Blocked by Sequence-Specific Methylation - Journal of Virology

JOURNAL OF VIROLOGY, Apr. 1991, p. 1735-1742

Vol. 65, No. 4

0022-538X/91/041735-08$02.00/0 Copyright © 1991, American Society for Microbiology

Adenovirus Type 2 VAI RNA Transcription by Polymerase III Is Blocked by Sequence-Specific Methylation RUTH JUTTERMANN,1 KEITCHI HOSOKAWA,2 STEFAN KOCHANEK,1

AND

WALTER DOERFLER1*

Institute of Genetics, University of Cologne, D-5000 Cologne 41, Federal Republic of Germany,' and Department of Biochemistry, Kawasaki Medical School, Kurashiki City, Okayama, Japan2 Received 1 October 1990/Accepted 31 December 1990

Sequence-specific methylation of the promoter and adjacent regions in mammalian genes transcribed by RNA polymerase II leads to the inhibition of these genes. So far, RNA polymerase ITT-transcribed genes have not been investigated in depth. We therefore studied methylation effects on the RNA polymerase ITTtranscribed VAI gene of adenovirus type 2 DNA. The VAI gene contains 20 5'-CG-3' dinucleotides, of which 4 (20%) can be methylated by Hpall (5'-CCGG-3') and Hhal (5'-GCGC-3'). Three of these 5'-CG-3' sequences are located close to the internal regulatory region of the VAI segment. An unmethylated, a 5'-CCGG-3'- and 5'-GCGC-3'-methylated, and a 5'-CG-3'-methylated pUC18 construct containing the VAI and VAII regions were transfected into mammalian cells. In many experiments, an inactivating effect of 5'-CCGG-3' and 5'-GC GC-3' DNA methylation on the VAI region was not observed. In contrast, methylation of all 20 5'-CG-3' sequences in the VAI region by a CpG-specific DNA methyltransferase from Spiroplasma species did interfere with VAI transcription. Transcription of the VAI- and VAII- and of the VAI-containing constructs was also shown to be inhibited in an in vitro cell-free transcription system after the constructs had been methylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences or at all 5'-CG-3' sequences. When an oligodeoxyribonucleotide which carried the internal control block A of the VAI region was methylated at three 5'-CG-3' sequences, the formation of a complex with HeLa nuclear proteins was abrogated. The results presented support the notion that the VAI gene transcribed by the DNA-dependent RNA polymerase III is also inactivated by methylation of the decisive 5'-CG-3' sequences.

synthesis in adenovirus-infected cells (44). VAI RNA prevents the activation of the interferon-inducible doublestranded RNA-dependent eIF-2a kinase, which inactivates the protein synthesis initiation factor eIF-2ot by phosphorylation (22, 31, 37, 39). Additional functions of VAI RNA in enhancing the stability of cytoplasmic mRNAs have also been discussed (41, 43). We used the Ad2 VAI region as a model to demonstrate that the sequence-specific methylation of an RNA polymerase ITT-transcribed gene leads to transcriptional inactivation upon transfection into mammalian cells and in in vitro

Sequence-specific methylation of 5'-CCGG-3' sequences at positions +6, +24, and -215 leads to the inactivation of the late E2A promoter of adenovirus type 2 (Ad2) DNA, as demonstrated by transient expression experiments and after the integration of a promoter-indicator gene construct into the DNA of mammalian cells (for reviews, see references 5, 6, and 7). Methylation of the +6 and +24 sites in the late E2A promoter abrogates the formation of a DNA-protein complex (18, 19), and the loss of this interaction is presumably responsible for promoter inhibition. The inhibitory effect of promoter methylations has been documented for a large number of DNA-dependent RNA polymerase II-transcribed eukaryotic genes. Much less work has been devoted to effects of sequence-specific methylation on genes that are transcribed by RNA polymerase I or III (3, 25, 27). In recent years, evidence has been adduced that eukaryotic RNA polymerases I, II, and III are rather closely related to each other. The sequences of the single-copy RNA polymerase IT and III genes of Saccharomyces cerevisiae, for example, show remarkable patterns of amino acid conservation (11). The Ad2 genome encodes the VAI and VAII RNAs, which are about 160 nucleotides in length. These RNAs have been termed virus-associated (VA) RNAs. This designation refers to their abundance in Ad2-infected cells but not in the intact virion. The VA RNAs are transcribed from the Ad2 genome by host RNA polymerase III. The VAI and VAII RNA regions on the Ad2 genome have internal and external control elements characteristic of class III genes and belong to the tRNA type of polymerase III genes (1, 9, 14, 29, 32, 33, 47). VAI RNA plays an important role in late protein *

transcription experiments. MATERIALS AND METHODS Plasmid constructs. The plasmid construct containing the VAI and VAII regions of Ad2 DNA was termed pAd2VAI+VAII and had a size of 3,445 bp. Its map is shown in Fig. 1. In the polycloning site (XbaI and SmaI), the plasmid contains the Ad2 DNA segment between nucleotides 10579 (Xbal) and 11338 (Nrul) (36), comprising the VAI and VAII regions. The construct pAd2VAI contained the Ad2 DNA sequence between nucleotides 10579 (Xbal) and 10812 (BalI). In vitro methylation of the pAd2VAI+VAII construct. The DNA was in vitro methylated (24) simultaneously with the HpaII and Hhal DNA methyltransferases (3 to 4 U/,ug of DNA) or with the prokaryotic CpG DNA methyltransferase from Spiroplasma species (34) (1 U/,ug of DNA) in 10 mM Tris-HCl (pH 7.9)-S50 mM NaCI-10 mM EDTA-1 mM dithiothreitol-80 ,uM S-adenosylmethionine (SAM) in a total reaction mixture of 10 FLl/p.g of DNA. The CpG DNA methyltransferase was a gift from New England BioLabs, Beverly,

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Plasmid used for transfections FIG. 1. Plasmid constructs containing the VAI and VAII regions of Ad2 DNA. (Top) Map of the VAI segment. The C residues in 5'-CG-3' dinucleotide combinations on either strand are indicated by open triangles. Most of the iandmarks of this region are described in the text. The DNA fragments protected by VAI RNA from transfected cells after Si nuclease treatment are also designated. (Bottom) Map of the 3,445-bp pAd2VAI+VAII plasmid, which carries the VAI and VAII regions (Ad2 nucleotides [nt] 10579 to 11338) in the XbaI-SmaI site of the polycloning segment of plasmid pUC18. The asterisk indicates the site of 32p labeling. lac i', po, and lacZ' refer to parts of the lactose operon of Escherichia coli.

Mass. The methylation reactions proceeded at 37°C for 16 to 20 h. Mock methylations were carried out under the same conditions except that SAM was omitted from the reaction mixture. At the end of the reaction, the DNA was reextracted by the sodium dodecyl sulfate-proteinase K-phenol method (42) and challenged for complete methylation by restriction with HpaII or HhaI, followed by Southern blot hybridization (24, 40) or by determination of the nucleotide sequence (26). Nuclease Si protection analyses. At 48 h posttransfection, cytoplasmic RNA was isolated (38) from cells transfected with the methylated, unmethylated, or mock-methylated construct. For the S1 nuclease reaction, 30 ,ug of the total cytoplasmic RNA was then annealed at 51°C to a VAI DNA fragment probe that was prepared in the following way. Plasmid pAd2VAI (map in Fig. 1) carries the nucleotide 10579 to 10812 fragment of Ad2 DNA with the VAI region. This plasmid was cut either with BamHI at Ad2 nucleotide 10680 or with AhaII at Ad2 nucleotide 10741. The linearized plasmid was then 32p labeled by using [y-32P]ATP and Escherichia coli polynucleotide kinase (35). Subsequently, the construct was cleaved with HindIll, yielding either a 125-bp BamHI-HindIII fragment or a 186-bp AhaII-HindIII fragment. These fragments were gel purified and used as hybridization probes. The S1 nuclease reaction was performed for 1 h under standard conditions at 30°C (28). Finally, the protected, 32P-labeled VAI DNA fragments

were analyzed by electrophoresis on 6% polyacrylamide gels containing 8 M urea. Appropriate 32P-labeled marker DNA fragments were coelectrophoresed. In vitro transcription of the pAd2VAI+VAII and pAd2VAI constructs in a cell-free transcription system with HeLa cell nuclear extracts. Experimental details of the cell-free in vitro transcription system were described previously (4). pAd2VAI+VAII or pAd2VAI was used as the template in amounts of 50 ng, 5 ng, or 500 pg. The HeLa cell nuclear extract contained 10 ,ug of protein per reaction. Subsequent to the in vitro transcription reaction, the RNA was extracted and analyzed by the S1 protection technique as described above. Band shift experiments. A double-stranded oligodeoxyribonucleotide was prepared by synthesizing both complements with the Ad2 DNA sequence between nucleotides 10605 and 10644 (36) in a 318A DNA synthesizer (Applied Biosystems). In some experiments, the three 5'-CG-3' sequence pairs in this oligodeoxyribonucleotide (Fig. 1) carried 5-methyldeoxycytidine (5-mC) instead of C residues. This sequence contained the internal control region A of the VAI gene. The top strand was 5'-end labeled by using polynucleotide kinase and [_Y_32P] ATP. About 10,000 cpm of this oligodeoxyribonucleotide was incubated with about 10 ,g of protein in HeLa nuclear extracts. Incubation was done at 0°C for 90 min under the conditions described elsewhere (18, 19), except that Mg2+ was omitted from the reaction mix. As

VOL. 65, 1991

5'-CG-3' METHYLATION BLOCKS VAI RNA TRANSCRIPTION

unspecific competitors, 0.5 ,ug each of poly(dA dT) and poly(dIT dC) were added. Specific competitors were 0.5, 5, 50, or 200 ng of the unmethylated oligodeoxyribonucleotide. The free 32P-labeled oligodeoxyribonucleotide was separated from the protein-complexed form by electrophoresis on 4% polyacrylamide gels, which were dried after electrophoresis and autoradiographed on Kodak XAR-5 films. Standard protocols. Southern blotting (40), transfection of mammalian cells by the calcium phosphate precipitation technique (13), nucleotide sequence determination (26), and the assay for ,-galactosidase in extracts of mammalian cells (15, 17) have been described previously. RESULTS Methylatable sites in the VAI and VAII regions of Ad2 DNA. The map in Fig. 1 (top) depicts structural details of the Ad2 DNA fragment from nucleotide 10579 (XbaI site) to 10812 (BaIT site) containing the VAI and VAII regions, as well as 30 nucleotides of the 5'-flanking region which are thought to contain regulatory signals for VAI transcription (9, 49). The pAd2VAI+VAII plasmid (Fig. 1, bottom) carrying the XbaI-NruI Ad2 DNA fragment was used for the transfection experiments. The nucleotide sequence of the VAI region (approximately Ad2 nucleotides 10610 to 10766) highlights all 5'-CG-3' sequences and the internal control region (boxes A and B) (9, 14) of this polymerase TTTtranscribed segment. The HpaII (5'-CCGG-3') and Hhal (5'-GCGC-3') sequences are also indicated. The XbaI-Nrul fragment in the pAd2VAI+VAII plasmid used for transfection contains 80 5'-CG-3' sequences. Of these dinucleotide combinations, only 21 (26.25%) can be methylated in vitro by HpaTI and HhaI. The VAI region by itself carries 20 5'-CG-3' sequences, of which only 4 (20%) can be methylated by the HpaII and Hhal DNA methyltransferase. In some experiments, the pAd2VAI construct was used, which contained the Ad2 fragment between nucleotides 10579 and 10812 (36). The plasmids were left unmethylated, methylated in vitro with the prokaryotic DNA methyltransferases HpaTT and Hhal or with the CpG DNA methyltransferase, or mock-methylated with one of the enzyme combinations in the absence of SAM. The plasmids thus treated were then transfected into mammalian cells or tested in a cell-free transcription system. In vitro methylation at 5'-CCGG-3' and 5'-GCGC-3' sequences in pAd2VAI+VAIH fails to inactivate the VAI region of Ad2 DNA. The pAd2VAI+VAII plasmid is transcribed into VAI RNA upon transfection into human HeLa cells in culture. By using the 32P-labeled BamHI-HindIII fragment (see Materials and Methods) as the probe and RNA isolated from HeLa cells infected with Ad2 at 24 h postinfection (p.i.) (Fig. 2, lane 3) or RNA isolated from HeLa cells at 48 h after the transfection with the pAd2VAI+VAII plasmid (Fig. 2, lanes 5 and 7), a 72- to 74-bp fragment was protected in S1 nuclease experiments. Since the probe is specific for VAI RNA, it can be concluded that this RNA is transcribed efficiently in Ad2-infected and in pAd2VAI+VAII-transfected HeLa cells. As a negative control, tRNA did not protect the same DNA fragment (Fig. 2, lane 2). All size determinations for protected DNA fragments are related to the 32P-labeled MspI fragments of pUC18 as the marker (Fig. 2, lane 1). The data collected in Fig. 2 also demonstrate that transcription of VAI RNA was not inhibited by in vitro methylation of the 5'-CG-3' combinations in the HpaII (5'-CCGG3') and HhaI (5'-GCGC-3') sequences at 48 h (Fig. 2, lanes 6

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FIG. 2. Unmethylated and 5'-CCGG-3' and 5'-GCGC-3' methylated pAd2VAI+VAII plasmids are transcribed after transfection into HeLa cells. Cell lines HeLa and 293 were transfected with the unmethylated (u) (lanes 4, 5, 7, and 9) or the 5'-CCGG-3' (HpaII)and 5'-GCGC-3' (HhaI)-methylated (m) construct (lanes 6, 8, and 10), and cytoplasmic RNA was extracted at 48 h (lanes 4 to 8) or 72 h (lanes 9 and 10) after transfection (h p.t.). The cells were growing in 10-cm-diameter plastic dishes, and 20 ,ug of DNA was transfected per dish. VAI RNA transcription was verified by S1 protection analyses of 30 ,ug of total cytoplasmic RNA (Materials and Methods). In the experiments described here, the 125-bp BamHI-HindIII fragment was used as the probe. RNA from HeLa cells infected with Ad2 virions was extracted at 24 h p.i. and served as a positive control (lane 3); yeast tRNA (lane 2) served as the negative control. The Mspl fragments of pUC18 DNA were labeled with polynucleotide kinase and [_-32P] ATP, heat denatured, and coelectrophoresed as size markers on a 6% polyacrylamide gel containing 8 M urea (lane 1). An autoradiogram of this gel is shown. Size marker lengths are indicated in nucleotides (nt). The positions of the VAI RNA protected fragment (72 to 74 nt) and the reannealed DNA probe (125 nt) are indicated by arrowheads.

and 8) or 72 h (Fig. 2, lane 10) after transfection. Similar results (not shown) were obtained after transfection of human KB cells. VAI RNA transcription was somewhat enhanced after transfection of the adenovirus type 5 (AdS)transformed human cell line 293 with the unmethylated pAd2VAI+VAII plasmid (Fig. 2, lane 4). Transcription of the adenovirus VAI region is controlled by genes encoded in the viral El region (2, 10, 20), and the human cell line 293 contains the left terminus of AdS DNA in an integrated form and expresses it constitutively (13). It is concluded that methylation of 20% of the 5'-CG-3' sites in the VAI region of Ad2 DNA does not inhibit its transcription 48 or 72 h after transfection of the methylated pAd2VAI+VAII construct into HeLa cells. In vitro-methylated pAd2VAI+VAII constructs are not detectably demethylated after transfection into HeLa cells. The unmethylated (Fig. 3, lane 4), mock-methylated (Fig. 3, lanes 5 and 7), and in vitro HpaIl- and HhaI-methylated

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