Adenovirus type 2 VAI RNA transcription by polymerase III is blocked by sequence-specific methylation

R Jüttermann, K Hosokawa, S Kochanek, W Doerfler
1991 Journal of Virology  
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
more » ... -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 VAIand VAIIand 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. 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 * Corresponding author. 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 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 reac-
doi:10.1128/jvi.65.4.1735-1742.1991 fatcat:h36q47autncp7a2temwkqddky4