Proc. NatL Acad. Sci. USA Vol. 79, pp. 6299-6303, October 1982 Cell Biology

Expression of transferred kinase genes is controlled by methylation' (gene transfer/DNA methylation/5-azacytidine/DNA rearrangement) BARBARA CHRISTY AND GEORGE SCANGOS* Department of Biology, The Johns Hopkins University, Charles and 34th Streets, Baltimore, Maryland 21218 Communicated by John W. Littlefield, July 19, 1982

ABSTRACT Plasmid pTKx-l, containing the herpes simplex activation ofendogenous viral genomes, and Compere and.Pal- virus gene for thymidine kdnase (TK.) inserted into the BamHI site miter (9) demonstrated that growth ofcultured cells in the pres- of plasmid pBR322, was introduced into Ltk- cells by calcium ence of5-azaCyd resulted in hypomethylation and activation of phosphate precipitation in the absence of carrier DNA. Line 101 the metallothionein-I gene. These studies suggest a causative is a TKV derivative ofLtk- that contains multiple copies ofpTKx-I role for methylation in the reduction of gene expression, but in a multimeric structure. A derivative of 101 that retained but no they could not conclusively demonstrate that reexpression of longer expressed the herpes simplex TK genes (termed lOlBUl) the genes resulted directly from demethylation of gene se- and derivatives ofline LOlBUl that reexpressed the genes (termed quences induced by 5-azaCyd. lOiHI, IOIHC, and IOIHG) were selected. The TK genes in We have transferred the herpes simplex virus (HSV) gene lOBUI were hypermethylated relative to those in the TKV parent and derivatives. Growth of lOlBUl in the presence of the meth- for (TK) into TK-deficient mouse fibroblast ylation inhibitor 5-azacytidine resulted in an average 13-fold in- Ltk- cells and have studied the modulation of expression of crease in the number of TKV reexpressors. DNA from IOlBUI TK in one cell line, termed 101. Derivatives of 101 were se- was inactive in secondary gene transfer, whereas DNA from 101 lected that retained but did not express the TK genes; subse- and from TKV reexpressors was active. These data support a caus- quently, derivatives ofthose were selected in which the genes ative relationship between DNA methylation and decreased gene were reexpressed. The powerful selections for and against the expression. All TKV reexpressors examined had DNA rearrange- expression of the TK gene, the use of the cloned gene as a nu- ments involving TK DNA. cleic acid probe, and the ability to use cellular DNA in second- ary gene transfer experiments allowed precise correlations be- The presence of methylated bases in the DNA ofboth prokar- tween gene expression and the biochemical state of the TK yotes and eukaryotes has been known for some time (1). In DNA. The experiments to be presented here demonstrate that mammalian DNA, the only detectable methylated base is 5- the extent ofmethylation ofthe TK DNA is inversely correlated methylcytosine (m5C) (2). From 2% to 7% ofcytosine residues with gene expression, and that growth of TK-deficient deriv- are found as 5-methylcytosine, and greater than 90% of those atives of 101 in the presence of5-azaCyd resulted in a 6- to 23- occur in the dinucleotide sequence CpG (3). The distribution fold increase in the number ofTKV derivatives. Furthermore, of 5-methylcytosine in the DNA is nonrandom, and the pat- we showthat DNA isolated from 101 and from reexpressingTK+ terns of methylation appear to be species specific and tissue derivatives was active in secondary gene transfer experiments, specific (1, 4). The pattern of methylation is passed on from a whereas DNA isolated from TK-deficient derivatives of 101, in cell to its daughters through the action of one or more "main- which the TK genes were heavily methylated, was inactive. tenance methylase" , which act on hemimethylated These data demonstrate that the activation/inactivation of the DNA shortly after replication (5). TK genes is effected directly through a change in the extent of Although the precise enzymatic mechanisms of DNA meth- DNA methylation of the TK gene. Finally, we show that DNA ylation are only beginning to be elucidated, a strong inverse rearrangements involving the TK genes were seen in each of correlation has been established between the amount of DNA three TKV reexpressors, raising the possibility that gene rear- methylation and the level ofgene expression. Hypomethylation rangement might be a mechanism of demethylation. ofseveral genes, including globin (6), ovalbumin (7), ribosomal DNA (8), and metallothionein (9), has been found in tissues in MATERIALS AND METHODS which the genes are expressed, whereas hypermethylation of Cell Culture. Cells were maintained in monolayer culture the same genes or their surrounding DNA has been found in at 37°C under 5% C02/95% air in Dulbecco's modified Eagle's tissues in which they are not expressed. Several investigators medium (GIBCO) supplemented with 5% fetal bovine serum have found that endogenous, unexpressed viral genomes often (GIBCO). TKV cells were maintained in Dulbecco's modified are heavily methylated but that expressed viral DNA is un- Eagle's medium supplemented with , aminop- methylated (10-14). In the case of adenovirus, portions of the terin, and thymidine (HAT medium) (16). DNA-mediated gene viral DNA can be methylated while other regions remain un- transfer was performed as described (17). methylated, and the level ofmethylation is inversely correlated 5-azaCyd Treatments. lOlBUl (TK-) cells were plated at with the level of expression ofparticular viral genes (15). 0.5_1 x 106 cells per 100-mm dish in nonselective medium; 24 Groudine et al. (12) demonstrated that inhibition of DNA hr after plating, the medium was replaced with fresh medium methylation by growth ofcells in the presence of 5-azacytidine containing 0, 2, 5, or 10 tzM 5-azaCyd (Sigma). Cells were (5-azaCyd) resulted in the hypomethylation and transcriptional Abbreviations: TK, thymidine kinase; HSV, herpes simplex virus; 5- The publication costs ofthis article were defrayed in part by page charge azaCyd, 5-azacytidine; HAT, hypoxanthine//thymidine; payment. This article must therefore be hereby marked "advertise- kb, -kilobase(s). ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 6299 Downloaded by guest on September 25, 2021 6300 Cell Biology: Christy and Scangos Proc. Natl. Acad. Sci. USA 79 (1982) treated with 5-azaCyd for 3-5 days, rinsed with phosphate-buff- ported by BamHI digestion, which generated predominant ered saline, and refed with selective HAT medium. The me- bands of 4.3 and 3.5 kb, which correspond to the pBR322 and dium was changed every 2-3 days, and colonies were counted TK moieties of the plasmid. The presence of additional bands after 14-21 days.in selective medium. The cell number at the indicates that deleted or rearranged copies (or both) ofpTKx-1 start of selection in HAT medium was determined in a hemocy- also were present. tometer. TK-Deficient Derivatives Retained but Did Not Express Biochemical Procedures. Plasmid DNA was isolated as de- the Viral TK Genes. When plated in medium containing 5-bro- scribed by Guerry et al. (18). High molecular weight cellular modeoxyuridine (BrdUrd) to select against expression of TK, DNA was isolated as described by Wigler et al. (19). Levels of derivatives of line 101 were isolated at a frequency of 10-6 TK activity were determined by the method of Mc- (not shown). One such derivative, 101BUL, was analyzed in Bride et al. (20). Restriction endonuclease digestions were per- detail. Line 101BUl had no detectable TK enzymatic activity formed as recommended by the suppliers (Bethesda Research (not shown) but retained the viral TK genes. Digestion of the Laboratories or New England BioLabs) with 10- to 30-fold ex- of 101 and 101BUl with Xba I, HindIII, BamHI (Fig. cess of enzyme. Hpa II digests were performed with a 30- to 1), and Pvu II (not shown) failed to detect any difference in the 50-fold excess of enzyme on independently isolated samples of pattern of TK-specific bands between the two lines. 101BU1 DNA to ensure complete digestion. Samples of DNA 101BUl cells were replated in HAT medium to select deriv- (10 kug) were electrophoresed in 0.7%, 1%, or 1.2% agarose atives that reexpressed the TK genes, and HATr derivatives (Bethesda Research Laboratories) containing 40mM Tris, 1 mM were isolated at a frequency of 5 X 10-7. Two independent re- EDTA, and 60 mM NaOAc (pH 7.9). DNA blotting and filter expressing cell lines, termed 101HI and 101HG, were charac- hybridization were performed by the method of Wahl et al. (21) terized further. A third TK' derivative, 101HC, was isolated as modified by Huttner et aL (22). pTKx-1 DNA or the 3.5-kil- after 5-azaCyd treatment of 101BUl cells. obase (kb) BamHI fragment ofpTKx-1 DNA was nick translated Methylation of TK DNA Was Inversely Correlated with to a specific activity of 0.7-3 x 108 dpm/,ug by a modification Expression of the TK Gene. DNAs isolated from lines 101, of the method of Rigby et al. (23) as described by Maniatis et lOlBUl, 101H1, 101HC, and 101HG were each digested with al. (24). Msp I and Hpa II, isoschizomers that recognize and cleave the same nucleotide sequence-C-C-G-G (26). Because Hpa II fails RESULTS to cleave the DNA when the internal C is methylated whereas Cell line 101 arose after treatment of Ltk- cells with the circu- Msp I is insensitive to methylation at that site, a comparison of lar plasmid pTKx-1 (25) in the absence of-carrier DNA. Prelim- the pattern of bands visualized on Southern blots after diges- inary characterization of several cell lines generated in this way tion with the two enzymes provides an indication of the extent demonstrated that most contained multiple copies of plasmid of DNA methylation. Plasmid pTKx-1 contains more than 40 pTKx-1 in a multimeric structure at one chromosomal locus (17). recognition sites for Msp I, generating fragments ranging in size Southern blot analysis of 101 DNA showed the appearance of from 9 to 600 base pairs. Digestion of 101 DNA with Msp I gen- one band homologous to the TK probe after Xba I digestion, erated a number ofpoorly resolved low molecular weight bands even when the DNA was electrophoresed through0.7% agarose (Fig. 2). Hpa II digestion yielded the same pattern of low mo- gels for 18 cm (Fig. 1). Xba I does not cleave plasmid pTKx-1 lecular weight bands and a number of high molecular weight (25), so that the sites that generated the single band must have bands, indicating that the TK genes in line 101 were differen- been in chromosomal DNA flanking the plasmid DNA. Mul- tially methylated. Some gene copies contained little or no meth- tiple bands were visualized after HindIII or BamHI digestion, ylation, whereas others were more heavily methylated. which cleaved pTKx-1 once or twice, respectively (Fig. 1). A Digestion of lOlBUl DNA with Msp I yielded the same pat- predominant band of 7.9 kb (the size of pTKxl) was seen after tern of low molecular weight bands as seen in 101 DNA, HindIII digestion, indicating that the TK sequences were or- whereas Hpa II digestion generated only high molecular weight ganized in a multimeric structure consisting predominantly of bands. The low molecular weight bands indicative ofunmethyl- full-length plasmid concatamers. This interpretation was sup- ated TK genes were not visualized. These data indicate that sites within the plasmid sequences that were not methylated in 101 had become methylated in the DNA of the line A B C D E A B C D E F A B C D E TK-deficient X!: e 101BUL. The low molecular weight bands reappeared in Hpa -I V *d_ eY. II digests of DNA isolated from the reexpressing cell lines 101H1, 101HC, and 101HG, indicating that at least one copy ofthe TK gene in the DNA ofthese lines had been demethylated. Sma I, another methylation-sensitive enzyme, recognized five sites within plasmid pTKx-1 to generate bands of 0.2, 0.25, 0.8, 1.6, and 5.0 kb (Fig. 3) (M. Wagner and W. Summers, Hind III personal communication). The 0.8- and 1.6-kb bands are de- XbaI BamHi rived entirely of TK DNA, and the sites that generate them lie within and on both sides ofthe structural gene. The 5.0-kb band FIG. 1. Xba I, HinduII, and BamHI digests of line 101 and deriv- consists of pBR322 and a small amount of TK DNA. The 0.8-, atives. DNA of lines 101 (lanes A), lOlBUl(lanes B), lOiH1 (lanesC), 1.6-, and 5.0-kb bands can be 101 101HC (lanes D), 1O1HG (lanes E), and LTK- (lane F) were digested seen clearly in DNA of line withXbaI,HindIII, orBamHI. The DNAs were separated in 0.7% agar- and the reexpressors 101H1, 101HC, and 101HG after Sma I ose gels, transferred tonitrocellulose, and hybridizedwith 32P-labeled digestion (Fig. 3) but are not detectable in 101BUl DNA. pTKx-1 as a probe. The band patterns of 101, lOlBUl and the reex- Digestion with Ava I, another methylation-sensitive enzyme pressing cell line lOHC (lanes A, B,-and D, respectively) are indistin- that recognizes eight sites in pTKx-1 (M. Wagner and W. Sum- ,guishable after HindIH andBamHI digestion. lOH1 and 101HG reex- mers, personal communication), also demonstrated that the TK pressor lines (lanes C and E) are missing bands inboththeHindI and DNAof101BU1 is more heavily BamHI digests. The arrows point to bands of 17, 7.9, and 3.5 kb in the methylated than is the TK DNA HindIII digests and to bands of 4.3 and 3.5 kb in the BamHI digests. in 101 and in the TKV reexpressors. Two bands of 0.64 and 0.78 The XbaI bandis >23 kb. kb, derived from within the TK gene, were present in 101 and Downloaded by guest on September 25, 2021 Cell Biology: Christy and Scangos Proc. Natl. Acad. Sci. USA 79 (1982) 6301

101 BU HI HC HG L.. _ iiU a = - U 0 t 11vn h h rn m I I I - m m m x fln h rm r rn% rn. -

jw-

I-I u jp- & 3.7 i l -

..

E... .ib dW ob - 1.3

J .78 _ 0.8 .64 A-. 4"W :'.-_,-,1; Sma I .-V:--w- Ava

Avo 1 1.3 - 0.78±0.643 1 0.784 |

..~~~~.~~,_~ ~~~~~~

Smo I FIG. 3. Sma I and Ava I digests of line 101 and derivatives. DNA of lines 101, lOlBU1, lOiH1, 101HC, 101HG, and LTK- were digested with the enzymes Sma I or Ava I, both of which are sensitive to on 1.2% FIG. 2. Msp I and Hpa II digests of line 101 and derivatives. DNA methylation at the CpG sequence. The DNAs were separated 101H1, 101HC, and 101HG were digested with agarose gels, transferred to nitrocellulose, and hybridized with 32P-la- of lines 101, 101BU1, TK fragment as a probe. A partial restriction map I m) and Hpa II (lanes h). LTK- (lane L) DNA was digested beled 3.5-kb HSV Msp (lanes showing the Sina I and Ava I cleavage sites within pTKx-1 is shown with Msp I. DNA (10 Mg) from each cell line was electrophoresed on with at the bottom of the figure. The 3.5-kb HSV TK fragment cloned into a 1.2% agarose gel, transferred to nitrocellulose, and hybridized line. The location and as a probe. Some of the TK sequences in 101 are theBamHI site of pBR322 is depicted as a heavy 32P-labeled pTKx-1 direction of transcription of TK mRNA are represented by the arrow. methylated, as shown by the many high molecular weight bands in the from cleav- Hpa II digest. Low molecular weight bands similar to those seen in the The 5.0-, 1.6-, and 0.8-kb bands in the Sma I digests result presence of unmethyl- age of pTKx-1 within and immediately flanking the TK gene. The Msp I digest are also present, demonstrating the in DNA from one or more TK genes. The TK sequences of bands are absent from lOlBUl DNA but are present all ated Hpa II sites within that the sites that these 101BUl are heavily methylated, and no low molecular weight bands cells expressing TK, indicating generated bands must have been methylated in the DNA of lOlBUl but unmeth- appear in the Hpa II digest. The low molecular weight bands are pres- ylated in the DNA of 101 and the reexpressing lines. Similarly the ent in both Msp I and Hpa II digests of 101H1 and 101HG, indicating from one copy gene was demethylated. In 101HC, the 0.64-, 0.78-, and 1.3-kb bands in theAva Idigest, derived cleavage that at least of the TK in DNA but presence of high molecular weight bands in the Hpa II digest indicates within the HSV TK fragment, are absent lOlBUl present that some methylation is present, but the low molecular weight bands in DNA of all of the TK' lines. seen in the Msp I digest are also present, indicating that the TK DNA in 101HC is less methylated than that in 101BUL. Arrows indicate marker bands of 9.7, 4.3, and 0.5 kb. lOlBUl after 5-azaCyd treatment, were hypomethylated rel- ative to the TK genes in lOlBUl. DNA ofLine lOlBUl Was Unable To Transfer TK Activity in the reexpressors but absent from lOlBUl (Fig. 3). These data in Secondary Gene Transfer. IfDNA methylation directly pre- demonstrate that at least one copy of the TK gene within the vents expression of the TK genes in line lOlBUl, then DNA DNA ofeach ofthe four TK+ lines contained unmethylated Hpa isolated from lOlBUl should not be active in secondary gene II, Smn I, and Ava I sites and that those sites were methylated lOlBUl. in the DNA of Table 1. Increase in TK reexpression after treatment Frequency of Reexpression of TK in Line lOlBUl Was In- with 5-azaCyd creased by Growth in the Presence of 5-azaCyd. 5-azaCyd is Ratio Colonies per a cytidine analog that is incorporated into DNA and inhibits positive/ Treatment of 101 cells with 5-azaCyd decreased 5-azaCyd total plates 106 cells methylation. treatment No With No With Fold the extent of DNA methylation as judged by ethidium bromide staining of Msp I- and Hpa II-digested DNA after electropho- Dose Time drug* drug* drug* drug* increase resis through 1% agarose gels (data not shown). Growth of 5 uM. 3 day 3/5 8/10 0.59 6.4 10.88 lOlBUl cells in 2, 5, or 10 ,uM 5-azaCyd for 3-5 days prior to 5 ,M 3 day 4/10 10/10 0.27 2.7 10.0 selection in HAT medium resulted in a 6- to 23-fold increase 5 ;LM 4 day 3/5 9/10 0.27 6.33 23.35 14.2 in the number of TK' colonies relative to untreated controls 5 uM 5 day 9/10 10/10 0.40 5.68 0.27 3.3 12.2 is re- 5 ,uM 5 day 4/10 10/10 (Table 1), which suggests that DNA methylation causally 5.9 in these cells. Max- 2 aM 3 day 4/10 10/10 0.27 1.6 lated to the elimination of gene expression 10 ,uM 4 day 9/10 10/10 0.40 2.9 6.6 imal increase was seen after treatment with 5 ,M 5-azaCyd for 3-4 days. The TK genes in line 1O1HC, which was derived from * 5-azaCyd treatment. Downloaded by guest on September 25, 2021 6302 Cell Biology: Christy and Scangos Proc. Natl. Acad. Sci. USA 79 (1982) Table 2. Relative efficiencies of transfer of TK activity of DNA previous studies that demonstrated demethylation specifically isolated from line 101 and derivatives of promoter regions (27). Gene transfer experiments, Our data suggest that the hypermethylation seen in line no. of colonies/no; of Petri plates 101BUl was relatively specific and did not involve the entire TK gene region. This is demonstrated by the pattern of high DNA source 1 2 3 4 Total molecular weight bands seen in the Hpa II digests of 101 and 101 105/5 191/5 1/5 58/5 355/20 101BUl DNAs. The patterns of high molecular weight bands lOlBUl 0/5 0/5 0/10 0/5 0/25 seen in the two lines were similar; the only major difference was O1H1 - 12/5 72/5 14/5 98/15 the absence of low molecular weight bands in 101BUl DNA. 101110 - - 161/5 70/5 231/10 The high molecular weight bands must have arisen from cleav- 1O1HG - 17/5 9/5 26/10 age at unmethylated sites within andflanking the TK genes. The presence ofdiscrete high molecular weight bands indicated that at least some ofthe TK genes were somewhat methylated, even transfer experiments. DNA isolated from 101, lOlBUl, and the in line 101, and that unmethylated sites were present, even in threereexpressors was applied to.Ltk cells. DNA from 101 and the DNA of line 101BUL. That the pattern ofbands was similar from each ofthe reexpressors generated TK' colonies, whereas indicated that the hypermethylation in 10lBUl involved only each of three preparations of lOlBUl DNA was inactive (Table some ofthe TK genes. The two lines have been maintained sep- 2). lOLHL and LO1HG suffered deletions of TK sequences, arately in culture for over 9 months, indicating that the pattern whereas LOLHC had a gene amplification (see below), so that ofmethylation of the TK DNA is stable over time. the relative number of colonies generated by each of the TKV Two aspects ofthe present study support acausal relationship lines correlated with the relative number of gene copies as between methylation and elimination ofgene expression. First, judged from autoradiographs (Figs. 1-3). growth ofline 101BUl in the presence of5-azaCyd, which de- DNA Rearrangements Accompanied Demethylation and creased DNA methylation in these cells, resulted in an increase Gene Reexpression. Each of the TK' reexpressors had altera- in the frequency ofreexpression ofthe TK genes, indicating that tions in the pattern or intensity of TK-specific bands after diges- a decrease in the extent of DNA methylation led to an increase tion with Pvu II (not shown), Xba I, HindIII, and BamHI (Fig. in gene expression. As. in previous studies where azacytidine 1). DNA of line lOLH1 contained an additional band of about reactivation has been noted (9, 12, 28), these data do not un- 10 kb after Xba I digestion -and two additional bands after ambiguously determine whether demethylation ofthe gene and HindIII digestion. Additionally, bands at 17 and 5 kb were re- flanking sequences, demethylation ofother sequences, or even duced in intensity relative to other bands after HindIII diges- mutational events induced by 5-azaCyd are the important tion, and at least two high molecular weight bands were un- events in gene reexpression. For this reason, DNA isolated from detectable after BamHI digestion. DNA of line 1OHC had a 101, 101BUI, and from each ofthe three reexpressors was used pattern of bands indistinguishable from that of line 101 except in secondary gene transfer. TheTK genes in 101BU1 DNAwere that the intensity ofthe bands, indicative ofthe numberof gene inactive, whereas those in 101 DNA were active. Because gene copies, was increased. DNA from line LOLHG was missing all transfer separates the TK DNA from most ofthe donor genome but the 7.9-kb and 3.5 kb HindIII bands and had alterations in and from other cellular constituents (29), these data-suggest that the high molecular weight region after BamHI digestion. In the difference lies within or near the TK genes themselves. The order to determine ifthe alterations in gene structure were cor- only detectable difference between the TK genes in lines 101 related' with changes in gene expression, 12 subclones of and 101BUl is the extent of DNA methylation. lOlBUl were isolated randomly and without selection for TK Each of three TK+ derivatives of line 101BUl had alter- expression. DNA from nine ofthe subelones was indistinguish- able from lOlBUl DNA after HindIII digestion, whereas DNA ations in the structure ofthe TK genes. In two cases a deletion from three of the subclones had changes in the pattern of TK- was evident, whereas in a third case a gene amplification was bands. detected. DNA from 9 of12 subclones of 101BUI isolated with- specific out selection had band patterns indistinguishable from 101BUl after HindIII digestion, whereas the pattern in 3 subelones dif- DISCUSSION fered. The presence ofdetectable rearrangements in the DNA We have characterized a cell line, 101, which contains and ex- of3 of12 random subclones indicates that rearrangement in the presses the HSV TK gene. Selection of 101 cells against expres- region ofthe introduced TK genes is a common event. We have sion of TK resulted in the appearance of derivatives that re- not determined yet whether the gene rearrangements seen in tained but did not express the TK genes. Derivatives of these the reexpressors provide a means of demethylation or are sep- subsequently were selected that reexpressed the TK gene. arate events. It may be that some rearrangements bring the In line 101 and its derivatives, expression of the TK gene is methylated TK genes intoproximity with hypomethylated DNA regulated by methylation. The TK genes in 101 are partially and, therefore, that the pattern ofmethylation ofthe TK genes methylated, but as seen from the Hpa II, Sma I, and Ava I di- is disrupted. Alternatively, demethylation may occur without gests, at least one TK gene contains sites for these enzymes that gene rearrangement but is not in itself sufficient for gene are unmethylated. In line lOlBUl, which does not express the reexpression, so that subsequent rearrangements are necessary TK genes, some ofthe Hpa II, Sna I, and Ava I sites that were for expression. Finally, DNA rearrangement may occur ran- unmethylated in 101 have become methylated, and those same domly and without relation to gene reexpression. sites have become demethylated in derivatives of lOlBUl that Gene transfer has been used previously to study the rela- reexpress the genes, thus demonstrating an inverse correlation tionship between DNA methylation and gene expression between DNA methylation and expression ofthe TK genes. The (30-32). In one study in which the chicken TK gene was meth- Sma I and Ava I digestions demonstrated that sites on the 5' ylated in vitro and used in gene transfer experiments, the fre- and 3' ends of the TK gene, as well as within structural gene quency of transfer was 5-33% of that with the unmethylated sequences, were unmethylated in the TKV lines and methylated gene. The authors estimated that the pattern ofmethylation of in lOlBUl, indicating that the pattern ofmethylation through- transferred genes was lost at about 9% per generation (32). A out the gene was altered. These results are in contrast to some second publication indicated that there was an initial demeth- Downloaded by guest on September 25, 2021 Cell Biology: Christy and Scangos Proc. Natd Acad. Sci. USA 79 (1982) 6303 ylation oftransferred genes, but the pattern subsequently was 3. Grippo, P., lacarino, M., Parisi, E. & Scarano, E. (1970)J. Mol maintained faithfully (30). Our data indicate that the frequen- Biw 35, 195-208. cy of transfer of methylated genes was reduced to less than 4. Ehrlich, M. & Wang, R. Y.-H. (1981) Science 212, 1350-1357. 5. Gruenbaum, Y., Cedar, H. & Razin, A. (1982) Nature (London) 1/200th that ofthe hypomethylated genes, and further, that the 295, 620-622. pattern ofmethylation ofthe transferred genes was altered suf- 6. van der Ploeg, L. H. T. & Flavell, R. A. (1980) Cell 18, 947-958. ficiently to alter gene expression only at a frequency of 5 x 7. Mandel, J. L. & Chambon, P. (1979) Nucleic Acids Res. 7, 10'. We feel that the differences between our results and the 2081-2103. previous studies stem from the fact that genes in the first studies 8. Bird, A., Taggert, M. & Macleod, D. (1981) Cell 26, 381-390. were methylated in vitro only at Hpa II sites, which represent, 9. Compere, S. J. & Palmiter, R. D. (1981) Cell 25, 233-240. 10. Desrosiers, R. C., Mulder, C. & Fleckenstein, B. (1979) Proc. on the average, only 6% of all potential methylation sites and Natl. Acad. Sci. USA 76, 3839-3843. may or may not be sites that are important for gene control. In 11. Vardimon, L., Neuman, R., Kuhlmann, I., Sutter, D. & Doer- the experiments reported here, the methylation was carried out fler, W. (1980) Nucleic Acids Res. 8, 2461-2473. by the normal cellular machinery in vivo so that the extent of 12. Groudine, M., Eisenman, R. & Weintraub, H. (1981) Nature methylation was likely to be greater, and the pattern of meth- (London) 292, 311-317. was likely to be that which abolished gene expression. 13. Stuhlmann, H., Jahner, D. & Jaenisch, R. (1981) Cell 26, ylation 221-232. Although the DNA of line 101BUl was unable to generate 14. Harbers, K., Schnieke, A., Stuhlmann, H., Jahner, D. & Jaen- TKV colonies in a number ofindependent gene transfer exper- isch, R. (1981) Proc. Nat. Acad. Sci. USA 78, 7609-7613. iments, DNA from line 101 and from each of the three reex- 15. Sutter, D. & Doerfler, W. (1980) Proc. Natl Acad. Sci. USA 77, pressors could generate TKV colonies. The frequency of colo- 253-256. nies generated by DNA oflines 101HI and 101HG was reduced 16. Littlefield, J. W. (1964) Science 145, 709-710. relative to that obtained with 101 and 101HC, suggesting that 17. Huttner, K. M., Barbosa, J. A., Scangos, G. A., Pratcheva, D. D. & Ruddle, F. H. (1981)J. CelL Biol 91, 153-156. they had fewer active gene copies. This interpretation is con- 18. Guerry, P., Leblanc, D. J. & Falkow, S. (1973)J. Bacteriot 116, sistent with the intensity and diversity of bands visualized on 1064-1066. Southern blots. 19. Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacy, Thus, our data demonstrate that changes in the pattern of E., Maniatis, T., Silverstein, S. & Axel, R. (1979) Cell 16, methylation that effected changes in gene expression occurred 777-785. throughout the TK gene. The secondary gene transfer experi- 20. McBride, D. W., Burch, J. W. & Ruddle, F. H. (1978) Proc. NatL Acad. Sci. USA 75, 914-918. ments demonstrated that methylation ofthe structural gene and 21. Wahl, G. M., Stem, M. & Stark, G. R. (1979) Proc. Natl Acad. offlanking DNAwas sufficient to abolish expression. However, Sci. USA 76, 5820-5824. our data do not permit a determination of whether there are 22. Huttner, K. M., Scangos, G. A. & Ruddle, F. H. (1979) Proc. particular sites ofmethylation within a gene that are important NatL Acad. Sci. USA 76, 5820-5824. for controlling gene expression or whether methylation of the 23. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) entire gene, perhaps altering the conformation of the DNA, is J. Mol, Biol 113, 237-251. 24. Maniatis, T., Jeffrey, A. & Kleid, D. G. (1975) Proc. Natl Acad. the important factor. Sci. USA 72, 1184-1188. 25. Enquist, L. W., VandeWoude, G. F., Wagner, M., Smiley, J. R. We thank K. M. Huttner for communication ofunpublished results; & Summers, W. C. (1979) Gene 7, 335-342. Dr. E. Moudrianakis, Dr. R. C. Huang, and G. Ketner for helpful dis- 26. Roberts, R. (1980) Gene 8, 329. cussions; and Diane Warr for typing ofthe manuscript. This work was 27. Breznik, T. & Cohen, J. C. (1982) Nature (London) 295, 255-257. supported by a biomedical research grant from the National Institutes 28. Taylor, S. M. & Jones, P. A. (1980) Cell 20, 85-93. 1173 the of 29. Scangos, G. & Ruddle, F. H. (1981) Gene 14, 1-10. of Health. This is contribution no. from Department 30. Stein, R., Greenbaum, Y., Pollack, Y., Razin, A. & Cedar, H. Biology. (1982) Proc. NatL Acad. Sci. USA 79, 61-65. 31. Pollack, Y., Stein, R., Razin, A. & Cedar, H. (1980) Proc. NatL 1. Razin, A. & Riggs, A. D. (1980) Science 210, 605-610. Acad. Sci. USA 77, 6463-6467. 2. Wyatt, G. R. (1951) Biochem. J. 48, 584-590. 32. Wigler, M., Levy, D. & Perucho, M. (1980) Cell 24, 33-40. Downloaded by guest on September 25, 2021