THE INITIATION OF DE-NOVO METHYLATION OF FOREIGN DNA INTEGRATED INTO A MAMMALIAN GENOME IS NOT EXCLUSIVELY TARGETED BY NUCLEOTIDE-SEQUENCE

The de novo methylation of foreign DNA integrated into the mammalian g enome is a fundamental process whose mechanism has not yet been elucid ated. We have studied de novo methylation in adenovirus type 12 (Ad12) genomes inserted into the genomes of Ad12-induced hamster tumor cells . De novo methylation of Ad12 DNA, which is not methylated in the viri on, is initiated in two paracentrally located regions and spreads from there across the integrated Ad12 genomes. (i) After extensive cultiva tion of cloned Ad12-induced hamster tumor cell lines, the same segment s in integrated Ad12 DNA in different cell lines become methylated or remain unmethylated, depending on their positions in the viral genome. (ii) When Ad12 DNA or Ad12 DNA fragments are transfected into hamster cells and permanent cell lines are established by selection for the c otransfected neomycin phosphotransferase gene, patterns of de novo met hylation in terminally or internally located segments of Ad12 DNA are different from those in Ad12-induced tumor cell lines, (iii) A detaile d study on the topology of the integrated viral genomes in the Ad12-tr ansformed hamster cell lines T637 and A2497-3 and in the Ad12-induced hamster tumors T191, T1111(1), and T181 has been performed. Some of th e integrated viral genomes are inserted into the cellular genome in an orientation colinear with the virion genome; others have been rearran ged. An originally internally located Ad12 DNA segment has become tran sposed to the left-terminal sequences of the viral genome in several c ell lines and tumors. In the complete Ad12 genomes, the internally loc ated PstI-D fragment becomes extensively methylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences. When this DNA segment has been juxtaposed to the left-terminal, hypomethylated fragment of Ad12 DNA in rearranged genomes, the PstI-D fragment remains unmethylated. We therefore reason that the initiation of de novo methylation in integrated Ad12 DNA can not be directed exclusively by the nucleotide sequence. Other paramete rs, such as site of integration, conformation of integrates, mode of c ell selection, or chromatin structure related to transcriptional activ ity, may play decisive roles.