The methylation state of the proviruses in avian sarcoma virus transformed chick and rat cells

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Biochimica et Biophysica Acta 910 (1987) 116-122

Elsevier BBA 91748

T h e methylation state of the proviruses in avian sarcoma virus transformed chick and rat cells Nitzan Roguel, Henia Moskowitz, Hanna

Relevy, Judith Hamburger

and Moshe Kotler Department of Molecular Genetics, The Hebrew University-Hadassah Medical School. Jerusalem (Israel)

(Received 16 June 1987)

Key words: Avian sarcoma virus; Methylation; Provirus; Transformation; (Chicken cell); (Rat cell)

The endogenous viruses in the avian cells are not completely methylated, nor are the Schmidt-Ruppin RSV-D (SLID) proviruses in the infected cells completely unmethylated. Avian sarcoma proviruses integrated in rat transformed cloned cells are heavily methylated, in these cells, a region in the 3' end of the e n v gene is unmethylated in all the s r c - c o n t a i n i n g proviruses but not in the transformed defective (td) proviruses. A possible role for the hypomethylation of the 3' end of the e n v region is proposed.

Introduction Mammalian cells infected with Schmidt-Ruppin RSV-D (SRD) virus, unlike infected avian cells, do not produce viral particles [1,2]. Most of the infected mammalian cells remain phenotypically unchanged, whereas others exhibit morphological transformation [3]. The transformed avian cells contain three viral m R N A s of 35 S, 28 S and 21 S that express the g a g / p o l , e n v and src peptides, respectively [4]. The 35 S and 28 S viral m R N A s are transcribed in the transformed m a m malian cells 100-1000-fold less, and the 21 S src m R N A is synthesized 20-50-fold less, than in the ASV-infected avian cells. In addition, the transformed mammalian cells contain aberrant forms of the viral m R N A , especially of the 21 S src m R N A [5]. In general, methylation and expression of eukaryotic cellular and viral genes are inversely

related [6,7]. However, recent studies have shown that in some cases methylated genes are efficiently transcribed [8]. The site of the methylated bases in the gene plays an important role in the regulation of gene expression [7,9,10]. In this report we demonstrate the following. (a) The ASV proviruses present in the transformed mammalian cells are hypermethylated while these proviruses, integrated in the avian transformed cells, are almost unmethylated. This phenomenon correlates well with the low levels of transcription of viral genes in the mammalian cells [11]. (b) The avian sarcoma proviruses present in cells cloned from a single transformed rat cell contain scattered unmethylated C C G G residues. (c) The proviral genomes containing the complete src gene have a hypomethylated region at the 5' end of the src gene. This region is hypermethylated at the 5' end of the src deleted gene.

Abbreviation: SRD, Schmidt-Ruppin RSV-D.

Materials and Methods Correspondence: M, Kotler, Department of Molecular Genetics, The Hebrew University-Hadassah Medical School, P.O. Box 1172, Jerusalem 91.010, Israel.

Cells, v i r u s e s a n d c u l t u r e conditions. Cells were grown in Dulbecco's modified Eagle's medium

0167-4781/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

117 supplemented with 5% fetal calf serum and 10% Tryptose phosphate broth (Difco). Primary cultures of chick fibroblasts were prepared from l l day-old chick embryos from SPAFAS eggs (R.F.D. No. 3, Norwich, CT) and rat embryonic fibroblasts were prepared from Sprague-Dawley rat embryos (Weizmann Institute, Rehovot, Israel). The SRD virus was cloned on chick embryo fibroblasts by limiting dilution before using. Rat embryo fibroblasts were infected with SRD virus (m.o.i. 1-2), and 15 days post-infection cells from several foci were transferred into separate soft agar cultures as described by Macpherson [12]. 20 days later, well separated colonies reached sufficient size (about 0.5-1.0 mm) and were isolated from the agar under an inverted microscope with Pasteur pipettes onto 24-well plates. Subclones were obtained by the same procedure. Purification of DNA. Total cellular DNA was purified by using sodium dodecyl sulfate (SDS)pronase digestion, extraction with phenol/chloroform and RNAase treatment. The aqueous phase was precipitated with 2.5 vol. of ethanol and 100 mM sodium acetate at - 20 ° C.

Restriction enzyme digestion and analysis of DNA. DNA samples were digested with EcoRI, Sail, BamHI, SstI (Bethesda Research Laboratories), HpalI and MspI (Boehringer) restriction endonuclease using conditions prescribed by the suppliers. DNA fragments were separated by electrophoresis on 0.8-1% agarose gels. The fractionated DNA was then denatured and transferred to nitrocellulose filters by blotting in 20 SSPE (1 × SSPE is 0.18 M N a C I / 1 0 mM NaH2PO4/0.1 mM N a E D T A (pH 7.0)), essentially as described by Southern [13]. Hybridization, probes and reagents. The SRA probe was rescued from the pSRA (pBR322 plasmid containing the SRA insert [14] kindly provided by M. Bishop) by Sail cleavage. The PvulI (800 bp) src and the BamHI gag (1300 bp) fragments were derived from src- or gag-containing plasmids (Fig. lc) by digestion with the appropriate restriction enzymes. The DNA fragments to be used were separated by electrophoresis and eluted from the agarose gels by adsorption to glass beads (Flint 325 Mesh Ceramic Supply of New York and New Jersey Inc.) as previously described [15]. The isolated fragments were

labelled by nick translation using 32p-deoxyribonucleotides (Amersham International, U.K.) and approx. 1.106 cpm//~g labelled DNA probe, as described by Maniatis et al. [16]. Results

The methylation state of endogenous and exogenous viruses in chick embryo fibroblasts The methylation state in avian sarcoma proviruses was determined by using the isoschizomeric MspI and HpalI enzymes. Although this technique reveals only a specific subset of the potential methylation sets in a given DNA sequence, it can be used as an indicator for the methylation state in a DNA region. As is evident in Fig. la, most of the SRD exogenous proviral DNAs present in the chick embryo fibroblasts are unmethylated in the C C G G sequences, since HpalI-digested D N A (slot 2) yields approximately the same bands as those seen in the MspI-digested DNA (slot 1). The DNA extracted from SRD-infected chick embryo fibroblasts cleaved with HpalI (Fig. la, slot 2) contains a greater amount of large DNA fragments homologous to the SRA probe than chick embryo fibroblast D N A treated similarly (Fig. la, slot 4). Thus, some of the SRD information in the infected chick embryo fibroblasts is methylated. This conclusion is based on the observation that the intensity of the hybridized radioactive probe is much lower in slot 4 than in slot 2 (Fig. l a represents one blot, but tracks 3 and 4 were exposed for 72 h, while 1 and 2 were exposed only 24 h). It can also be seen that the majority of the endogenous proviral D N A sequences are methylated but some fractions of the proviral genome are in an unmethylated state (Fig. la, slots 3 and 4). To ensure complete digestion, the enzymes were used in excess and, in some parallel experiments, plasmid DNA was mixed with cellular D N A in order to monitor complete digestion (not shown). The excess of large DNA fragments in the SRD-infected cells indicates that some of the integrated exogenous proviral DNA underwent methylation. Comparison of the methylation state of proviruses in the SRD transformed rat and chick cells To compare the extent of methylation of SRD

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transferred for propagation as monolayers. D N A was extracted from morphologically transformed cultures and then analyzed by digestion with restriction enzymes. The cloned ASV-transformed rat cells used here contained about 15 proviruses per cell, as revealed by hybridization with LTR and 3' src probes [17]. ASV-infected chick embryo fibroblasts contain 10-100 proviral copies per cell [4]. Cleavage of the ASV-transformed rat cell D N A with MspI and HpaII (Fig. lb, slots 2 and 3) shows that most of the proviruses are methylated in the C C G G residues, since a large fraction of the viral D N A appears in the top of the slot containing HpaII cleaved D N A (Fig. lb, slot 2). However, some of the bands identified after digestion of the same D N A with MspI (Fig. lb, slot 3) can also be seen in track 2 containing HpaII-cleaved DNA, but at very low intensity. Similar results were obtained with D N A preparations extracted from uncloned transformed cells and with four independent cloned cells (not shown). Thus, SRD-transformed rat cells contain small amounts of unmethylated proviral DNA. It can be concluded that the majority of the SRD proviral information present in the non-permissive cells is in a methylated state, while that present in the permissive chick embryo fibroblasts is essentially unmethylated.

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Fig. 1. The methylation state of proviral DNA in uninfected and infected chick embryo fibroblasts and SRD-transformed rat embryo fibroblasts. (a) SRD-transformed chick cells DNA cleaved with Mspl (lane 1) and Hpall (lane 2). Lanes 3 and 4 show DNA extracted from uninfected chick cells cleaved with the same isoschizomericenzymes. Lanes 3 and 4 were exposed for 72 h, lanes 1 and 2 for 24 h only. (b) SRD-transformed rat cells DNA cleaved with HpalI (lane 2) and with Mspl (lane 3). Lane 1 pSRA DNA cleaved with EcoRI used as a marker. (c) The cleavage site of EcoRI and SstI at the SRD proviral DNA. Bars at the bottom represent fragments derived from the pSRA that were used as probes in this report. Hybridization was performed with the SRA fragment.

proviral D N A integrated into rat embryo fibroblasts to that of proviruses present in the infected chick embryo fibroblasts, cultures of rat cells were infected with the same SRD cloned virus (m.o.i. 1-2). Transformed cells were cloned in soft agar and colonies that originated from single cells were

Unmethylated sites at specific regions in the proviral DNAs To determine whether proviruses in mammalian cells contain specific regions that are preferentially unmethylated, cloned cell D N A was digested with EcoRI alone and simultaneously HpaII or MspI (Fig. 2) and blots were hybridized with SRA, src 800 bp, or gag 1200 bp probes. The EcoRI endonuclease cut four times in the proviral D N A (Fig. 2a, slot 2) and gave rise to three internal D N A fragments of 3650, 2950, 2330 bp, and an additional band of 1100 bp. The 1100 bp fragment results from the deleted src gene in part of the proviruses. That is the reason for the lower intensity of the 2950 bp src-containing fragment in comparison to the 3650 bp and 2330 bp bands. D N A extracted from cloned SRD-transformed rat cells was simultaneously digested with EcoRI and MspI nucleases. All the EcoRI fragments were cleaved and migrated toward the bottom of

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Fig. 2. A specific unmethylated fragment present in most of the SRD proviruses in SRD-transformed rat cells. (a) Hybridization was performed with SRA probe. Lane 1, pSRA DNA used as a marker; lane 2, EcoRI alone; lane 3, EcoR1 and Mspl; and lane 4, EcoR1 and HpaII together. (b) Hybridization was performed with 800 bp src. Lane 1, pSRA DNA used as a marker; lane 2, EcoRI alone; lane 3, EcoRI and MspI; lane 4, EcoRI and HpaII together. (c) Hybridization was performed with 1300 bp gag probe. Lane 1, pSRA DNA used as a marker; lane 2, EcoRI alone; lane 3, EcoRI and HpaII; lane 4, EcoRI and Mspl together.

the gel (Fig. 2a, slot 3). However, a combination of EcoRl and HpaII did not change the location of the 3650, 2330 and 1100 bp fragments, but did reduce the size of the 2950 bp fragment, which migrated lower in the gel (Fig. 2a, slot 4). This observation is supported by additional hybridizations carried out with the src and gag probes (Fig. 2b and 2c). The SstI endonuclease cut twice in the proviral DNA (Fig. lc) and gave rise to an internal DNA fragment of about 6500 bp, which is common to all proviruses present in the SRD-transformed cells (Fig. 3). Simultaneous digestion with HpaII and SstI enzymes resulted in the generation of two bands, both of which migrated faster than the band which resulted by the SstI digestion alone. These results were confirmed by using the 1300 bp gag probe (not shown).

Unmethylated region upstream of the src gene The SRD-transformed rat cell DNA was cleaved with the following enzyme combinations: (1)

EcoRI; (2) EcoRI and SstI; (3) EcoRI, SstI and HpalI; and (4) EcoRI, SstI and MspI. The cleavage products were then separated by gel electrophoresis, blotted and hybridized with the 2950 bp src-containing probe (Fig. 4). The 2950 bp and the 1100 bp fragments resulted from EcoRI digestion. Two bands, of 2300 bp and 600 bp, appeared after combined cleavage of EcoRI and Sstl (Fig. 4, slots 1 and 2). The 600 bp fragment appears intense, since it contains sequences derived from both the sre-containing and the 1100 bp fragments of the transformed defective (td) viruses. Two MspI restriction sites are present in this fragment located about 40 and 210 bp downstream of the EcoRI cleavage site (our unpublished results and which are correlated to Ref. 18). Simultaneous digestion of the cellular DNA with EcoRI, SstI and HpaII (slot 3) clearly shows that the intensity of the 600 bp band is reduced and becomes relatively similar to that of the 1100 bp region. An additional band of about 400 bp appears in this track. Slot 4 shows that all the provirus bands

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Fig. 4. The 3' end region of the env gene is unmethylated in src-containing proviruses. Lane 1, SRD transformed rat cells DNA cleaved with EcoRI. Lane 2, SRD-transformed rat cells DNA simultaneously cleaved with EcoRl and SstI. Lane 3, EcoRl, Sstl and Hpall. Lane 4, EcoRl, SstI and Mspl. Hybridization was performed with the 2950 bp EcoRl fragment.

Fig. 3. The methylation state of the internal SstI fragment of the provirus. Cleavage of SRD-transformed rat cells DNA with Sstl (lane 1), with Sstl and HpalI (lane 2). Hybridization was performed with SRA probe.

visualized after cleavage with E c o R l and S s t I are displaced to the bottom of the gel after additional cleavage with M s p I . These results and those described above strongly suggest that the region upstream of the src gene 3' end of the eno gene is hypomethylated.

Discussion

Infection of permissive avian cells (producers) and non-permissive mammalian cells (nonproducers) with SRD results in the formation of proviruses which are integrated into the host cell DNA. It was previously shown [19-22], and confirmed here, using other host cells and different strains of viruses, that most of the C C G G sequences present in the integrated ASV proviruses in mammalian cells are methylated, while the

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majority of these sequences are unmethylated in the ASV-infected avian cells. The results presented in this report clearly show that the methylation of proviruses is not an allor-none phenomenon; that is, the endogenous viruses in the avian cells are not completely methylated, nor are the SRD proviruses in the infected cells completely unmethylated. The SRD proviruses present in transformed mammalian cells, although methylated in most of their C C G G sequences, contain a region at the 3' end of the env gene which is unmethylated. Our findings are in accordance with previous findings [22,23], but have established three contributions: (1) the unmethylated region is defined by the E c o R I and S s t I cleavage sites (the S s t I site in the env gene is present in the SRD provirus used in our laboratory); (2) this specific region is unmethylated in most or even all of the s r c - c o n t a i n i n g proviruses integrated in a single cell (about 15); and (3) this region is methylated in proviruses containing src-deleted gene (td proviruses). The presence of unmethylated sequences in the env-src region was described before in transformed cells containing a single provirus [23,24]. It could be assumed that this unmethylated region is essential for the src gene expression, so that only cells with unmethylated sequences were selected. The cells analyzed in our laboratory carry about 15 s r c - c o n t a i n i n g proviruses, most of which exhibit the unmethylated region. The selection for the transformed cell cannot be the reason for the demethylation of this region in all proviruses. Moreover, the finding that the env-src region is methylated in the td proviruses, but not in the s r c - c o n t a i n i n g proviruses, may suggest that the downstream sequences of a gene may determine the methylation state of this region. Recently, it was demonstrated that introduction of an in vitro methylated actin gene into cultured myotubes resulted in demethylation of this gene, while the same DNA inserts remained highly methylated after transfection of fibroblasts. It was suggested that the methylation state of a gene is determined by tissue specific mechanisms [24]. This type of putative tissue specific control could account for cell regulation of the methylation state of retroviral DNA as demonstrated for SRD-infected avian and mammalian ceils. How-

ever, the host cells is not the sole factor controlling the methylation state of the viral DNA. In the same SRD-infected chick embryo fibroblasts most of the exogenous viruses are hypomethylated and the endogenous viruses are hypermethylated (Fig. la). The SRD and the endogenous viruses have some genetic information in common. The differences are mainly located in the U3 region of the LTR, and in the env region. The src gene is present only in the SRD genome. Thus, it seems that variations in the proviral information may affect the level of methylation. Alternatively, it is possible that the endogenous viruses achieve their heavy methylation state by a selection process during evolution. References 1 2 3 4

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11 12 13 14 15 16 17 18 19 20

Boettiger, D. (1974) Cell 3, 71-74 Kotler, M. (1971) J. Gen. Virol. 12, 199-206 Altaner, C. and Temin, H.M. (1970) Virology 40, 118-134 Varmus, H.E. and Swanstrom, R. (1982) in RNA Tumor Viruses (Weiss, R., Teich, N., Varmus, H. and Coffin, J., eds.), Cold Spring Harbor, New York Quintrell, N., Hughes, S.H., Varmus, H.EI and Bishop, J.M. (1980) J. Mol. Biol. 143, 363-393 Doerfler, W. (1983) Annu. Rev. Biochem. 52, 93-124 Cedar, H. (1984) in DNA Methylation (Razin, A., Cedar, H. and Riggs, A., eds.), Springer-Verlag, New York Tanaka, K., Appella, E. and Jay, G. (1983) Cell 35,457-465 Doerfler, W., Langner, K.D., Kruczer, I., Vardimon, L. and Renz, D. (1984) in DNA Methylation (Razin, A., Cedar, H. and Riggs, A., eds.) Springer-Verlag, New York Kun, C. and Shen, J. (1984) in DNA Methylation (Razin, A. Cedar, H. and Riggs, A., eds.), Springer-Verlag, New York Deng, C.T., Stehelin, D., Bishop, J.M. and Varmus, H.E. (1977) Virology 46, 313-330 Macpherson, I. and Montagnier, L. (1964) Virology 23, 291-294 Southern, E. (1975) J. Mol. Biol. 98, 503-517 DeLorbe, W.J., Luciw, P.A., Goodman, H.M., Varmus, H.E. and Bishop, J.M. (1980) J. Virol. 36, 50-61 Vogelstein, B. and GiUespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, 615-619 Maniatis, T., Fritch, E.F. and Sambrook, J. (1982) Molecular Cloning Cold Spring Harbor Laboratories, New York Roguel, N., Relevi, H., Hamburger, J. and Kotler, M. (1986) Biochim. Biophys. Acta 908, 12-20 Schwartz, D.E., Tizard, R. and Gilbert, W. (1983) Cell 32, 853-869 Groudine, M., Eisenman, R. and Weintraub, H. (1981) Nature 292, 311-317 Hu, W.S., Fanning, T.G. and Cardiff, R.G. (1984) J, Virol. 49, 66-71

122 21 Katz, R.A., Mitsialis, S.A. and Guntaka, R.V. (1983) J. Gen. Virol. 64, 429-435 22 Searle, S., Gillespie, D.A.F., Chiswell, D.J. and Wyke, J. (1984) Nucleic Acids Res. 12, 5193-5211

23 Catala, F. (1986) Nucleic Acids Res. 30, 2481-2495 24 Yisraeli, J., Adelstein, R.S., Melloul, D., Nudel, M., Yaffe D. and Cedar, H. (1986) Cell 46, 409-416