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Mouse mammary tumor virus dna methylation: Tissue-specific variation
VIROLOGY
136. 69-7’7 (1964)
Mouse Mammary Tumor Virus DNA Methylation:
Tissue-Specific
Variation
TRUDY BREZNIK,* VICKI TRAINA-DORGE,t MIGUEL GAMA-SOSA,# CHARLES W. GEHRKE,§ MELANIE EHRLICH,* DAN MEDINA,** JANET S. BUTEL,tt AND J. CRAIG COHEN**’ Departments of +Micmbiology, #Medicine, and #Biochemistry, Lxnbsiana State University Medical Center, New Orleans, Lvukinna 70112,Departments of fMimobiology and $Bimhemistry, Tulune University Medical Center, New Orhns, Louisiana 7011.2,%Dq.nxtment CLfBiochemists, Experhnmti Station Chemical Labomhh Univertit~ of Missouri, Columbia, Missouri 65201,and Departments of **Cell Biology and “;t Virologll and Epidemidogy, Baylor College of Medicine, Houston, Team 77030
Received January 3, 1984 accepted March
16,
1984
Mouse mammary tumor virus-specific DNA sequences endogenous to the BALB/c mouse are shown to exhibit variable levels of methylation in a tissue-specific manner. In DNA from both lactating mammary gland and spleen, MMTV-specific sequences were hypomethylated at specific HpuII and HhaI sites. These variably methylated sites were found in the terminal repetitive sequences of the endogenous viral genomes. The specific hypomethylation of a HpoII site in Mb-9 is associated with expression of a 1.6 kb transcript in the lactating mammary gland.
aL, 1982; Clough et a.& 1982; Creusot et &, 1982; Conklin et a& 1982; Fradin et cd, 5-Methylcytosine (5mC) is the predom- 1982; Simon et al, 1983; Busslinger et cd, inant modified base found in the DNA of 1983; Wigler et cd, 1981). higher eukaryotes (Ehrlich and Wang, Mouse mammary tumor virus (MMTV) 1981; Razin and Riggs, 1980). Methylation is one of the eukaryotic viruses for which occurs after DNA replication by sequence- there is evidence that site-specific DNA specific transfer of the methyl group from methylation is linked to transcriptional S-adenosylmethionine to cytosine. There is inactivity (Cohen, 1980). MMTV is a retmuch recent evidence that one of the func- rovirus associated with mammary adenotions of DNA methylation in eukaryotes carcinomas in mice. This virus may be is to participate in the control of gene reg- transmitted either endogenously through ulation and cell differentiation. Studies the mouse genome where it exists as a stasupporting this hypothesis have shown an ble genetic element (Cohen and Varmus, inverse correlation between methylation of 1979; Traina-Dorge and Cohen, 1983) or a number of eukaryotic or eukaryotic viral exogenously through mother’s milk (Cohen genes and their transcriptional activity et d, 1979b). The BALB/c inbred mouse (Guntaka et d, 1980;Weintraub et &, 1981, strain lacks milk-borne MMTV and has a Compere and Palmiter, 1981; Jones et al, tumor incidence of ~1% at 18 months of 1981; Stuhlman et aL, 1981; Youssuofian et age. This strain can be foster-nursed on al, 1982). Also, various procedures leading females from the C3H strain which conto decreases in methylation of certain host tains the milk-borne virus; the resulting or viral genes in vertebrate cells have led substrain, BALB/cfC3H, has a tumor into the increased expression of these genes cidence approaching 100% (Andervont and, correspondingly, increased methyl1941,1945). Genetically transmitted, virusation to decreased expression (Groudine et specific sequences in tumors from these al, 1981; Sager and Kovac, 1981; Stewart mice are highly methylated, while viral sequences acquired by milk-borne infection 1 To whom reprint requests should be addressed. are hypomethylated. The former viral INTRODUCTION
et
69
0042-6822184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reprcduetion in any form reserved.
BREZNIK
70
DNA sequences appear to be inactive in transcription whereas the latter are transcriptionally active (Varmus et &, 1973). In this report we examine the location of hypomethylated sites in the endogenous MMTV sequences of different tissues of the BALB/c mouse strain. We also compare this tissue-specific hypomethylation of proviral sequences with their transcription and with the overall levels of 5mC in host DNA from different tissues. METHODS
Tissue.s. All tissues were taken from 4to 5-month-old virgin or primiparous BALB/c 4- to 5-month-old virgin or primiparous BALB/cMed female mice. The mice were raised in a closed conventional mouse colony in the Department of Cell Biology, Baylor College of Medicine. The mammary tumor incidence in breeding BALB/cMed female mice is approximately 1% at 18 months (Pauley et d, 1979). Restriction enzyme digestion DNA samples (10 pg) isolated from BALB/c mouse tissues as previously described (Cohen et al, 1979b) were digested with either EcoRI, MspI, HpaII, or HhaI (New England Biolabs). Specific restriction enzyme buffers used were as recommended by the manufacturer. For double digestions DNA (20 c(g) was cleaved with a fourfold excess of EcoRI for 4 hr and then extracted with chloroform-isoamyl alcohol (24:1, v/v) and precipitated with ethanol. DNA was resuspended in the appropriate buffer and 10 pg digested for 4 hr with a threefold excess of either M.1, &&I, or HhaI using the recommended conditions. All restriction enzyme digests were monitored for completeness prior to electrophoresis using pBR313 or X bacteriophage DNA as an internal control in a duplicate sample digest. Gel ekctrop~esis and blot h&ridizatim Electrophoresis through either 0.8 or 1.0% agarose gels containing 0.5 X TEA (TEA = 0.04 M Tris-acetate, pH 8.1, 0.02 M sodium acetate, 18 mM NaCl, and 2 mM EDTA) was used to fractionate restriction endonuclease-digested DNA. X bacteriophage q-labeled DNA digested with Hind111 was electrophoresed in a parallel lane and used as a molecular weight stan-
ET AL.
dard. DNAs were transferred using the method of Southern (1975), modified as previously described by Cohen et aL (1979b). A complementary q-labeled DNA representative of the entire MMTV genome (cDNArep) was synthesized by nick-translation of a pBR322 plasmid clone of unintegrated MMTV(C3H) DNA, generously provided by J. Majors, University of California, San Francisco. Filter hybridization was carried out as described by Cohen (1980). Densitometric scanning of autoradiograms in the linear response range was performed on a computer-assisted, E-C Apparatus Corp. densitometer. Determination of the nuckoside content of DNA. The DNA samples were quantitatively hydrolyzed to deoxynucleosides and the major and minor base composition determined by high-performance liquid chromatography (HPLC) on a reversedphase column (Supelcosil LC-18DB; Supelco) by a modification (Gehrke et al, in preparation) of our previous method (Kuo et a+!,1980). From 2 to 5 pg of hydrolyzed DNA was chromatographed in the presence of 8-bromoguanosine as an internal marker by dual wavelength monitoring of absorbance at 254 and 280 nm. Isolation of cellular RNA. Whole cell RNA from normal tissue and lactating mammary gland was prepared by the guanidinium thiocyanate procedure of Chirgwin et al. (1979). Briefly, excised tissues were immediately homogenized with a mechanical homogenizer in 4 M guanidinium thiocyanate, 0.5% sodium N-lauroyl sarcosine, 25 mM sodium citrate, pH 7.0, and 0.1 M 2-mercaptoethanol. Cellular RNA was isolated by a 12-16 hr centrifugation at 150,000 g through a 5.7 M CsCl cushion with 25 mM sodium citrate, pH 5.0. The RNA pellet was dissolved in the guanidinium thiocyanate solution, extracted once with an equal volume of chloroform:l-butanol (4:1), and the final organic phase extracted with an equal volume of 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, and 0.1% SDS. Aqueous phases were combined and .RNA precipitated with ethyl alcohol. RNA samples were stored in 70% ethyl alcohol at -20’.
MMTV
DNA
METHYLATION
RNA dot blot. The preparation of RNA dot blots was based on the method of Thomas (1980). Briefly, RNA samples were spotted onto dried nitrocellulose sheets previously equilibrated with 20x SSC (3 M NaCI, 0.3 M sodium citrate), air-dried, and heated in vacua at 80” for 2 hr to immobilize RNA, and hybridized as described previously for DNA blots (Cohen, 1980). Serial dilutions of RNA were made with a yeast RNA diluent and MMTV virion RNA standards were included on each filter. Preparation of finwuddehyde gels. RNA samples (lo-12 1.18of total cellular RNA per gel slot) were denatured in 6% formaldehyde and 50% formamide, in MOPS buffer (20 mM morpholinopropanesulfonic acid; 50 mM sodium acetate; 1 mM EDTA, pH 7.0). Each sample was heated at 65” for 5 min, cooled in an ice bath, and electrophoresed in duplicate 1.2% agarose gel in MOPS buffer and 6% formaldehyde. One gel was stained with ethidium bromide for ultraviolet visualization of intact cellular ribosomal RNA species. The other gel was sequentially soaked for 15 min each in 50 mM NaOH, 1 mM NaCl, 0.1 M Tris-HCl pH 7.4, and 20x SSC, and then blotted 1216 hr onto a nitrocellulose filter in 20X SSC. The filter was then baked for 2 hr at 80” in a vacuum oven.
71 RESULTS
Tissue-&e&@ &Ltion
Variations in DNA Meth-
The BALB/c mouse strain transmits three MMTV genetic loci designated Mtv6, Mtv-8, and Mtv-9, which have been characterized extensively by mapping with restriction endonucleases (Fig. 1; Cohen et al, 1979a; Traina et aL, 1981). Two proviruses (Mtv-8 and 9) appear to be full length, while the third (Mtv-6) is a subgenomic fragment. Because EcoRI cleaves once within the MMTV genome, two virus-specific fragments are generated for each full length provirus. The subgenomic Mtv-6 locus is included in the 16.7 kilobase pair (kb) EcoRI fragment. The 5’ and 3’ halves of Mtv-8 are found in 8.4- and 6.7-kb EcoRI fragments, respectively, and the 5’ and 3’ ends of Mtv-9 appear in 7.8- and lO.O-kb fragments, respectively (Fig. 1). The presence of 5-methylcytosine in virus-specific sequences was examined by secondary digestion after EcoRI with HpaII or HhaI, which are inhibited by the presence of 5mC in the CG sequence of their recognition sites. Particularly useful is HpaII, which recognizes the DNA sequence 5’-CCGG-3’ but is inhibited if the internal cytosine is methylated, unlike its isoschizomer MspI 16.1
‘1.11
10.0
FIG. 1. Restriction endonuclease map of MMTV-specific genetic loci endogenous to the BALB/c mouse strain. Boxes represent long terminal repeated sequences. R, EuJRI site, H, HpaII site, and Hh, HhaI site.
BREZNIK
+ 10.0
l 6.417.6 + 6.7
+ 4.0 FIG. 2. Densitometry scan of autoradiograms of EcoRI-digested lactating mammary gland DNA (A), and EcoRVHpaII double digestion of the same DNA (B). Ten micrograms of DNA was digested and DNA fragments separated on 0.8% agarose gels followed by densitometric scanning as described under Materials and Methods. Numbers above peaks indicate the size of the DNA fragment in kb determined by inclusion of restriction endonuclease digested, radiolabeled bacteriophage DNA in a parallel lane.
ET AL.
(LMG; Fig. 2) a 4.0-kb virus-specific fragment was found in the EcoRI-HpuII double digestion that was absent in the digest with EcoRI alone. None of the EcoRI-generated, virus-specific fragments was lost completely. In the linear range of the densitometer the 16.7-kb fragment was not detectable although it was present visually on the autoradiogram. Of importance, however, the 10-O-kb fragment was significantly reduced in intensity when compared to the 8.4-, 7.8-, and 6.7-kb fragments in the double digest. The lO.O-kb fragment was mapped to the 3’ end of the Mtv-9 locus (Fig. 1; Cohen et al, 1979a). Therefore, the 4.0-kb EcoRI-HpuII fragment is derived from the 3’ end of Mtv-9. Examination of the restriction endonuclease map of Mtv9 (Fig. 1) reveals that undermethylation of the HpuII site in the 3’LTR would result in the 4.0-kb fragment seen in DNA from lactating mammary gland. Similar findings were obtained with DNA from tissues of animals at various parity and time of lactation. An autoradiogram of EcoRI-HhaI double digestions of the various tissues studied is presented in Fig. 3. Digestions of DNA ABCDE
(Waalwijk and Flavell, 1978). In addition, HhaI which cleaves DNA at 5’-GCGC-3’ but not 5’-G5mCGC-3’ is useful in identifying tissue-specific variations in the methylation of certain genes if homologous nonmethylated DNA sequences are available for comparison. Previously published results have demonstrated the presence of 5’-CCGG-3’ sites in all E’coRI fragments using EcoRI-MspI double digestion (Breznik and Cohen, 1982). Double digestion with EcoRI and HpuII of DNA from liver, brain, kidney, lung, and spleen (data not shown) exhibited no novel bands (bands other than those seen typically following EcoRI digestion) and indicated that all MspI/HpaII sites in, and adjacent to the endogenous MMTV loci were methylated. However, as shown in the densitometric scan of the electrophoresed EcoRI and EcoRI/HpaII digestion of DNA from lactating mammary gland
-7.1 -5.6
-5.6 -3.6
-3.6
- 2.7
-2.7
FIG. 3. EcoRI/HhaI double digestions of DNA from normal organs of the BALB/c mouse strain. DNA samples from liver (A), kidney (B), brain (C), lung (D), spleen (E), and lactating mammary gland (F) were digested and electrophosed on agarose gels as described in Fig. 2.
MMTV
DNA
METHYLATION
from liver, kidney, brain, and lung (lanes A-D, respectively) showed that HhaI caused no change in the EcoRI fragments and generated no new virus-specific fragments, indicating that all sites for this DNA methylation-sensitive endonuclease are methylated. However, DNA from spleen and lactating mammary gland (lanes E and F, respectively) contained HhaI sites that are hypomethylated, because new, lower molecular weight fragments were observed in the double digest. Similar results were observed previously with LMG DNA from other inbred mice (Cohen, 1980). The predominent fragments generated by EcoRI-HhaI double digestion of both LMG and splenic DNA were 5.6 to 2.7 kb in size. These fragments are the size expected if the HhuI site in the 3’ and 5’ LTRs is hypomethylated in either Mtv-8 or -9 (Fig. 1; Donehower et aL, 1981). The other virus-specific fragments, 3.6 and 7.1 kb, generated by double digestion exhibited an intensity disproportionately low compared to their molecular weights indicating the presence of nonviral sequences. Therefore, these fragments must represent the virushost junction fragments. The size of these fragments corresponds to that expected from the 3’ end of Mtv-8 and -9 if HhaI cleaved with DNA within the 3’ LTR. Densitometric analysis of the EcoRIHhd digestions (Fig. 4; only the region from 5.6 to 16.7 kb is included in this scan) of DNA from LMG and spleen and integration of peak areas for the reference EcoRI fragments (16.7, 10.0, 8.4, and 7.8 combined, and 6.7 kb) confirmed the origin of these double digestion products. The relative integral value (i.e., percentage of total proviral sequences) for the 6.7- and lO.O-kb EcoRI fragments from the 3’ end of Mtv-8 and -9 decreased after secondary HhaI digestion in DNA from both LMG and spleen. Thus, the HhuI site in the 3’ LTR of these two MMTV-specific loci are hypomethylated in the majority of cells of the LMG and spleen. The 8.4-kb EcoRI fragment derived from the 5’ end of Mtv8 was not altered by HhaI but it was apparent by visual inspection of the densitometric tracing of the autoradiogram that
6.1 3.1 7.7
73
170 9.0 6.6
53.7 32.6 225
21.2 125 6.6
A 6 C
FIG. 4. Densitometry scan of autoradiograms of double-digested DNA from spleen and lactating mammary glands of BALB/c mice. EcoRI-digested lactating mammary gland (A), EcoRI/HhaI double digestion of LMG DNA (B), and EcoRI/HhaI double digestion of splenic DNA (C) were analyzed on a computer assisted densitometer after gel analysis presented in Fig. 3. Only the region of the gel from 16.7 to 5.6 kb is included for ease of interpretation. Numbers above peaks indicate the size of the DNA fragment in kb. The relative integral value calculated as a percentage of the total viral sequences was determined. These values are presented below the densitometric tracings for the five EcoRI reference fragments derived from genetically transmitted MMTV sequences. The 8.4- and 7.8-kb fragments could not be resolved in the peak integration procedure.
the 7.8-kb virus-specific fragment from the 5’ end of Mtv-9 did decrease in intensity in the double digestions. Therefore, the 5’ LTR of Mtv-8 is not hypomethylated, while this site in Mtv-9 is undermethylated in a subset of the cells in the lactating mammary gland and in most of the cells of the spleen. The subgenomic MMTV-specific locus, Mtv-6, may be undermethylated at HhaI sites in DNA from LMG (Fig. 4). The relative integral value of the 16.7-kb EcoRI fragment derived from this locus does decrease after subsequent digestion with Hhd. In addition, in overexposed autoradiograms (not shown) MMTV-specific fragments at 2.2 and 1.6 kb that could be derived from Mtv-6 were visualized in
74
BREZNIK
EcoRI-HhaI double digestion of DNA from LMG. However, because the restriction endonuclease map of this locus is not known, it is uncertain whether the small degree of undermethylation of Hhd sites of Mtv6 locus in LMG is significant or simply reflects the difficulty in quantitative transfer of high molecular weight fragments from agarose gels. Total 5mC Content of Normal BALB/c sues
Tti-
Variations in cytosine methylation at specific locations in MMTV-specific sequences could reflect different extents of methylation at a limited number of genomic regions or large, generally distributed differences in the 5mC content throughout the genomes of the tissues examined. To evaluate the latter possibility, the overall 5mC content of these DNA was determined by HPLC analysis of DNA hydrolyzed enzymatically to deoxynucleosides. The mol% 5mC in DNA from tissue samples derived from three to eight mice were 0.92 f 0.04 for mammary gland, 0.87 & 0.05 for spleen, 0.88 + .Ol for kidney, 0.90 + 0.01 for lung, and 0.90 f 0.02 for liver DNAs. No significant differences in overall methylation of spleen DNA or lactating mammary gland DNA and other tissue DNAs were observed. Therefore, the tissuespecific differences which were seen in the methylation of MMTV-specific loci do not represent differences in the overall genomic 5mC content of the tissues used in this study. However, considerable tissue specificity has been observed in other in wivo derived murine cell populations as well as in tissues from various other mammals (Ehrlich et d, 1982; Gama-Sosa et al, 1983a, b).
ET AL.
standards, the lactating mammary gland was found to contain between 10 and 20 MMTV genome equivalents per cell, whereas the brain, kidney, spleen, lung, heart, and liver had less than 10 genome equivalents per cell, which was the lower limit of this assay. In addition, RNA samples were electrophoresed under formaldehyde denaturing conditions in 1.2% agarose, transferred to nitrocellulose, and hybridized with radiolabeled MMTV genomic sequences. Ethidium bromide staining confirmed the presence of similar amounts of intact 28 S and 18 S ribosomal RNA bands, in the samples from each tissue tested. Formaldehyde gels (data not shown) revealed the presence of an MMTV-specific RNA transcript of 1.6 kb only in RNA extracted from the lactating mammary gland. No other MMTVrelated transcripts were detected. DISCUSSION
In tumors induced by exogenous infection with MMTV those proviral sequences acquired via reverse transcription of virion RNA were not methylated although the genetically transmitted viruses remained methylated (Cohen, 1980). In tumors from animals not infected with the milk-borne virus hypomethylation of the genetically transmitted viruses was observed in tumors from both BALB/c and C3H/He mouse strains (Breznik and Cohen, 1982) as well as in the GR mouse strain (unpublished observation of authors). With the exception of mammary tumors from the BALB/c strain, the undermethylation of these viral sequences correlated with virus-specific RNA expression in the tumor. In this study hypomethylation of 5’CCGG-3’ and 5’-GCGC-3’ sites in the genetically transmitted, MMTV-specific seTranscriptional Activity of MMTV Se- quences in some normal tissues of the BALB/c mouse strain was demonstrated. quSplenic DNA contained a HhaI site in 3’ Virus-specific RNA in total RNA ex- LTR of the Mtv-8 locus that was undertracted from the various tissues studied methylated. However, this occurred in a was examined by dot blot hybridization. subset of splenic cells or in only one chroNo virus-specific RNA was detected in any mosomal homolog because the 6.7-kb &&RI of the tissues except in lactating mammary fragment decreased in intensity but did gland. When compared with MMTV RNA not disappear (Fig. 4). Similar data were
MMTV
DNA
METHYLATION
obtained with HhaI digestion of DNA from lactating mammary gland. The HhuI sites in both LTRs of Mtv-9 (7.8 and lO.O-kb EcoRI fragments) and the subgenomic viral locus Mtv-6 (16.7-kb EcoRI fragment) were also undermethylated in the lactating mammary gland DNA and to a lesser degree in DNA from the spleen (Fig. 4); again these EcoRI fragments decreased in intensity but did not completely disappear on subsequent digestion with HhaI (Fig. 4). The extent of virus-specific hypomethylation was less than that previously observed in DNA from mammary tumors. Although splenic DNA exhibited no detectable hypomethylation of HpuII sites in virus-specific sequences, DNA from lactating mammary gland contained a H&I site that appeared to be undermethylated in 3’ LTR of Mtv-9 in either one homolog or a subset of cells, because the lO.O-kb EcoRI fragment (Fig. 2) decreased in intensity. Our results are consistent with DNA sequencing data (Donehower et a& 1981) that revealed a site for HpaII cleavage approximately 1300 basepairs from the 5’ end of the MMTV LTR. Hypomethylation at this site in the 3 LTR should yield a virus-specific fragment of 4.0 kb, which is what was observed (Fig. 2). The remaining viral sequences would not be detected because only 50 basepairs of viral sequences would be present in this fragment. Although many studies of DNA methylation in vertebrate DNA have revealed an inverse relationship between DNA methylation and transcriptional activity of a specific gene counter examples to this correlation often have been found (Ehrlich and Wang, 1981; Cooper, 1983). Etkind and Sarkar (1983) demonstrated tissue specific hypomethylation of MMTV-specific sequences in splenic DNA of the C3Hf mouse strain. In the present study, confirmation of these tissue-specific differences was obtained, additional sites of hypomethylation in the lactating mammary gland identified; and undermethylated sites mapped to specific location in the viral loci. Methylation of MMTV-specific loci at HhuI sites was not correlated with changes in their transcriptional activity, because greater hypomethylation of the two full-length pro-
75
viruses was observed in DNA from spleen, nonexpressing tissue, than in DNA from lactating mammary gland, the source of the 1.6-kb MMTV-specific transcript (Fig. 4). However, undermethylation at the HpaII site in the 3’ LTR of Mtv-9 (Fig. 2) was the only verifiable variation in methylation associated with expression of an RNA transcript of 1.6 kb. Recently, Wheeler et al. (1983) described a single 1.6kb transcript in BALB/c lactating mammary glands. This transcript was shown to contain sequences derived from the transcriptional leader and the U3 region of the proviral DNA, a region that encompasses the long terminal repeat open reading frame. It was therefore proposed to encode the putative “orf” gene product (Dickson and Peters, 1981). Another report of this single RNA species in BALB/c tissues was published by van Ooyen et al. (1983). They observed the same transcript in the lactating mammary glands of C3H/ He and GRn/A mice along with sequences related to the gag-p01 region of MMTV genomic RNA. If one accepts the hypothesis that undermethylation may be a marker for regions of DNA in which transcription is actively occurring, these results suggest that Mtv-9 is the template for transcription of the 1.6-kb transcripts observed in RNA extracted from normal lactating mammary gland of the BALB/c mouse strain. ACKNOWLEDGMENTS This work WBSsupported by USPHS Grant numbew ROl-CA-34823 (JCC), ROl-CA-19942 (ME), ROl-CA25215 (JSB), and ROl-CA-33369 (JSB), and research contract NOl-CP-91020 (JSB and DM). JCC is supported by the Optimist Leukemia Foundation of Louisiana, Inc. We gratefully acknowledge the technical assistance of F. S. Kittrell. REFERENCES ANDERVONT, H. B. (1941). Note on the transfer of the strain C3H milk influence through successive generations of strain C mice. J. N&L Cancer Znet 2, 307-308. ANDERVONT, H. B. (1945). Fate of C3H milk influence in strains C and C67 black. J. NatL Cancer Inst. 5, 383-390. BREZNIK, T., and COHEN, J. C. (1982). Altered methylation of endogenous viral promoter sequences
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terial methylase are non-infectious. Nature &ondon) 304,275-277. SOUTHERN,E. M. (19’75). Detection of Specific sequences among DNA fragments separated by gel electrophoresis. J. Mel Bid 98,503-507. STEWART,C., STUHLMANN,H., JAHNER,D., and JAENISCH, R. (1982). De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells. Proc Nuti Ad Soi USA 79,4098-4102.
STUHLMANN,H., JAHNER,D., and JAENISCH,R. (1981). Infectivity and methylation of the retroviral genomes is correlated with expression in the animal. CeU 26, 221-232. THOMAS,P. S. (1980).Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Nat1 Acad Sci USA 77,5201-5205. TRAINA, V. L., TAYLOR,B. A., and COHEN,J.C. (1981). Genetic mapping of endogenous mouse mammary tumor viruses: locus characterization, segregation, and chromosomal distribution. J. Vird 40,735-744. TRAINA-DORGE,V. L., and COHEN,J. C. (1983). The molecular genetics of mouse mammary tumor virus. C??.wrent Topics Micro Imm 196.35-56. VAN OOYEN,A. J. J., MICHALIDES, R. J. A. M., and NIJSSE, R. (1983). Structural analysis of a 1.7 kilo-
METHYLATION
77
base mouse mammary tumor virus-specific RNA. J. ViroL 46, 362-370.
VARMUS,H. E., QUINTRELL,N., MEDIEROS,E., BISHOP, J. M., NOWINSKI,R. C., and SARKAR,N. H. (19’73). Transcription of mouse mammary tumor virus genes in tissues from high and low tumor incidence mouse strains. J. MoLBioL 79, 663-679. WAALWIJK,C., and F'LAVELL, R. A. (1978). DNA methylation at a CCGG sequence in the large intron of the rabbit b-globin gene: tissue specific variations. NucKc
Acids Res. 5,4631-4641.
WEINTRA~ H., LARSON,A., and GROUDINE,M. (1981). Alpha-globin gene switching during the development of chicken embryos: Expression and chromosome structure. CeU 24.333-344. WHEELER,D. A., BUTEL, J. S., MEDINA, D., CARDIFF, R. D., and HAGER, G. L. (1983). Transcription of mouse mammary tumor virus: Identification of a candidate mRNA for the long terminal repeat gene product. J. I&X! 46, 42-49. WIGLER, M., LEVY, D., and PERUCHO,M. (1981). The somatic replication of DNA methylation. Cell 24, 33-40. YOUSSOUFIAN, H., HAMMER,S. M., HIRSCH,M. S., and MULDER,C. (1982). Methylation of the viral genome in an in vitro model of herpes simplex virus latency. Proc. NatL Acad Sci USA 79.2207-2210.
136. 69-7’7 (1964)
Mouse Mammary Tumor Virus DNA Methylation:
Tissue-Specific
Variation
TRUDY BREZNIK,* VICKI TRAINA-DORGE,t MIGUEL GAMA-SOSA,# CHARLES W. GEHRKE,§ MELANIE EHRLICH,* DAN MEDINA,** JANET S. BUTEL,tt AND J. CRAIG COHEN**’ Departments of +Micmbiology, #Medicine, and #Biochemistry, Lxnbsiana State University Medical Center, New Orleans, Lvukinna 70112,Departments of fMimobiology and $Bimhemistry, Tulune University Medical Center, New Orhns, Louisiana 7011.2,%Dq.nxtment CLfBiochemists, Experhnmti Station Chemical Labomhh Univertit~ of Missouri, Columbia, Missouri 65201,and Departments of **Cell Biology and “;t Virologll and Epidemidogy, Baylor College of Medicine, Houston, Team 77030
Received January 3, 1984 accepted March
16,
1984
Mouse mammary tumor virus-specific DNA sequences endogenous to the BALB/c mouse are shown to exhibit variable levels of methylation in a tissue-specific manner. In DNA from both lactating mammary gland and spleen, MMTV-specific sequences were hypomethylated at specific HpuII and HhaI sites. These variably methylated sites were found in the terminal repetitive sequences of the endogenous viral genomes. The specific hypomethylation of a HpoII site in Mb-9 is associated with expression of a 1.6 kb transcript in the lactating mammary gland.
aL, 1982; Clough et a.& 1982; Creusot et &, 1982; Conklin et a& 1982; Fradin et cd, 5-Methylcytosine (5mC) is the predom- 1982; Simon et al, 1983; Busslinger et cd, inant modified base found in the DNA of 1983; Wigler et cd, 1981). higher eukaryotes (Ehrlich and Wang, Mouse mammary tumor virus (MMTV) 1981; Razin and Riggs, 1980). Methylation is one of the eukaryotic viruses for which occurs after DNA replication by sequence- there is evidence that site-specific DNA specific transfer of the methyl group from methylation is linked to transcriptional S-adenosylmethionine to cytosine. There is inactivity (Cohen, 1980). MMTV is a retmuch recent evidence that one of the func- rovirus associated with mammary adenotions of DNA methylation in eukaryotes carcinomas in mice. This virus may be is to participate in the control of gene reg- transmitted either endogenously through ulation and cell differentiation. Studies the mouse genome where it exists as a stasupporting this hypothesis have shown an ble genetic element (Cohen and Varmus, inverse correlation between methylation of 1979; Traina-Dorge and Cohen, 1983) or a number of eukaryotic or eukaryotic viral exogenously through mother’s milk (Cohen genes and their transcriptional activity et d, 1979b). The BALB/c inbred mouse (Guntaka et d, 1980;Weintraub et &, 1981, strain lacks milk-borne MMTV and has a Compere and Palmiter, 1981; Jones et al, tumor incidence of ~1% at 18 months of 1981; Stuhlman et aL, 1981; Youssuofian et age. This strain can be foster-nursed on al, 1982). Also, various procedures leading females from the C3H strain which conto decreases in methylation of certain host tains the milk-borne virus; the resulting or viral genes in vertebrate cells have led substrain, BALB/cfC3H, has a tumor into the increased expression of these genes cidence approaching 100% (Andervont and, correspondingly, increased methyl1941,1945). Genetically transmitted, virusation to decreased expression (Groudine et specific sequences in tumors from these al, 1981; Sager and Kovac, 1981; Stewart mice are highly methylated, while viral sequences acquired by milk-borne infection 1 To whom reprint requests should be addressed. are hypomethylated. The former viral INTRODUCTION
et
69
0042-6822184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reprcduetion in any form reserved.
BREZNIK
70
DNA sequences appear to be inactive in transcription whereas the latter are transcriptionally active (Varmus et &, 1973). In this report we examine the location of hypomethylated sites in the endogenous MMTV sequences of different tissues of the BALB/c mouse strain. We also compare this tissue-specific hypomethylation of proviral sequences with their transcription and with the overall levels of 5mC in host DNA from different tissues. METHODS
Tissue.s. All tissues were taken from 4to 5-month-old virgin or primiparous BALB/c 4- to 5-month-old virgin or primiparous BALB/cMed female mice. The mice were raised in a closed conventional mouse colony in the Department of Cell Biology, Baylor College of Medicine. The mammary tumor incidence in breeding BALB/cMed female mice is approximately 1% at 18 months (Pauley et d, 1979). Restriction enzyme digestion DNA samples (10 pg) isolated from BALB/c mouse tissues as previously described (Cohen et al, 1979b) were digested with either EcoRI, MspI, HpaII, or HhaI (New England Biolabs). Specific restriction enzyme buffers used were as recommended by the manufacturer. For double digestions DNA (20 c(g) was cleaved with a fourfold excess of EcoRI for 4 hr and then extracted with chloroform-isoamyl alcohol (24:1, v/v) and precipitated with ethanol. DNA was resuspended in the appropriate buffer and 10 pg digested for 4 hr with a threefold excess of either M.1, &&I, or HhaI using the recommended conditions. All restriction enzyme digests were monitored for completeness prior to electrophoresis using pBR313 or X bacteriophage DNA as an internal control in a duplicate sample digest. Gel ekctrop~esis and blot h&ridizatim Electrophoresis through either 0.8 or 1.0% agarose gels containing 0.5 X TEA (TEA = 0.04 M Tris-acetate, pH 8.1, 0.02 M sodium acetate, 18 mM NaCl, and 2 mM EDTA) was used to fractionate restriction endonuclease-digested DNA. X bacteriophage q-labeled DNA digested with Hind111 was electrophoresed in a parallel lane and used as a molecular weight stan-
ET AL.
dard. DNAs were transferred using the method of Southern (1975), modified as previously described by Cohen et aL (1979b). A complementary q-labeled DNA representative of the entire MMTV genome (cDNArep) was synthesized by nick-translation of a pBR322 plasmid clone of unintegrated MMTV(C3H) DNA, generously provided by J. Majors, University of California, San Francisco. Filter hybridization was carried out as described by Cohen (1980). Densitometric scanning of autoradiograms in the linear response range was performed on a computer-assisted, E-C Apparatus Corp. densitometer. Determination of the nuckoside content of DNA. The DNA samples were quantitatively hydrolyzed to deoxynucleosides and the major and minor base composition determined by high-performance liquid chromatography (HPLC) on a reversedphase column (Supelcosil LC-18DB; Supelco) by a modification (Gehrke et al, in preparation) of our previous method (Kuo et a+!,1980). From 2 to 5 pg of hydrolyzed DNA was chromatographed in the presence of 8-bromoguanosine as an internal marker by dual wavelength monitoring of absorbance at 254 and 280 nm. Isolation of cellular RNA. Whole cell RNA from normal tissue and lactating mammary gland was prepared by the guanidinium thiocyanate procedure of Chirgwin et al. (1979). Briefly, excised tissues were immediately homogenized with a mechanical homogenizer in 4 M guanidinium thiocyanate, 0.5% sodium N-lauroyl sarcosine, 25 mM sodium citrate, pH 7.0, and 0.1 M 2-mercaptoethanol. Cellular RNA was isolated by a 12-16 hr centrifugation at 150,000 g through a 5.7 M CsCl cushion with 25 mM sodium citrate, pH 5.0. The RNA pellet was dissolved in the guanidinium thiocyanate solution, extracted once with an equal volume of chloroform:l-butanol (4:1), and the final organic phase extracted with an equal volume of 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, and 0.1% SDS. Aqueous phases were combined and .RNA precipitated with ethyl alcohol. RNA samples were stored in 70% ethyl alcohol at -20’.
MMTV
DNA
METHYLATION
RNA dot blot. The preparation of RNA dot blots was based on the method of Thomas (1980). Briefly, RNA samples were spotted onto dried nitrocellulose sheets previously equilibrated with 20x SSC (3 M NaCI, 0.3 M sodium citrate), air-dried, and heated in vacua at 80” for 2 hr to immobilize RNA, and hybridized as described previously for DNA blots (Cohen, 1980). Serial dilutions of RNA were made with a yeast RNA diluent and MMTV virion RNA standards were included on each filter. Preparation of finwuddehyde gels. RNA samples (lo-12 1.18of total cellular RNA per gel slot) were denatured in 6% formaldehyde and 50% formamide, in MOPS buffer (20 mM morpholinopropanesulfonic acid; 50 mM sodium acetate; 1 mM EDTA, pH 7.0). Each sample was heated at 65” for 5 min, cooled in an ice bath, and electrophoresed in duplicate 1.2% agarose gel in MOPS buffer and 6% formaldehyde. One gel was stained with ethidium bromide for ultraviolet visualization of intact cellular ribosomal RNA species. The other gel was sequentially soaked for 15 min each in 50 mM NaOH, 1 mM NaCl, 0.1 M Tris-HCl pH 7.4, and 20x SSC, and then blotted 1216 hr onto a nitrocellulose filter in 20X SSC. The filter was then baked for 2 hr at 80” in a vacuum oven.
71 RESULTS
Tissue-&e&@ &Ltion
Variations in DNA Meth-
The BALB/c mouse strain transmits three MMTV genetic loci designated Mtv6, Mtv-8, and Mtv-9, which have been characterized extensively by mapping with restriction endonucleases (Fig. 1; Cohen et al, 1979a; Traina et aL, 1981). Two proviruses (Mtv-8 and 9) appear to be full length, while the third (Mtv-6) is a subgenomic fragment. Because EcoRI cleaves once within the MMTV genome, two virus-specific fragments are generated for each full length provirus. The subgenomic Mtv-6 locus is included in the 16.7 kilobase pair (kb) EcoRI fragment. The 5’ and 3’ halves of Mtv-8 are found in 8.4- and 6.7-kb EcoRI fragments, respectively, and the 5’ and 3’ ends of Mtv-9 appear in 7.8- and lO.O-kb fragments, respectively (Fig. 1). The presence of 5-methylcytosine in virus-specific sequences was examined by secondary digestion after EcoRI with HpaII or HhaI, which are inhibited by the presence of 5mC in the CG sequence of their recognition sites. Particularly useful is HpaII, which recognizes the DNA sequence 5’-CCGG-3’ but is inhibited if the internal cytosine is methylated, unlike its isoschizomer MspI 16.1
‘1.11
10.0
FIG. 1. Restriction endonuclease map of MMTV-specific genetic loci endogenous to the BALB/c mouse strain. Boxes represent long terminal repeated sequences. R, EuJRI site, H, HpaII site, and Hh, HhaI site.
BREZNIK
+ 10.0
l 6.417.6 + 6.7
+ 4.0 FIG. 2. Densitometry scan of autoradiograms of EcoRI-digested lactating mammary gland DNA (A), and EcoRVHpaII double digestion of the same DNA (B). Ten micrograms of DNA was digested and DNA fragments separated on 0.8% agarose gels followed by densitometric scanning as described under Materials and Methods. Numbers above peaks indicate the size of the DNA fragment in kb determined by inclusion of restriction endonuclease digested, radiolabeled bacteriophage DNA in a parallel lane.
ET AL.
(LMG; Fig. 2) a 4.0-kb virus-specific fragment was found in the EcoRI-HpuII double digestion that was absent in the digest with EcoRI alone. None of the EcoRI-generated, virus-specific fragments was lost completely. In the linear range of the densitometer the 16.7-kb fragment was not detectable although it was present visually on the autoradiogram. Of importance, however, the 10-O-kb fragment was significantly reduced in intensity when compared to the 8.4-, 7.8-, and 6.7-kb fragments in the double digest. The lO.O-kb fragment was mapped to the 3’ end of the Mtv-9 locus (Fig. 1; Cohen et al, 1979a). Therefore, the 4.0-kb EcoRI-HpuII fragment is derived from the 3’ end of Mtv-9. Examination of the restriction endonuclease map of Mtv9 (Fig. 1) reveals that undermethylation of the HpuII site in the 3’LTR would result in the 4.0-kb fragment seen in DNA from lactating mammary gland. Similar findings were obtained with DNA from tissues of animals at various parity and time of lactation. An autoradiogram of EcoRI-HhaI double digestions of the various tissues studied is presented in Fig. 3. Digestions of DNA ABCDE
(Waalwijk and Flavell, 1978). In addition, HhaI which cleaves DNA at 5’-GCGC-3’ but not 5’-G5mCGC-3’ is useful in identifying tissue-specific variations in the methylation of certain genes if homologous nonmethylated DNA sequences are available for comparison. Previously published results have demonstrated the presence of 5’-CCGG-3’ sites in all E’coRI fragments using EcoRI-MspI double digestion (Breznik and Cohen, 1982). Double digestion with EcoRI and HpuII of DNA from liver, brain, kidney, lung, and spleen (data not shown) exhibited no novel bands (bands other than those seen typically following EcoRI digestion) and indicated that all MspI/HpaII sites in, and adjacent to the endogenous MMTV loci were methylated. However, as shown in the densitometric scan of the electrophoresed EcoRI and EcoRI/HpaII digestion of DNA from lactating mammary gland
-7.1 -5.6
-5.6 -3.6
-3.6
- 2.7
-2.7
FIG. 3. EcoRI/HhaI double digestions of DNA from normal organs of the BALB/c mouse strain. DNA samples from liver (A), kidney (B), brain (C), lung (D), spleen (E), and lactating mammary gland (F) were digested and electrophosed on agarose gels as described in Fig. 2.
MMTV
DNA
METHYLATION
from liver, kidney, brain, and lung (lanes A-D, respectively) showed that HhaI caused no change in the EcoRI fragments and generated no new virus-specific fragments, indicating that all sites for this DNA methylation-sensitive endonuclease are methylated. However, DNA from spleen and lactating mammary gland (lanes E and F, respectively) contained HhaI sites that are hypomethylated, because new, lower molecular weight fragments were observed in the double digest. Similar results were observed previously with LMG DNA from other inbred mice (Cohen, 1980). The predominent fragments generated by EcoRI-HhaI double digestion of both LMG and splenic DNA were 5.6 to 2.7 kb in size. These fragments are the size expected if the HhuI site in the 3’ and 5’ LTRs is hypomethylated in either Mtv-8 or -9 (Fig. 1; Donehower et aL, 1981). The other virus-specific fragments, 3.6 and 7.1 kb, generated by double digestion exhibited an intensity disproportionately low compared to their molecular weights indicating the presence of nonviral sequences. Therefore, these fragments must represent the virushost junction fragments. The size of these fragments corresponds to that expected from the 3’ end of Mtv-8 and -9 if HhaI cleaved with DNA within the 3’ LTR. Densitometric analysis of the EcoRIHhd digestions (Fig. 4; only the region from 5.6 to 16.7 kb is included in this scan) of DNA from LMG and spleen and integration of peak areas for the reference EcoRI fragments (16.7, 10.0, 8.4, and 7.8 combined, and 6.7 kb) confirmed the origin of these double digestion products. The relative integral value (i.e., percentage of total proviral sequences) for the 6.7- and lO.O-kb EcoRI fragments from the 3’ end of Mtv-8 and -9 decreased after secondary HhaI digestion in DNA from both LMG and spleen. Thus, the HhuI site in the 3’ LTR of these two MMTV-specific loci are hypomethylated in the majority of cells of the LMG and spleen. The 8.4-kb EcoRI fragment derived from the 5’ end of Mtv8 was not altered by HhaI but it was apparent by visual inspection of the densitometric tracing of the autoradiogram that
6.1 3.1 7.7
73
170 9.0 6.6
53.7 32.6 225
21.2 125 6.6
A 6 C
FIG. 4. Densitometry scan of autoradiograms of double-digested DNA from spleen and lactating mammary glands of BALB/c mice. EcoRI-digested lactating mammary gland (A), EcoRI/HhaI double digestion of LMG DNA (B), and EcoRI/HhaI double digestion of splenic DNA (C) were analyzed on a computer assisted densitometer after gel analysis presented in Fig. 3. Only the region of the gel from 16.7 to 5.6 kb is included for ease of interpretation. Numbers above peaks indicate the size of the DNA fragment in kb. The relative integral value calculated as a percentage of the total viral sequences was determined. These values are presented below the densitometric tracings for the five EcoRI reference fragments derived from genetically transmitted MMTV sequences. The 8.4- and 7.8-kb fragments could not be resolved in the peak integration procedure.
the 7.8-kb virus-specific fragment from the 5’ end of Mtv-9 did decrease in intensity in the double digestions. Therefore, the 5’ LTR of Mtv-8 is not hypomethylated, while this site in Mtv-9 is undermethylated in a subset of the cells in the lactating mammary gland and in most of the cells of the spleen. The subgenomic MMTV-specific locus, Mtv-6, may be undermethylated at HhaI sites in DNA from LMG (Fig. 4). The relative integral value of the 16.7-kb EcoRI fragment derived from this locus does decrease after subsequent digestion with Hhd. In addition, in overexposed autoradiograms (not shown) MMTV-specific fragments at 2.2 and 1.6 kb that could be derived from Mtv-6 were visualized in
74
BREZNIK
EcoRI-HhaI double digestion of DNA from LMG. However, because the restriction endonuclease map of this locus is not known, it is uncertain whether the small degree of undermethylation of Hhd sites of Mtv6 locus in LMG is significant or simply reflects the difficulty in quantitative transfer of high molecular weight fragments from agarose gels. Total 5mC Content of Normal BALB/c sues
Tti-
Variations in cytosine methylation at specific locations in MMTV-specific sequences could reflect different extents of methylation at a limited number of genomic regions or large, generally distributed differences in the 5mC content throughout the genomes of the tissues examined. To evaluate the latter possibility, the overall 5mC content of these DNA was determined by HPLC analysis of DNA hydrolyzed enzymatically to deoxynucleosides. The mol% 5mC in DNA from tissue samples derived from three to eight mice were 0.92 f 0.04 for mammary gland, 0.87 & 0.05 for spleen, 0.88 + .Ol for kidney, 0.90 + 0.01 for lung, and 0.90 f 0.02 for liver DNAs. No significant differences in overall methylation of spleen DNA or lactating mammary gland DNA and other tissue DNAs were observed. Therefore, the tissuespecific differences which were seen in the methylation of MMTV-specific loci do not represent differences in the overall genomic 5mC content of the tissues used in this study. However, considerable tissue specificity has been observed in other in wivo derived murine cell populations as well as in tissues from various other mammals (Ehrlich et d, 1982; Gama-Sosa et al, 1983a, b).
ET AL.
standards, the lactating mammary gland was found to contain between 10 and 20 MMTV genome equivalents per cell, whereas the brain, kidney, spleen, lung, heart, and liver had less than 10 genome equivalents per cell, which was the lower limit of this assay. In addition, RNA samples were electrophoresed under formaldehyde denaturing conditions in 1.2% agarose, transferred to nitrocellulose, and hybridized with radiolabeled MMTV genomic sequences. Ethidium bromide staining confirmed the presence of similar amounts of intact 28 S and 18 S ribosomal RNA bands, in the samples from each tissue tested. Formaldehyde gels (data not shown) revealed the presence of an MMTV-specific RNA transcript of 1.6 kb only in RNA extracted from the lactating mammary gland. No other MMTVrelated transcripts were detected. DISCUSSION
In tumors induced by exogenous infection with MMTV those proviral sequences acquired via reverse transcription of virion RNA were not methylated although the genetically transmitted viruses remained methylated (Cohen, 1980). In tumors from animals not infected with the milk-borne virus hypomethylation of the genetically transmitted viruses was observed in tumors from both BALB/c and C3H/He mouse strains (Breznik and Cohen, 1982) as well as in the GR mouse strain (unpublished observation of authors). With the exception of mammary tumors from the BALB/c strain, the undermethylation of these viral sequences correlated with virus-specific RNA expression in the tumor. In this study hypomethylation of 5’CCGG-3’ and 5’-GCGC-3’ sites in the genetically transmitted, MMTV-specific seTranscriptional Activity of MMTV Se- quences in some normal tissues of the BALB/c mouse strain was demonstrated. quSplenic DNA contained a HhaI site in 3’ Virus-specific RNA in total RNA ex- LTR of the Mtv-8 locus that was undertracted from the various tissues studied methylated. However, this occurred in a was examined by dot blot hybridization. subset of splenic cells or in only one chroNo virus-specific RNA was detected in any mosomal homolog because the 6.7-kb &&RI of the tissues except in lactating mammary fragment decreased in intensity but did gland. When compared with MMTV RNA not disappear (Fig. 4). Similar data were
MMTV
DNA
METHYLATION
obtained with HhaI digestion of DNA from lactating mammary gland. The HhuI sites in both LTRs of Mtv-9 (7.8 and lO.O-kb EcoRI fragments) and the subgenomic viral locus Mtv-6 (16.7-kb EcoRI fragment) were also undermethylated in the lactating mammary gland DNA and to a lesser degree in DNA from the spleen (Fig. 4); again these EcoRI fragments decreased in intensity but did not completely disappear on subsequent digestion with HhaI (Fig. 4). The extent of virus-specific hypomethylation was less than that previously observed in DNA from mammary tumors. Although splenic DNA exhibited no detectable hypomethylation of HpuII sites in virus-specific sequences, DNA from lactating mammary gland contained a H&I site that appeared to be undermethylated in 3’ LTR of Mtv-9 in either one homolog or a subset of cells, because the lO.O-kb EcoRI fragment (Fig. 2) decreased in intensity. Our results are consistent with DNA sequencing data (Donehower et a& 1981) that revealed a site for HpaII cleavage approximately 1300 basepairs from the 5’ end of the MMTV LTR. Hypomethylation at this site in the 3 LTR should yield a virus-specific fragment of 4.0 kb, which is what was observed (Fig. 2). The remaining viral sequences would not be detected because only 50 basepairs of viral sequences would be present in this fragment. Although many studies of DNA methylation in vertebrate DNA have revealed an inverse relationship between DNA methylation and transcriptional activity of a specific gene counter examples to this correlation often have been found (Ehrlich and Wang, 1981; Cooper, 1983). Etkind and Sarkar (1983) demonstrated tissue specific hypomethylation of MMTV-specific sequences in splenic DNA of the C3Hf mouse strain. In the present study, confirmation of these tissue-specific differences was obtained, additional sites of hypomethylation in the lactating mammary gland identified; and undermethylated sites mapped to specific location in the viral loci. Methylation of MMTV-specific loci at HhuI sites was not correlated with changes in their transcriptional activity, because greater hypomethylation of the two full-length pro-
75
viruses was observed in DNA from spleen, nonexpressing tissue, than in DNA from lactating mammary gland, the source of the 1.6-kb MMTV-specific transcript (Fig. 4). However, undermethylation at the HpaII site in the 3’ LTR of Mtv-9 (Fig. 2) was the only verifiable variation in methylation associated with expression of an RNA transcript of 1.6 kb. Recently, Wheeler et al. (1983) described a single 1.6kb transcript in BALB/c lactating mammary glands. This transcript was shown to contain sequences derived from the transcriptional leader and the U3 region of the proviral DNA, a region that encompasses the long terminal repeat open reading frame. It was therefore proposed to encode the putative “orf” gene product (Dickson and Peters, 1981). Another report of this single RNA species in BALB/c tissues was published by van Ooyen et al. (1983). They observed the same transcript in the lactating mammary glands of C3H/ He and GRn/A mice along with sequences related to the gag-p01 region of MMTV genomic RNA. If one accepts the hypothesis that undermethylation may be a marker for regions of DNA in which transcription is actively occurring, these results suggest that Mtv-9 is the template for transcription of the 1.6-kb transcripts observed in RNA extracted from normal lactating mammary gland of the BALB/c mouse strain. ACKNOWLEDGMENTS This work WBSsupported by USPHS Grant numbew ROl-CA-34823 (JCC), ROl-CA-19942 (ME), ROl-CA25215 (JSB), and ROl-CA-33369 (JSB), and research contract NOl-CP-91020 (JSB and DM). JCC is supported by the Optimist Leukemia Foundation of Louisiana, Inc. We gratefully acknowledge the technical assistance of F. S. Kittrell. REFERENCES ANDERVONT, H. B. (1941). Note on the transfer of the strain C3H milk influence through successive generations of strain C mice. J. N&L Cancer Znet 2, 307-308. ANDERVONT, H. B. (1945). Fate of C3H milk influence in strains C and C67 black. J. NatL Cancer Inst. 5, 383-390. BREZNIK, T., and COHEN, J. C. (1982). Altered methylation of endogenous viral promoter sequences
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BREZNIK
during mammary carcinogeneeis. Nature (Lo&m) 295,255-257. BUSSLINGER, M., HURST, J., and FLAVELL, R. A. (1983). DNA methylation and the regulation of globin gene expression. Ceu 34, 197-256. CHIRCWIN,J. M., PRZYBYLA,A. E., MACDONALD,R. J., and RLFFER,W. J. (1979). Isolation of biologically active RNA from sources enriched in ribonucleaae. Biochemistry 18.5294-5299. CHRISTY,B., and SCANGOS,G. (1982). Expression of transferred thymidine kinase genes is controlled by methylation. Proc NatL AazaXci USA 79.629% 6303. CLOUCH,D., KUNDEL,L. M., and DAWN, R. L. (1982). 5-azacytidine-induced reactivation of a herpes aimplex thymidine kinaae gene. Science 216, 70-73. COHEN, J. C. (1989). Methylation of milk-borne and genetically transmitted mouse mammary tumor virus proviral DNA. CeU 19, 653-662. COHEN, J. C.. MAJORS,J. E., and VARMUS, H. E. (19’79). Organization of mouse mammary tumor virus-epechic DNA endogenous to BALB/c mice. .I. Vird 32, 483-496. COHEN,J. C., SHANK, P. R., MORRIS,V. L., CARDIFF, R., and VARMUS,H. E. (1979). Integration of the DNA of mouse mammary tumor virus in virusinfected normal and neoplaatic tissues of the mouse. CkU 16,333-345. COHEN, J. C., and VARMUS,H. E. (1979). Endogenous mammary tumour virus DNA varies among wild mice and segregates during inbreeding. Natzlre (Lvndon) 278.418423. COMPERE,S. J., and PALMITER, R. D. (1981). DNA methylation controls the inducibility of the mouse metallothioneinn-1 gene in lymphoid cells. Cell 26, 233-240. CONKLIN,K. F., COFFIN,J. M., ROBINSON,H. L., GROUDINE, M., and EISENYAN. R. (1982). Role of methylation in the induced and spontaneous expression of the avian endogenous virus ev-1. Mel Cell Bid 2, 638-652. COOPER,D. N. (1983). Eucaryotic DNA methylation. Human
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CREUSOT,F., Acs. G., and CHRISTMAN,J. K. (1982). Inhibition of DNA methyltransferaae and induction of friend erythroleukemia cell different&ion by 5azacytidine and 5-aza-Z-deoxycytidine. J. Bid Chem 257, 2041-2048. DICKSON, C., and PETERS,G. (1981). Protein-coding potential of mouse mammary tumor virus genome RNA ae examined by in vitro tranelation. J. Vied 37, 36-47. DONEHOWER,L. A., HUNG, A. L., and HAGER, G. (1981).Regulatory and coding potential of the mouse mammary tumor virus long terminal redundancy. J. Vird
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