Adenovirus type 2 VAI RNA transcription by polymerase III is blocked by sequence-specific methylation.
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 III-transcribed genes have not been investigated in depth. We therefore studied methylation effects on the RNA polymerase III-transcribed VAI gene of adenovirus type 2 DNA. The VAI gene contains 20 5'-CG-3' dinucleotides, of which 4 (20%) can be methylated by HpaII (5'-CCGG-3') and HhaI (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'-GCGC-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.
Jüttermann
R
R
Institute of Genetics, University of Cologne, Federal Republic of Germany.
Hosokawa
K
K
Kochanek
S
S
Doerfler
W
W
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D006224:Cricetinae; D004279:DNA, Viral / Q000737:chemistry; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D005814:Genes, Viral; D006367:HeLa Cells / Q000382:microbiology; D006801:Humans; D008745:Methylation; D008957:Models, Genetic; D012320:RNA Polymerase III / Q000096:biosynthesis / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry; D013149:Spiroplasma / Q000201:enzymology; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D004279:DNA, Viral; D012367:RNA, Viral; C052835:adenovirus associated RNA; C037664:DNA modification methylase HhaI; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases; D012320:RNA Polymerase III