The initiation of de novo methylation of foreign DNA integrated into a mammalian genome is not exclusively targeted by nucleotide sequence

Abstract

The de novo methylation of foreign DNA integrated into the mammalian genome is a fundamental process whose mechanism has not yet been elucidated. 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 virion, is initiated in two paracentrally located regions and spreads from there across the integrated Ad12 genomes. (i) After extensive cultivation of cloned Ad12-induced hamster tumor cell lines, the same segments 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 cotransfected neomycin phosphotransferase gene, patterns of de novo methylation in terminally or internally located segments of Ad12 DNA are different from those in Ad12-induced tumor cell lines. (iii) A detailed study on the topology of the integrated viral genomes in the Ad12-transformed hamster cell lines T637 and A2497-3 and in the Ad12-induced hamster tumors T191, T1111(1), and T181 has been performed. Some of the integrated viral genomes are inserted into the cellular genome in an orientation colinear with the virion genome; others have been rearranged. An originally internally located Ad12 DNA segment has become transposed to the left-terminal sequences of the viral genome in several cell lines and tumors. In the complete Ad12 genomes, the internally located 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 cannot be directed exclusively by the nucleotide sequence. Other parameters, such as site of integration, conformation of integrates, mode of cell selection, or chromatin structure related to transcriptional activity, may play decisive roles.