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

Abstract

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 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.

5S rRNA genes expression is not inhibited by DNA methylation in Arabidopsis

“…Methylation appears to have a different impact on polymerase III genes transcription according to the nature of their promoter sequences. The plant results with Brassica SINE S1 and Arabidopsis 5S rRNA genes are in agreement with mammalian reports, where SINEs (Liu and Schmid, 1993), viral genes (Juttermann et al, 1991) and tRNA Lys gene transcription (Besser et al, 1990) were inhibited by methylation, although the transcription of a Xenopus 5S rRNA gene was not (Besser et al, 1990).…”

“…The impact of DNA methylation on pol III transcription is not clear and relies on a small number of reports, all of which (except The Plant Journal (2002) 29 (3), 313±323 ã 2002 Blackwell Science Ltd one in Brassica) use animal systems. Most of these studies were done on mammalian SINEs (Liu and Schmid, 1993), viral genes (Juttermann et al, 1991) and a tRNA Lys gene (Besser et al, 1990), and report a more-or-less severe transcriptional inhibition following methylation. In Brassica, using an in vitro assay, Arnaud et al (2001) reported a transcriptional inhibition that is directly correlated to the level of the template methylation (a Brassica SINE).…”

Proposed roles for DNA methylation inAlutranscriptional repression and mutational inactivation

“…Previously, effects of methylation on the transcription of Alu repeats and tRNA genes were not detected in in vitro transcription assays (5,20). However, in a recent study (21), the effects of methylation on the in vitro transcription of the VAI RNA gene were reliably detected at lower template concentrations than those employed in these previous studies. Methylation inhibits transcription of this gene both in vivo and in vitro.…”

“…Here we used low rather than high template concentrations, nuclear rather than cytoplasmic extracts, very CpG rich young Alu templates rather than a single older Alu, and either general methylation by CpG methylase or methylation targeted specifically to the A box rather than the untargeted methylation of a few restriction sites. While each of these technical differences undoubtedly contributes somewhat to the detection of methylation induced repression, the use of low template concentrations is critical (21).…”

“…These findings imply that either a methylation sensitive repressor is present in limiting amounts or that positive transcription factors have a lower affinity toward methylated templates. In the case of the VAI RNA gene, the inhibition of transcription has been attributed to methylation interfering with complex formation by positive transcription factors (21). A repressor that binds specific to methylated DNA has been identified for Pol II transcription units but its effect on Pol HI templates has not been reported (22,23).…”