hypothesis.

A C K N O W L E D G E M E N T S

This work was supported by a grant to MS from the Medical Research

Council and the National Cancer Institute of Canada.

R E F E R E N C E S

1. Razin, A. & Szyf, M. (1984) DNA methylation patterns: formation and

function. Biochim. Biophys. Acta 782, 331±342.

Fig. 7. Negative feedback regulation of dnmt1

expression by methylation, a model. An AP-1

regulatory element of dnmt1, flanked by a CG

rich sequence upstream to the proximal promoter,

is heavily methylated (methylated CGs are

indicated as filled circles adjacent to the AP-1

consensus sites) in P19 cells and in many tissues

in vivo. This methylation prevents access of AP-1

to this region resulting in low basal

transcriptional activity of the dnmt1 gene

(indicated by the small horizontal arrows

representing transcription initiation positions).

When the cell is challenged by a DNA

methyltransferase inhibitor, this region is

demethylated (demethylated CGs are indicated as

open circles) and this is followed by interaction

of AP-1 with the regulatory region (depicted as

Fos and Jun dimers interacting with the

regulatory region) and induction of dnmt1 mRNA

expression (indicated by the large horizontal

arrows). Induction of dnmt1 results in high levels

of DNA methyltransferase activity and

remethylation of the regulatory region resulting

in a return to basal tone of dnmt1 expression.


2. Razin, A. & Riggs, A.D. (1980) DNA methylation and gene function.

Science 210, 604±610.

3. Siegfried, Z. & Cedar, H. (1997) DNA Methylation: a molecular lock.

Curr. Biol. 7, 305±307.

4. Szyf, M. (1996) The DNA methylation machinery as a target for

anticancer therapy. Pharmacol. Ther. 70, 1±37.

5. Jones, P.L., Veenstra, G.J.C., Wade, P.A., Vermaak, D., Kass, S.U.,

Landsberger, N., Strouboulis, J. & Wolffe, A.P. (1998) Methylated

DNA and MeCP2 recruit histone deacetylase to repress transcription.

Nat. Genet. 1 (G9), 187±191.

6. Nan, X., Campoy, F.J. & Bird, A. (1997) MeCP2 is a transcriptional

repressor with abundant binding sites in genomic chromatin. Cell 88,

471±481.

7. Wu, J.C. & Santi, D.V. (1985) High level expression and purification of

the HhaI methyltransferase. Prog. Clinic. Biol. Res. 198, 119±129.

8. Bestor, T.H., Laudano, A., Mattaliano, R. & Ingram, V. (1988) Cloning

and sequencing of a cDNA encoding DNA methyltransferase of mice

cells. J. Mol. Biol. 203, 971±983.

9. Yen, R.W., Vertino, P.M., Nelkin, B.D., Yu, J.J., el-Deiry, W.,

Cumaraswamy, G., Lennon, G., Trask, B.J., Celano, P. & Baylin,

S.B. (1992) Isolation and characterization of the cDNA encoding

human DNA methytransferase. Nucleic Acids Res. 20, 2287±2291.

10. Li, E., Bestor, T.H. & Jaenisch, R. (1992) Targeted mutation of the

DNA methyltransferase gene results in embryonic lethality. Cell 69,

915±926.

11. Jones, P.A. (1985) Altering gene expression with 5-azacytidine. Cell

40, 485±486.

12. Szyf, M., Rouleau, J., Theberge, J. & Bozovic, V. (1992) Ras induces

a general DNA demethylation activity in mouse embryonal P19

cells. J. Biol. Chem. 267, 12831±12836.

13. Ramchandani, S., MacLeod, A.R., Pinard, M., von Hofe, E. & Szyf, M.

(1997) Inhibition of tumorigenesis by a cytosine-DNA methyltrans-

ferase antisense oligonucleotide. Proc. Natl Acad. Sci. USA 94,

684±689.

14. Szyf, M. (1994) DNA methylation properties: consequences for

pharmacology. Trends Pharmacol. Sci. 7, 233±238.

15. MacLeod, A.R., Rouleau, J. & Szyf, M. (1995) Regulation of DNA

methylation by the Ras signaling pathway. J. Biol. Chem. 270,

11327±11337.

16. Rouleau, J., MacLeod, A.R. & Szyf, M. (1995) Regulation of the DNA

methyltransferase by the Ras-AP-1 signaling pathway. J. Biol. Chem.

270, 1595±1601.

17. Yang, J., Deng, C., Hemati, N., Hanash, S.M. & Richardson, B.C.

(1997) Effect of mitogenic stimulation and DNA methylation

on human T cell DNA methyltransferase expression and

activity. J. Immunol. 159, 1303±1309.

18. Szyf, M., Bozovic, V. & Tanigawa, G. (1991) Growth regulation of

mouse DNA methyltransferase. J. Biol. Chem. 266, 10027±10030.

19. Cole, T.J., Blendy, J.A., Monaghan, A.P., Krieglstein, K., Schmid, W.,

Aguzzi, A., Fantuzzi, G., Hummler, E., Unsicker, K. & Schutz, G.

(1995) Targeted disruption of the glucocorticoid receptor gene blocks

adrenergic chromaff in cell development and severely retards lung

maturation. Genes Dev. 9, 1608±1621.

20. Gasson, J.C., Ryd en, T. & Bourgeois, S. (1983) Role of de novo DNA

methylation in the glucocorticoid resistance of a T lymphoid cell line.

Nature 302, 621±623.

21. Christman, J.K., Sheikhnejad, G., Dizik, M., Abileah, S. & Wainfan, E.

(1993) Reversibility of changes in nucleic acid methylation and gene

expression induced in rat liver by severe dietary methyl deficiency.

Carcinogenesis 14, 551±555.

22. Rouleau, J., Tanigawa, G. & Szyf, M. (1992) The mouse DNA

methyltransferase 5 0 region: a unique housekeeping gene promoter.

J. Biol. Chem. 267, 7368±7377.

23. Yasumura, Y., Buonsassisi, V. & Sato, G. (1966) Clonal analysis of

differentiated function in animal.

24. McBurney, M.W., Jones-Villeneuve, E.M.V., Edwards, M.K.S. &

Anderson, P.J. (1982) Control of muscle and neuronal differentiation

in a cultured embryonal cell line. Nature 299, 165±167.

25. de Groot, R.P., van der Saag, P.T. & Krujjer, W. (1990) Ectopic

expression of c-jun leads to differentiation of P19 embryonal

carcinoma cells. EMBO J. 9, 1831±1837.

26. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A.,

Seidman, J.G. & Struhl, K. (1988) Current Protocols in Molecular

Biology. Wiley and Sons, New York.

27. Szyf, M., Kaplan, F., Mann, V., Giloh, H., Kedar, E. & Razin, A. (1985)

Cell cycle-dependent regulation of eukaryotic DNA methyltrans-

ferase levels. J. Biol. Chem. 260, 8653±8656.

28. Lewis, J.D., Meehan, R., Henzel, W., Maurer-Fogy, I., Jeppesen, P.,

Klein, F. & Bird, A. (1992) Purification, sequencing and cellular

localization of a novel chromosomal protein that binds to methylated

DNA. Cell 6, 905±914.

29. Becker, P.B.R., uppert, S. & Schutz, G. (1987) Genomic footprinting

reveals cell-type specific DNA binding of ubiquitous factors. Cell 51,

435±443.

30. Penix, L.A., Sweetser, M.T., Weaver, W.M., Hoeffler, J.P., Kerpolla,

T.K. & Wilson, C.B. (1996) The proximal regulatory element of the

interferon-gamma promoter mediates selective expression in T cells.

J. Biol. Chem. 271, 31964±31972.

31. Nur, I., Szyf, M., Razin, A., Glaser, G., Rottem, S. & Razin, S. (1985)

Prokaryotic and eukaryotic traits of DNA methylation in spiro-

plasmas (mycoplasmas). J. Bacteriol. 164, 19±24.

32. Buschhausen, G., Wittig, B., Graessmann, M. & Graessmann, A.

(1987) Chromatin structure is required to block transcription of the

methylated herpes simplex virus thymidine kinase gene. Proc. Natl

Acad. Sci. USA 84, 1177±1181.

33. Kass, S.U., Landsberger, N. & Wolffe, A.P. (1997) DNA methylation

directs a time-dependent repression of transcription initiation.

Current Biol. 7, 157±165.

34. Merteineit, C., Yoder, J., Taketo, T., Laird, D.W., Trasler, J. & Bestor,

T. (1988) Sex-specific exons control DNA methyltransferase in

mammalian germ cells. Development 125, 889±897.

Feedback regulation of DNA methyltransferase gene expression by methylation

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