Effect of in vitro DNA methylation on ,-globin gene expression

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Selection was achieved by growing in hypoxanthine/aminopterin/thymidine. Fifty to100 colonies per plate were pooled, except where indicated in the text, and.
Proc. Natl. Acad. Sci. USA Vol. 85, pp. 4638-4642, July 1988 Biochemistry

Effect of in vitro DNA methylation on ,-globin gene expression JOEL YISRAELI*, DALE FRANK, AHARON RAZIN, AND HOWARD CEDAR Department 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 methylated at every cytosine residue and was inserted into mouse fibroblasts by DNA-mediated gene transfer, the transcription of the gene was strongly inhibited. This methylation also prevented expression and induction of the gene in mouse erythroleukemia cells. By using partially methylated hybrid molecules, it was shown that methylation-sensitive negative regulatory elements are located in both the 5' and 3' ends of the 13-globin gene but not in the 90-base-pair region usually associated with promoter activity. To further investigate the role of DNA methylation in the regulation of the f3-globin gene, 50-base-pair poly(dG-dC) tracts were introduced into various sites in a mouse-human hybrid gene, and these inserts were methylated by means of the Hha I methylase. Heavy methylation of these artificially added sites had no effect on either transcription initiation or elongation, suggesting that DNA modification operates through fixed endogenous sites in the gene domain.

the negative regulatory elements present in the region of this gene.

MATERIALS AND METHODS Cell Growth and Transfection. Thymidine kinase deficient (tk-) L cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum (5). tk mouse erythroleukemia cells (MEL) were grown in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum and were induced by treatment with hexamethylenebisacetamide for 3-4 days (12). DNA-mediated gene transfer into mouse Ltk - or MEL tk - cells was performed as described (5) by using 50-200 ng of the selectable marker (the thymidine kinase gene) and 1-2 ,ug of the cotransfected methylated or unmethylated DNA construct per plate. Selection was achieved by growing in hypoxanthine/aminopterin/thymidine. Fifty to 100 colonies per plate were pooled, except where indicated in the text, and were 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) human genomic ,-globin Pst I fragment cloned into M13mp8 in both orientations. Fully methylated DNA was prepared from these constructs by second-strand synthesis of single-stranded phage molecules using the 15-mer universal primer and 5-methyl-2'-deoxycytidine triphosphate (m'dCTP) instead of dCTP, as previously described (13). Partially methylated DNA molecules were constructed by using globin restriction fragments as primer in the same reaction. In this way, the region covered by the primer remains unmethylated, while the rest of the molecule becomes methylated during secondstrand synthesis. 5' unmethylated globin DNA was made by using the 5' Pst I/Nco I fragment as primer, whereas 3' unmethylated DNA was made with the 3' Nco I/Pst I fragment (see Fig. 2). Since DNA polymerase can, under certain conditions, displace or digest regions containing the unmethylated primers, these hybrid constructs were tested for their topographical methyl specificity as described (5). Enzymatic Methylation of /3-Globin Plasmids. Plasmids pHl6 and pH,89, which contain the genomic Bgl II and HindIII fragments of the /3-globin gene, were obtained from T. Maniatis (Harvard University, Cambridge, MA). Globin constructs containing poly(dG-dC) tracts were made by transferring a 50-base-pair (bp) poly(dG-dC) fragment with BamHI linkers (A. Rich, Massachusetts Institute of Technology, Cambridge, MA) from pBR322 into two sites in the mouse-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. The poly(dG-dC) fragment was inserted at either the Bgl II site (p/3Z1) or the BamHI site (pf3Z2) (see Fig 3). These constructs were methylated using Hha I methylase and were tested for

DNA methylation has been implicated as one of the factors that influences tissue-specific gene expression (1). This conclusion was suggested by the fact that these genes tend to be undermethylated in the tissue of expression but highly methylated in other tissues and sperm DNA. Convincing evidence that DNA methylation plays a causative role in the repression of gene activity comes from studies showing that the transcription of in vitro-methylated DNA sequences is inhibited when the sequences are inserted into various cell types in culture. Furthermore, several genes that are normally inactive can be activated following treatment of cells with 5-azacytidine, a potent demethylating agent. Despite considerable effort to understand the specificity of this modification, the mechanism of action has not yet been fully elucidated. Although experiments with some genes show that DNA methylation has powerful effects at selective individual sites in the 5' region (2-4), other studies indicate that methylation may work over the entire gene domain (5), probably by interfering with the formation of a normal active chromosomal conformation (6). The sequences involved in the expression and positive regulation of the human f3-globin gene have been well characterized (7), but little is known about the role of DNA methylation in this process. Since CpG sequences are underrepresented in ,3-globin genes (8, 9), few methyl-sensitive restriction enzyme sites are available for study. Analysis of these sites, which are mostly found in flanking sequences, shows that the activity of globin is clearly correlated with undermethylation in a tissue- and stage-specific manner (10, 11). In this paper, we have used in vitro-methylated /3-globin DNA to study the causative effects of this modification on its expression in mouse fibroblasts and its regulation in mouse erythroleukemia cells. The results show that methylation inhibits transcriptional initiation but not elongation. The localization of this effect reveals interesting information on

Abbreviations: tk-, thymidine kinase deficient; m'dCTP, 5-methyl2'-deoxycytidine triphosphate. *Present address: Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ยง1734 solely to indicate this fact.

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Biochemistry: Yisraeli et aL complete methylation as previously described (15). It should be noted that the chimeric globin gene has no intrinsic Hha I sites, and only the poly(dG-dC) sequences can be modified by Hha I methylase. DNA and RNA Analysis. DNA was purified from mouse L cells or MEL cells (5) and was subjected to Southern blot analysis using gel-purified restriction fragment probes labeled by nick-translation (5) to a specific activity of2-5 x 10' cpm/,ug. S1 nuclease hypersensitivity analysis of supercoiled plasmids and integrated DNA in isolated nuclei was performed as described (16). RNA was isolated by guanidine isothiocyanate extraction followed by ultracentrifugation through a CsCl cushion and was subjected to S1 nuclease analysis (12) by using a 3' end-labeled human Pst I/EcoRI probe (see Fig. 3). The RNA dot blot was prepared by formaldehyde denaturation and was hybridized to the indicated nick-translated probes.

Proc. Nadl. Acad. Sci. USA 85 (1988)

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RESULTS To study the effect of DNA methylation on eukaryotic genes, we previously developed a convenient method for methylating all CpG residues by a combined in vitro/in vivo procedure (13). Gene sequences are cloned into the single-stranded phage vector M13, and the complementary strand is synthesized in the presence of m'dCTP instead of dCTP. Although the resulting molecules are methylated nonphysiologically at every cytosine residue on the newly synthesized strand, when they are introduced into cultured animal cells by DNA-mediated gene transfer, the intracellular CpG-specific maintenance methylase perpetuates all of the CpG methylations, while cytosine modification at other sites is diluted out through generations of growth. The M31 construct containing the complete human f8globin gene cloned into M13mp8 was fully methylated by using the M13 15-mer universal primer. Unmethylated controls were synthesized with the same primers by using dCTP as the substrate in the DNA polymerase reaction. These methylated and nonmethylated constructs were inserted separately into tk - mouse L cells by cotransfection using the Herpes thymidine kinase gene as the selective marker, and hypoxanthine/aminopterin/thymidine-resistant colonies were isolated or pooled 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 an EcoRI/Pst I 3' probe, which protects the last 212 bp of the transcript. Total CpG methylation of the (-globin gene markedly inhibited expression of 3-globin in both pools and clones of L cells in comparison to the nonmethylated constructs (see Fig. 1). Since DNA methylation appears to inhibit the basal level of transcription of (3-globin, we then asked whether methylated genes can be induced in differentiating Friend erythroleukemia cells. S1 nuclease analysis of RNA produced in transfected Friend cells containing the exogenous globin gene showed that the methylated gene was expressed poorly in these cells and did not respond to hexamethylenebisacetamide induction despite the fact that the unmethylated control was induced 20-fold and the endogenous globin gene was induced 10- to 20-fold in the same cells (data not shown). Thus, DNA methylation appears to inhibit globin transcription and prevent stage-specific induction. Southern blot analysis using Hpa II showed that the M13 sequences and one site at the extreme 3' end of the globin gene retained their methylation even after 50 generations ofgrowth either before or after induction (Fig. 1). Other CpG sites within the gene domain probably stayed modified, but no restriction enzymes are available to analyze these sequences. The lack of basal activity in L cells and inducibility in erythroleukemia cells associated with the methylated molecules was not due to any

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FIG. 1. Expression of methylated 3-globin in fibroblasts and erythroleukemia cells. (a) S1 nuclease analysis of total RNA (30 ,g) extracted from cells containing transfected M13 3-globin constructs by using the 3' globin probe, which protects the last 212 bp of the transcript. 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 from individual methylated clones. Lanes G-R contain RNA from transfected MEL cells. The level of induction of unmethylated exogenous globin genes in a pBR vector (pHP6 or pHB9) in pooled clones is shown in lanes G and H and also lanes Q and R as well as in cells containing an unmethylated M13 construct (lanes 0 and P). In each case, the RNA from uninduced cells is shown in the left lane of each pair (lanes G, Q, and 0). Lanes K-N show RNA from induced clones containing methylated M13 globin constructs. In all methylated clones or pooled clones tested, the level of globin RNA was 15-30 times less than that observed for the cells containing unmethylated globin DNA. A long exposure of RNA extracted from uninduced (lane I) and induced (lane J) pooled clones containing methylated globin is also shown. The position of the protected 212-bp transcript is indicated at left. (b) DNA from pooled L-cell colonies (lanes C and D) or pooled erythroleukemia colonies (lanes A and B) containing the methylated M13 globin construct was digested with Pst I (lanes A and C) or EcoRI/BamHI (lanes B and D) and was analyzed by blot hybridization by using the human P-globin 4.4-kb Pst I fragment as probe. Fragment sizes (in kb) are indicated at left. (c) DNA from pooled transfected erythroleukemia colonies containing the methylated M13 globin construct was digested with Hpa II (lanes A, C, and E) or Msp I (lanes B, D, and F) and was analyzed by blot hybridization using a nick-translated probe of M13 sequences. Lanes A and B, DNA from uninduced cells. DNA from cells induced for 45 hr (lanes C and D) or 90 hr (lanes E and F) is also shown. The 1.6-kb and 0.8-kb fragments indicated at left are the expected digestion products of M13 DNA.

rearrangement that might have affected gene transcription. Restriction analysis of both isolated clones and pools of methylated globin gene-containing cells showed that they all contain 5-10 intact copies (Fig. 1). This transfection experiment was repeated numerous times using several different MEL cell lines, and in over 20 individual clones and pools, we never observed any induction of the methylated f3-globin gene. Since DNA methylation has such a profound effect on globin-gene expression, we attempted to pinpoint the site of action of this modification. This was accomplished by synthesizing partially methylated hybrid (3-globin gene con-

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structs using M13 templates and specific restriction fragments to direct the placement of methyl moieties to specific gene domains. Molecules methylated only in the 5' upstream region, for example, were synthesized with m5dCTP using the 2.3-kb Pst I/Nco I restriction fragment as primer (see Fig. 2). Complementary molecules, in which the 3' end was exclusively methylated, were obtained by using the 2.1-kb Nco I/Pst I fragment as primer on an M13 construct containing the f-globin gene in the opposite orientation. These partially methylated hybrid constructs and nonmethylated control constructs were inserted separately into mouse L cells by DNA-mediated gene transfer, and pools of >100 colonies per experiment were grown and analyzed for globingene expression by S1 nuclease digestion. As shown in Fig. 2, methylation of either the 5' or 3' regions of the gene were sufficient to inhibit p-globin expression by >20-fold. This inhibition could only be due to the effect of regional methylation and not to differences in copy number or gene arrangement, since Southern blots indicated that all cell populations contained an average of about one intact copy of the globin gene per cell. The results suggest that globin gene expression is indeed subject to control by DNA methylation and that the sequences responsive to this modification are distributed 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 globin constructs containing methylatable inserts. To this end, a 50-bp alternating (dG-dC)25 oligomer containing BamHI linkers was cloned into the plasmid p.3MH20 (Fig. 3), a mousehuman 8-globin chimera, at two locations in the transcribed region of the gene: the Bgl II site (+ 27) in the first mouse exon and the BamHI site (+ 500) in the second human exon. These constructs were methylated in vitro at the 50 Hha I sites of the CpG tract using Hha I methylase. Since the entire globin domain contains no intrinsic Hha I sites, methyl moieties were thus limited to the regions of the alternating CpG tracts. The methylated, the nonmethylated, and the original pf3MH20 constructs were introduced separately into tk- L cells by DNA-mediated gene transfer, and pools of colonies were grown to mass culture. We assayed globin RNA levels by using S1 nuclease analysis with a 3' specific probe. As shown in Fig. 3, there was no significant difference in the level of transcription between methylated, nonmethylated, and control constructs (see legend to Fig. 3). To verify that RNA elongation was not affected by methylation of the 50-bp CpG tracts, we compared the relative amounts of transcripts synthesized from upstream 5' sequences to the mRNA levels transcribed from sequences downstream of the tracts by using dot blot analysis with two specific probes. As shown in Fig. 3, the same relative levels of transcription occur both upstream and downstream of the 50-bp CpG tracts. Therefore, it appears that nonspecific methylated residues in the 3' region per se, as well as alternating

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FIG. 2. Analysis of L cells transfected with partially methylated hybrid globin genes. (a) Methylated and partially methylated hybrid M13 molecules were synthesized in the presence of trace amounts of radioactive nucleotides. The resulting double-stranded M13 molecules were purified, subjected to restriction enzyme analysis and gel electrophoresis, and visualized by autoradiography. The doublestrand DNA shown in lane 1 was made using the 2.1-kb Pst I/Nco I flanking primer. By using m'dCTP as substrate, the entire molecule, except for the primer, should be methylated. To test whether the primer indeed remained unmodified, and thus unlabeled, the M13 duplex was digested with Bgl II/Nco I/EcoRI (lane 2). Restricted duplex DNA made with the 15-mer universal M13 primer is shown in lane 3. Note that the 1.6-kb Bgl II/Nco I fragment (indicated by arrow), which is totally contained within the primer, is poorly labeled relative to the fully methylated molecule, indicating that the synthesis leakage into the primer was minimal. Hybrid constructs in which the 3' end was kept unmethylated were made using M13-containing globin in the opposite orientation with the 2.3-kb Nco I/Pst I primer and was tested by the same assay (data not shown). A A HindIII digest is shown in lane 4. (b) RNA (30 ,g) from cells transfected with unmethylated globin DNA (lane 1), 5' unmethylated DNA (lane 2), or 3' unmethylated DNA (lane 3) was analyzed by S1 nuclease analysis. The relatively low level of RNA observed in the unmethylated control (lane 1) is due to the very low p8-globin gene copy number in all transfectants produced in this experiment. Cell pools from each transfection contained an average of one copy per cell. (c) Map of genomic 4.4-kb Pst I fragment containing the human f-globin gene.

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FIG. 3. Expression of poly(dG-dC)-containing globin constructs in L cells. (Upper Left) S1 nuclease analysis of RNA (30 ,ug) from L cells 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 of pBR322 is shown in lane 8. (Upper Right) The same RNAs (6 Ag) were assayed on dot blots by using either a 5' specific probe (probe B) or a 3' specific probe (probe A). The control slot contains untransfected L-cell RNA. The ratio of hybridization to the 5' probe to hybridization to the 3' probe was the same for all samples. (Lower) The map shows the globin-containing region of pJBMH20 indicating the relevant restriction enzyme sites. The 5' HindIII/BamHI portion of the construct is from mouse /3-globin, whereas the 3' BamHI/Pst I portion contains human B-globin sequences. In p3Z1, a 50-bp poly(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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FIG. 4. Methylation analysis of transfected L-cell DNA. DNA (20 jg) from L cells transfected with various poly(dG-dC)-containing globin constructs was digested with Pst I/HindIII with or without Hha I and was subjected to gel electrophoresis and blot hybridization by using a 3' specific human 3-globin probe (probe A in Fig. 3). Each construct was analyzed in pairs in which the first sample (left) was digested 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 I site within the HindIII/Pst I fragment. The Southern blot digestion patterns of the original plasmid constructs pfZ1 (lane 1), pBZ2 (lane 2), and p,8MH20 (lane 3) are also shown. In this particular blot, digestion of lane 6 with H/ha I was only 90%o complete, but other blots indicated that this DNA is completely unmethylated in this region. Sizes (in kb) are indicated at left.

purine/pyrimidine tracts, affect neither RNA polymerase propagation nor initiation of transcription in the P-globin gene. The methylation state of the (dG-dC)25 tracts in vivo was determined by Nha I digestion and Southern blot analysis (Fig. 4) of transfected cells. The tract in the + 27 construct was found to be completely methylated, whereas the tract in the + 500 construct underwent =40% demethylation. Kinetic studies with Hha I show that this is due to the loss of one methyl group per 50 Hha I sites on 40o of the molecules, rather than a total demethylation of all 50 Hha I sites (data not shown). Since CpG tracts of this nature are known to undergo conversion to Z-DNA in supercoiled plasmids in vitro, we investigated the topological state of the poly(dG-dC)-containing globin DNA in vitro and in vivo. S1 nuclease hypersensitive sites were identified in both constructs at the apparent B-DNA-Z-DNA junctions (17) in supercoiled plasmids, as expected, but no S1 nuclease hypersensitive sites were found in either the methylated or nonmethylated constructs in nuclei from transfected cells

(data not shown).

DISCUSSION These experiments demonstrate that DNA methylation can inhibit 8-globin gene expression in mouse fibroblast and erythroleukemia cells. Despite the relatively low number of CpG residues in the gene domain (there are 15 CpG residues scattered through the gene domain from -1500 to + 1950), total methylation of these sites was sufficient to inhibit transcription by a factor of -20. This repression must involve regulatory sequences both in the 5' flanking region and the sequences covering the gene body and 3' flanking region, since localized methylations of either of these regions have similar effects. This disperse inhibition is characteristic of other genes that have been investigated, such as herpes thymidine kinase (5), but it is not an exclusive effect, since some 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,

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activity was assayed using a chloramphenicol acetyltransferase-linked construct, and thus the effect of in vitro DNA methylation on the 3' sequences was never properly investigated. The strong effect of DNA methylation within the f-globin 5' flanking sequences is rather surprising, since this region contains very few CpG residues and none within the first 120 bp upstream of the start of transcription. By using deletion and site-specific mutation analysis on (3-globin genes from several mammalian sources, it has been shown that only 90 nucleotides in the 5' region are necessary and sufficient for full transcriptional activity (7). It should be pointed out that this type of "reverse genetics" highlights the regions that are required for the positive regulation of the gene but does not relate to sequence domains that may have a negative regulatory role. Our studies show that methylation of sequences beyond the promoter region can inhibit gene expression and suggest the presence of a distal 5' negative regulatory element. A similar situation exists for the human y-globin gene, where there appears to be multiple methyl-sensitive sites both in the promoter region and in further upstream sequences (19). Mutation analysis in the /-globin structural gene and 3' flanking sequences does not show any specific sequence necessary for basal gene transcription, but these regions may indeed contain potential inhibitory elements that are responsive to DNA modification. Sequences in the 3' region are also involved in the induction response seen in erythroleukemia cells (12, 20, 21). Furthermore, DNA undermethylation at at least one site in the 3' flanking region is strongly correlated with gene expression in vivo (10, 22). Mouse erythroleukemia represent cells arrested in a late stage of erythroid development. Upon treatment with various chemical agents, they can be induced to produce the a- and f-globin chains and other genes characteristic of transcriptionally active erythroid precursors (23). Both a- and f-globin genes are DNase I sensitive and are undermethylated at a few specific sites, even in the erythroleukemia cell before induction (24). Since globin from other human and mouse tissues and sperm DNA are fully methylated at all assayable sites (11), the evolution of the erythroleukemia cell must involve specific demethylation events in consort with changes in gene conformation. Our results show that although an undermethylated exogenous f-globin gene can be induced in these cells, DNA modification at every CpG residue totally prevents this transcriptional activation. This suggests that DNA methylation at sites within the gene domain inhibits some aspect of the induction or transcription process and that these methyl 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 of several muscle-specific genes, including a-actin. In this case, methylated actin genes, which are inhibited in fibroblasts, are fully active in induced myoblasts following their introduction into these cells by DNA-mediated gene transfer. This activation is accompanied by a highly specific demethylation of the exogenous gene, which closely mimics the demethylation that occurs to the endogenous gene during muscle development (15). Interestingly, this terminal myoblast differentiation is accompanied by a marked change in the DNase I sensitivity of several muscle-specific genes (26), in contrast to the induction of MEL. Erythroleukemia cells represent a late stage in terminal differentiation, which may have lost the ability to "turn on" inactive globin genes and are thus unable to cope with a methylated gene. In this regard, it is surprising that an inactive, presumably methylated, /3globin gene in mouse fibroblasts could be efficiently activated following fusion with induced erythroleukemia cells (27). It is difficult to compare these two experimental approaches, however, since in the latter case the degree of DNA methylation of the

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globin gene has not been ascertained and the activation may also be of a transient nature. Despite the striking effect of DNA methylation at specific CpG residues in the entire globin gene domain, the insertion of a fully methylated 50-bp repeat of CpG had no effect on the activity of the gene. This suggests that the effect of DNA methylation on the natural CpG residues within the gene sequence cannot be mimicked by the addition of irrelevant methylated moieties. This is consistent with the idea that transcriptional inhibition is mediated through the interaction of proteins with various cis-acting, well-defined, negative regulatory elements that are influenced by DNA methylation. DNA methylation can alter protein binding to specific sites (28) and can modify the usual chromosomal conformation associated with the active domains (6). Since this latter effect may be limited to the sequences in the immediate vicinity of the methyl moieties, it is doubtful that methylation of the artificial CpG tracts within the globin gene would influence the protein conformation in the regulatory regions and thereby affect transcription. By carefully analyzing the relative transcriptional efficiency of 5' and 3' sequences, it was shown that fully methylated CpG tracts within either the first or second exon have no effect on the rate of transcriptional elongation. One problem in the interpretation of these results is that shorter transcripts might not be properly processed or could be degraded. If this were the case, however, we would detect a lower level of steady-state RNA produced from methylated constructs. Our results strongly suggest, therefore, that the mere presence of a large tract of methyl moieties is not sufficient to hinder the progress of RNA polymerase. When present in supercoiled plasmids, CpG tracts can easily flip into a Z-DNA conformation, and this altered secondary structure represents a strong barrier for the elongation of Escherichia coli RNA polymerase in vitro (29). The CpG-containing supercoiled plasmids used in our studies also contained a potential Z-DNA segment as shown by S1 nuclease digestion, yet these CpG tracts had no effect on either transcriptional initiation or elongation in vivo. Even though DNA methylation has a marked stimulatory effect on the ability of CpG tracts to convert to the Z form (30), this modification had no detectable influence on the transcriptional process. All of these results strongly suggest, but by no means prove, that neither the unmethylated nor the fully methylated CpG alternating sequence exists in the Z conformation in vivo. Attempts to directly detect such an altered conformation by S1 nuclease digestion of transfected cell nuclei confirmed the absence of Z-DNA. Thus, despite the existence of Z-DNA in vitro, there is, as yet, no compelling biophysical or biological evidence that this structure exists in vivo even under the most favorable conditions. We thank R. Flavell, F. Grosveld, M. Busslinger, T. Maniatis, and A. Rich for kindly supplying the vectors used in this study. We are

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