Proc. Natl. Acad. Sci. USAVol. 85, pp. 4638-4642, July 1988Biochemistry

Effect of in vitro DNA methylation on ,-globin gene expressionJOEL YISRAELI*, DALE FRANK, AHARON RAZIN, AND HOWARD CEDARDepartment of Cellular Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel 91010

Communicated by Richard Axel, March 4, 1988

ABSTRACT When the human j3-globin gene was methyl-ated at every cytosine residue and was inserted into mousefibroblasts by DNA-mediated gene transfer, the transcriptionof the gene was strongly inhibited. This methylation alsoprevented expression and induction of the gene in mouseerythroleukemia cells. By using partially methylated hybridmolecules, it was shown that methylation-sensitive negativeregulatory elements are located in both the 5' and 3' ends of the13-globin gene but not in the 90-base-pair region usuallyassociated with promoter activity. To further investigate therole ofDNA methylation in the regulation of the f3-globin gene,50-base-pair poly(dG-dC) tracts were introduced into varioussites in a mouse-human hybrid gene, and these inserts weremethylated by means of the Hha I methylase. Heavy methyl-ation of these artificially added sites had no effect on eithertranscription initiation or elongation, suggesting that DNAmodification operates through fixed endogenous sites in thegene domain.

DNA methylation has been implicated as one of the factorsthat influences tissue-specific gene expression (1). Thisconclusion was suggested by the fact that these genes tend tobe undermethylated in the tissue of expression but highlymethylated in other tissues and sperm DNA. Convincingevidence that DNA methylation plays a causative role in therepression of gene activity comes from studies showing thatthe transcription of in vitro-methylated DNA sequences isinhibited when the sequences are inserted into various celltypes in culture. Furthermore, several genes that are nor-mally inactive can be activated following treatment of cellswith 5-azacytidine, a potent demethylating agent.

Despite considerable effort to understand the specificity ofthis modification, the mechanism of action has not yet beenfully elucidated. Although experiments with some genesshow that DNA methylation has powerful effects at selectiveindividual sites in the 5' region (2-4), other studies indicatethat methylation may work over the entire gene domain (5),probably by interfering with the formation of a normal activechromosomal conformation (6).The sequences involved in the expression and positive

regulation of the human f3-globin gene have been wellcharacterized (7), but little is known about the role ofDNAmethylation in this process. Since CpG sequences are un-derrepresented in ,3-globin genes (8, 9), few methyl-sensitiverestriction enzyme sites are available for study. Analysis ofthese sites, which are mostly found in flanking sequences,shows that the activity of globin is clearly correlated withundermethylation in a tissue- and stage-specific manner (10,11). In this paper, we have used in vitro-methylated /3-globinDNA to study the causative effects of this modification on itsexpression in mouse fibroblasts and its regulation in mouseerythroleukemia cells. The results show that methylationinhibits transcriptional initiation but not elongation. Thelocalization of this effect reveals interesting information on

the negative regulatory elements present in the region of thisgene.

MATERIALS AND METHODSCell Growth and Transfection. Thymidine kinase deficient

(tk-) L cells were grown in Dulbecco's modified Eagle'smedium supplemented with 10% newborn calf serum (5). tk -mouse erythroleukemia cells (MEL) were grown in Dul-becco's modified Eagle's medium supplemented with 15%fetal calf serum and were induced by treatment with hexa-methylenebisacetamide for 3-4 days (12). DNA-mediatedgene transfer into mouse Ltk - or MEL tk - cells wasperformed as described (5) by using 50-200 ng of theselectable marker (the thymidine kinase gene) and 1-2 ,ug ofthe cotransfected methylated or unmethylated DNA con-struct per plate. Selection was achieved by growing inhypoxanthine/aminopterin/thymidine. Fifty to 100 coloniesper plate were pooled, except where indicated in the text, andwere grown to mass culture.M13 Methylation of Human fi-Globin. The M13 constructs

M,l1 and MB2 were obtained from R. Flavell (Biogen,Cambridge, MA) and contain the 4.4-kilobase (kb) humangenomic ,-globin Pst I fragment cloned into M13mp8 in bothorientations. Fully methylated DNA was prepared from theseconstructs by second-strand synthesis of single-strandedphage molecules using the 15-mer universal primer and5-methyl-2'-deoxycytidine triphosphate (m'dCTP) instead ofdCTP, as previously described (13). Partially methylatedDNA molecules were constructed by using globin restrictionfragments as primer in the same reaction. In this way, theregion covered by the primer remains unmethylated, whilethe rest of the molecule becomes methylated during second-strand synthesis. 5' unmethylated globin DNA was made byusing the 5' Pst I/Nco I fragment as primer, whereas 3' un-methylated DNA was made with the 3' Nco I/Pst I fragment(see Fig. 2). Since DNA polymerase can, under certainconditions, displace or digest regions containing the un-methylated primers, these hybrid constructs were tested fortheir topographical methyl specificity as described (5).Enzymatic Methylation of /3-Globin Plasmids. Plasmids

pHl6 and pH,89, which contain the genomic Bgl II andHindIII fragments of the /3-globin gene, were obtained fromT. Maniatis (Harvard University, Cambridge, MA). Globinconstructs containing poly(dG-dC) tracts were made bytransferring a 50-base-pair (bp) poly(dG-dC) fragment withBamHI linkers (A. Rich, Massachusetts Institute of Tech-nology, Cambridge, MA) from pBR322 into two sites in themouse-human chimeric globin-gene plasmid ppMH20 (T.Maniatis) (14). This gene contains the 5' end of mouse,8-globin gene fused to the 3' end of the human gene. Thepoly(dG-dC) fragment was inserted at either the Bgl II site(p/3Z1) or theBamHI site (pf3Z2) (see Fig 3). These constructswere methylated using Hha I methylase and were tested for

Abbreviations: tk-, thymidine kinase deficient; m'dCTP, 5-methyl-2'-deoxycytidine triphosphate.*Present address: Department of Biochemistry and Molecular Biol-ogy, Harvard University, Cambridge, MA 02138.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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complete methylation as previously described (15). It shouldbe noted that the chimeric globin gene has no intrinsic HhaI sites, and only the poly(dG-dC) sequences can be modifiedby Hha I methylase.DNA and RNA Analysis. DNA was purified from mouse L

cells or MEL cells (5) and was subjected to Southern blotanalysis using gel-purified restriction fragment probes la-beled by nick-translation (5) to a specific activity of2-5 x 10'cpm/,ug. S1 nuclease hypersensitivity analysis of supercoiledplasmids and integrated DNA in isolated nuclei was per-formed as described (16). RNA was isolated by guanidineisothiocyanate extraction followed by ultracentrifugationthrough a CsCl cushion and was subjected to S1 nucleaseanalysis (12) by using a 3' end-labeled human Pst I/EcoRIprobe (see Fig. 3). The RNA dot blot was prepared byformaldehyde denaturation and was hybridized to the indi-cated nick-translated probes.

RESULTSTo study the effect ofDNA methylation on eukaryotic genes,we previously developed a convenient method for methylat-ing all CpG residues by a combined in vitro/in vivo procedure(13). Gene sequences are cloned into the single-strandedphage vector M13, and the complementary strand is synthe-sized in the presence of m'dCTP instead of dCTP. Althoughthe resulting molecules are methylated nonphysiologically atevery cytosine residue on the newly synthesized strand,when they are introduced into cultured animal cells byDNA-mediated gene transfer, the intracellular CpG-specificmaintenance methylase perpetuates all of the CpG methyla-tions, while cytosine modification at other sites is diluted outthrough generations of growth.The M31 construct containing the complete human f8-

globin gene cloned into M13mp8 was fully methylated byusing the M13 15-mer universal primer. Unmethylated con-trols were synthesized with the same primers by using dCTPas the substrate in the DNA polymerase reaction. Thesemethylated and nonmethylated constructs were insertedseparately into tk - mouse L cells by cotransfection using theHerpes thymidine kinase gene as the selective marker, andhypoxanthine/aminopterin/thymidine-resistant colonieswere isolated orpooled together and grown into mass culture.To determine the level of globin expression in these lines,total RNA was subjected to S1 nuclease analysis using anEcoRI/Pst I 3' probe, which protects the last 212 bp of thetranscript. Total CpG methylation of the (-globin genemarkedly inhibited expression of 3-globin in both pools andclones of L cells in comparison to the nonmethylated con-structs (see Fig. 1).

Since DNA methylation appears to inhibit the basal levelof transcription of (3-globin, we then asked whether methyl-ated genes can be induced in differentiating Friend erythro-leukemia cells. S1 nuclease analysis of RNA produced intransfected Friend cells containing the exogenous globin geneshowed that the methylated gene was expressed poorly inthese cells and did not respond to hexamethylenebisaceta-mide induction despite the fact that the unmethylated controlwas induced 20-fold and the endogenous globin gene wasinduced 10- to 20-fold in the same cells (data not shown).Thus, DNA methylation appears to inhibit globin transcrip-tion and prevent stage-specific induction. Southern blotanalysis usingHpa II showed that the M13 sequences and onesite at the extreme 3' end of the globin gene retained theirmethylation even after 50 generations ofgrowth either beforeor after induction (Fig. 1). Other CpG sites within the genedomain probably stayed modified, but no restriction enzymesare available to analyze these sequences. The lack of basalactivity in L cells and inducibility in erythroleukemia cellsassociated with the methylated molecules was not due to any

aA B C D E F G H

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FIG. 1. Expression of methylated 3-globin in fibroblasts anderythroleukemia cells. (a) S1 nuclease analysis of total RNA (30 ,g)extracted from cells containing transfected M13 3-globin constructsby using the 3' globin probe, which protects the last 212 bp of thetranscript. Lanes A-F show RNA from transfected fibroblasts.Lanes: A, pooled clones containing unmethylated M13 M,81 DNA;B, pooled clones containing methylated M13 M(31 DNA; C and D,RNA from individual unmethylated clones; E and F, RNA fromindividual methylated clones. Lanes G-R contain RNA from trans-fected MEL cells. The level ofinduction ofunmethylated exogenousglobin genes in a pBR vector (pHP6 or pHB9) in pooled clones isshown in lanes G and H and also lanes Q and R as well as in cellscontaining an unmethylated M13 construct (lanes 0 and P). In eachcase, the RNA from uninduced cells is shown in the left lane of eachpair (lanes G, Q, and 0). Lanes K-N showRNA from induced clonescontaining methylated M13 globin constructs. In all methylatedclones or pooled clones tested, the level of globin RNA was 15-30times less than that observed for the cells containing unmethylatedglobin DNA. A long exposure of RNA extracted from uninduced(lane I) and induced (lane J) pooled clones containing methylatedglobin is also shown. The position of the protected 212-bp transcriptis indicated at left. (b) DNA from pooled L-cell colonies (lanes C andD) or pooled erythroleukemia colonies (lanes A and B) containing themethylated M13 globin construct was digested with Pst I (lanesA andC) or EcoRI/BamHI (lanes B and D) and was analyzed by blothybridization by using the human P-globin 4.4-kb Pst I fragment asprobe. Fragment sizes (in kb) are indicated at left. (c) DNA frompooled transfected erythroleukemia colonies containing the methyl-ated M13 globin construct was digested with Hpa II (lanes A, C, andE) or Msp I (lanes B, D, and F) and was analyzed by blothybridization using a nick-translated probe ofM13 sequences. LanesA and B, DNA from uninduced cells. DNA from cells induced for 45hr (lanes C and D) or 90 hr (lanes E and F) is also shown. The 1.6-kband 0.8-kb fragments indicated at left are the expected digestionproducts of M13 DNA.

rearrangement that might have affected gene transcription.Restriction analysis of both isolated clones and pools ofmethylated globin gene-containing cells showed that they allcontain 5-10 intact copies (Fig. 1). This transfection exper-iment was repeated numerous times using several differentMEL cell lines, and in over 20 individual clones and pools, wenever observed any induction of the methylated f3-globingene.

Since DNA methylation has such a profound effect onglobin-gene expression, we attempted to pinpoint the site ofaction of this modification. This was accomplished by syn-thesizing partially methylated hybrid (3-globin gene con-

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4640 Biochemistry: Yisraeli et al.

structs using M13 templates and specific restriction frag-ments to direct the placement of methyl moieties to specificgene domains. Molecules methylated only in the 5' upstreamregion, for example, were synthesized with m5dCTP usingthe 2.3-kb Pst I/Nco I restriction fragment as primer (see Fig.2). Complementary molecules, in which the 3' end wasexclusively methylated, were obtained by using the 2.1-kbNco I/Pst I fragment as primer on an M13 construct con-taining the f-globin gene in the opposite orientation. Thesepartially methylated hybrid constructs and nonmethylatedcontrol constructs were inserted separately into mouse Lcells by DNA-mediated gene transfer, and pools of >100colonies per experiment were grown and analyzed for globin-gene expression by S1 nuclease digestion. As shown in Fig.2, methylation of either the 5' or 3' regions of the gene weresufficient to inhibit p-globin expression by >20-fold. Thisinhibition could only be due to the effect of regional meth-ylation and not to differences in copy number or genearrangement, since Southern blots indicated that all cellpopulations contained an average of about one intact copy ofthe globin gene per cell. The results suggest that globin geneexpression is indeed subject to control by DNA methylationand that the sequences responsive to this modification aredistributed over the entire gene domain.To further pinpoint the mechanism of methylation and to

determine whether heavy modification anywhere in the gene

a

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domain would have an inhibitory effect, we made globinconstructs containing methylatable inserts. To this end, a50-bp alternating (dG-dC)25 oligomer containing BamHI link-ers was cloned into the plasmid p.3MH20 (Fig. 3), a mouse-human 8-globin chimera, at two locations in the transcribedregion of the gene: the Bgl II site (+ 27) in the first mouseexon and the BamHI site (+ 500) in the second human exon.These constructs were methylated in vitro at the 50 Hha Isites of the CpG tract using Hha I methylase. Since the entireglobin domain contains no intrinsic Hha I sites, methylmoieties were thus limited to the regions of the alternatingCpG tracts. The methylated, the nonmethylated, and theoriginal pf3MH20 constructs were introduced separately intotk- L cells by DNA-mediated gene transfer, and pools ofcolonies were grown to mass culture. We assayed globinRNA levels by using S1 nuclease analysis with a 3' specificprobe. As shown in Fig. 3, there was no significant differencein the level of transcription between methylated, nonmeth-ylated, and control constructs (see legend to Fig. 3). To verifythat RNA elongation was not affected by methylation of the50-bp CpG tracts, we compared the relative amounts oftranscripts synthesized from upstream 5' sequences to themRNA levels transcribed from sequences downstream of thetracts by using dot blot analysis with two specific probes. Asshown in Fig. 3, the same relative levels of transcriptionoccur both upstream and downstream of the 50-bp CpGtracts. Therefore, it appears that nonspecific methylatedresidues in the 3' region per se, as well as alternating

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Probe ProbeB A* pfZIMe

* ppZI

* * pPZ2 Me

-w 0 *p PZ2

Pst I Bgl II Nco I EcoRI PstI

1 kb

FIG. 2. Analysis of L cells transfected with partially methylatedhybrid globin genes. (a) Methylated and partially methylated hybridM13 molecules were synthesized in the presence of trace amounts ofradioactive nucleotides. The resulting double-stranded M13 mole-cules were purified, subjected to restriction enzyme analysis and gelelectrophoresis, and visualized by autoradiography. The double-strand DNA shown in lane 1 was made using the 2.1-kb Pst I/NcoI flanking primer. By using m'dCTP as substrate, the entire molecule,except for the primer, should be methylated. To test whether theprimer indeed remained unmodified, and thus unlabeled, the M13duplex was digested with Bgl II/Nco I/EcoRI (lane 2). Restrictedduplex DNA made with the 15-mer universal M13 primer is shownin lane 3. Note that the 1.6-kb Bgl II/Nco I fragment (indicated byarrow), which is totally contained within the primer, is poorly labeledrelative to the fully methylated molecule, indicating that the synthe-sis leakage into the primer was minimal. Hybrid constructs in whichthe 3' end was kept unmethylated were made using M13-containingglobin in the opposite orientation with the 2.3-kb Nco I/Pst I primerand was tested by the same assay (data not shown). A A HindIIIdigest is shown in lane 4. (b) RNA (30 ,g) from cells transfected withunmethylated globin DNA (lane 1), 5' unmethylated DNA (lane 2),or 3' unmethylated DNA (lane 3) was analyzed by S1 nucleaseanalysis. The relatively low level ofRNA observed in the unmethy-lated control (lane 1) is due to the very low p8-globin gene copynumber in all transfectants produced in this experiment. Cell poolsfrom each transfection contained an average of one copy per cell. (c)Map ofgenomic 4.4-kb Pst I fragment containing the human f-globingene.

0 * pPMH20

CONTROL

Probe B

HindMBgmI

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FIG. 3. Expression of poly(dG-dC)-containing globin constructsin L cells. (Upper Left) S1 nuclease analysis ofRNA (30 ,ug) from Lcells transfected with methylated (lanes 1 and 2) or unmethylated(lane 3) p,8Z1, methylated (lanes 4 and 5) or unmethylated (lane 6)p,3Z2, and unmethylated pfBMH20 (lane 7). An Msp I digest ofpBR322 is shown in lane 8. (Upper Right) The same RNAs (6 Ag)were assayed on dot blots by using either a 5' specific probe (probeB) or a 3' specific probe (probe A). The control slot containsuntransfected L-cell RNA. The ratio of hybridization to the 5' probeto hybridization to the 3' probe was the same for all samples. (Lower)The map shows the globin-containing region of pJBMH20 indicatingthe relevant restriction enzyme sites. The 5' HindIII/BamHI portionof the construct is from mouse /3-globin, whereas the 3' BamHI/PstI portion contains human B-globin sequences. In p3Z1, a 50-bppoly(dG-dC) segment was inserted into p.3MH20 at the Bgl II site,whereas in pBZ2 this same fragment was inserted at the BamHI site.

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2.8-2.5- _2.0- * -

FIG. 4. Methylation analysis of transfected L-cell DNA. DNA(20 jg) from L cells transfected with various poly(dG-dC)-containingglobin constructs was digested with Pst I/HindIII with or withoutHha I and was subjected to gel electrophoresis and blot hybridizationby using a 3' specific human 3-globin probe (probe A in Fig. 3). Eachconstruct was analyzed in pairs in which the first sample (left) wasdigested with Hha I, while the second sample (right) was not. Lanes:4 and 5, methylated pSZ1; 6 and 7, unmethylated pfBZ1; 8 and 9,methylated p,8Z2; 10 and 11, unmethylated pSZ2; 12 and 13,unmethylated p,8mH20. Note that p(3MH20 does not have an Hha Isite within the HindIII/Pst I fragment. The Southern blot digestionpatterns of the original plasmid constructs pfZ1 (lane 1), pBZ2 (lane2), and p,8MH20 (lane 3) are also shown. In this particular blot,digestion oflane 6 with H/ha I was only 90%o complete, but other blotsindicated that this DNA is completely unmethylated in this region.Sizes (in kb) are indicated at left.

purine/pyrimidine tracts, affect neither RNA polymerasepropagation nor initiation of transcription in the P-globingene. The methylation state of the (dG-dC)25 tracts in vivowas determined by Nha I digestion and Southern blotanalysis (Fig. 4) of transfected cells. The tract in the + 27construct was found to be completely methylated, whereasthe tract in the + 500 construct underwent =40% demethyl-ation. Kinetic studies with Hha I show that this is due to theloss of one methyl group per 50 Hha I sites on 40o of themolecules, rather than a total demethylation of all 50 Hha Isites (data not shown). Since CpG tracts of this nature areknown to undergo conversion to Z-DNA in supercoiledplasmids in vitro, we investigated the topological state of thepoly(dG-dC)-containing globin DNA in vitro and in vivo. S1nuclease hypersensitive sites were identified in both con-structs at the apparent B-DNA-Z-DNA junctions (17) insupercoiled plasmids, as expected, but no S1 nucleasehypersensitive sites were found in either the methylated ornonmethylated constructs in nuclei from transfected cells(data not shown).

DISCUSSIONThese experiments demonstrate that DNA methylation caninhibit 8-globin gene expression in mouse fibroblast anderythroleukemia cells. Despite the relatively low number ofCpG residues in the gene domain (there are 15 CpG residuesscattered through the gene domain from -1500 to + 1950),total methylation of these sites was sufficient to inhibittranscription by a factor of -20. This repression must involveregulatory sequences both in the 5' flanking region and thesequences covering the gene body and 3' flanking region,since localized methylations of either of these regions havesimilar effects. This disperse inhibition is characteristic ofother genes that have been investigated, such as herpesthymidine kinase (5), but it is not an exclusive effect, sincesome genes have methyl-sensitive sites only in their 5' region.This is true, for instance, in the case of the hamster aprt gene(5, 18), simian virus 40 early region (4), human y-Yglobin (2),and several adenovirus promoters (3). It should be noted,however, that in the case of the adenovirus promoters,

activity was assayed using a chloramphenicol acetyltrans-ferase-linked construct, and thus the effect of in vitro DNAmethylation on the 3' sequences was never properly inves-tigated.The strong effect ofDNA methylation within the f-globin

5' flanking sequences is rather surprising, since this regioncontains very few CpG residues and none within the first 120bp upstream of the start of transcription. By using deletionand site-specific mutation analysis on (3-globin genes fromseveral mammalian sources, it has been shown that only 90nucleotides in the 5' region are necessary and sufficient forfull transcriptional activity (7). It should be pointed out thatthis type of "reverse genetics" highlights the regions that arerequired for the positive regulation of the gene but does notrelate to sequence domains that may have a negative regu-latory role. Our studies show that methylation of sequencesbeyond the promoter region can inhibit gene expression andsuggest the presence of a distal 5' negative regulatoryelement. A similar situation exists for the human y-globingene, where there appears to be multiple methyl-sensitivesites both in the promoter region and in further upstreamsequences (19). Mutation analysis in the /-globin structuralgene and 3' flanking sequences does not show any specificsequence necessary for basal gene transcription, but theseregions may indeed contain potential inhibitory elements thatare responsive to DNA modification. Sequences in the 3'region are also involved in the induction response seen inerythroleukemia cells (12, 20, 21). Furthermore, DNA un-dermethylation at at least one site in the 3' flanking region isstrongly correlated with gene expression in vivo (10, 22).Mouse erythroleukemia represent cells arrested in a late

stage oferythroid development. Upon treatment with variouschemical agents, they can be induced to produce the a- andf-globin chains and other genes characteristic of transcrip-tionally active erythroid precursors (23). Both a- and f-globingenes are DNase I sensitive and are undermethylated at a fewspecific sites, even in the erythroleukemia cell before induc-tion (24). Since globin from other human and mouse tissuesand sperm DNA are fully methylated at all assayable sites(11), the evolution of the erythroleukemia cell must involvespecific demethylation events in consort with changes in geneconformation. Our results show that although an under-methylated exogenous f-globin gene can be induced in thesecells, DNA modification at every CpG residue totally pre-vents this transcriptional activation. This suggests that DNAmethylation at sites within the gene domain inhibits someaspect ofthe induction or transcription process and that thesemethyl moieties must be removed prior to gene activation.This system contrasts sharply to that of L8 rat myoblasts,which can be induced to differentiate into myotubules in vitro(25), a process that is accompanied by the induction ofseveral muscle-specific genes, including a-actin. In this case,methylated actin genes, which are inhibited in fibroblasts, arefully active in induced myoblasts following their introductioninto these cells by DNA-mediated gene transfer. This acti-vation is accompanied by a highly specific demethylation ofthe exogenous gene, which closely mimics the demethylationthat occurs to the endogenous gene during muscle develop-ment (15). Interestingly, this terminal myoblast differentia-tion is accompanied by a marked change in the DNase Isensitivity of several muscle-specific genes (26), in contrastto the induction of MEL. Erythroleukemia cells represent alate stage in terminal differentiation, which may have lost theability to "turn on" inactive globin genes and are thus unableto cope with a methylated gene. In this regard, it is surprisingthat an inactive, presumably methylated, /3globin gene inmouse fibroblasts could be efficiently activated followingfusion with induced erythroleukemia cells (27). It is difficultto compare these two experimental approaches, however,since in the latter case the degree ofDNA methylation of the

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globin gene has not been ascertained and the activation mayalso be of a transient nature.

Despite the striking effect ofDNA methylation at specificCpG residues in the entire globin gene domain, the insertionofa fully methylated 50-bp repeat ofCpG had no effect on theactivity of the gene. This suggests that the effect of DNAmethylation on the natural CpG residues within the genesequence cannot be mimicked by the addition of irrelevantmethylated moieties. This is consistent with the idea thattranscriptional inhibition is mediated through the interactionof proteins with various cis-acting, well-defined, negativeregulatory elements that are influenced by DNA methylation.DNA methylation can alter protein binding to specific sites(28) and can modify the usual chromosomal conformationassociated with the active domains (6). Since this latter effectmay be limited to the sequences in the immediate vicinity ofthe methyl moieties, it is doubtful that methylation of theartificial CpG tracts within the globin gene would influencethe protein conformation in the regulatory regions andthereby affect transcription.By carefully analyzing the relative transcriptional effi-

ciency of 5' and 3' sequences, it was shown that fullymethylated CpG tracts within either the first or second exonhave no effect on the rate of transcriptional elongation. Oneproblem in the interpretation of these results is that shortertranscripts might not be properly processed or could bedegraded. If this were the case, however, we would detect alower level of steady-state RNA produced from methylatedconstructs. Our results strongly suggest, therefore, that themere presence of a large tract of methyl moieties is notsufficient to hinder the progress of RNA polymerase.When present in supercoiled plasmids, CpG tracts can

easily flip into a Z-DNA conformation, and this alteredsecondary structure represents a strong barrier for theelongation of Escherichia coli RNA polymerase in vitro (29).The CpG-containing supercoiled plasmids used in our studiesalso contained a potential Z-DNA segment as shown by S1nuclease digestion, yet these CpG tracts had no effect oneither transcriptional initiation or elongation in vivo. Eventhough DNA methylation has a marked stimulatory effect onthe ability of CpG tracts to convert to the Z form (30), thismodification had no detectable influence on the transcrip-tional process. All of these results strongly suggest, but by nomeans prove, that neither the unmethylated nor the fullymethylated CpG alternating sequence exists in the Z confor-mation in vivo. Attempts to directly detect such an alteredconformation by S1 nuclease digestion of transfected cellnuclei confirmed the absence of Z-DNA. Thus, despite theexistence of Z-DNA in vitro, there is, as yet, no compellingbiophysical or biological evidence that this structure exists invivo even under the most favorable conditions.

We thank R. Flavell, F. Grosveld, M. Busslinger, T. Maniatis, andA. Rich for kindly supplying the vectors used in this study. We are

especially grateful to M. Chou, A. Rosenthal, and S. Wright, whoperformed some of the DNA transfections of methylated constructsinto MEL cells for use in these experiments. We thank CarolineGopin for her help in the preparation ofthe manuscript. This researchwas supported by a grant from the National Institutes of Health.

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