Insertion of foreign DNA into an established mammalian genome can alter the methylation of cellular DNA sequences

The insertion of adenovirus type 12 (Ad12) DNA into the hamster genome and the transformation of these cells by Ad12 can lead to marked alterations in the levels of DNA methylation in several cellular genes and DNA segments. Since such alterations in DNA methylation patterns are likely to affect the transcription patterns of cellular genes, it is conceivable that these cha nges have played a role in the generation or the maintenance of the Ad12-tr ansformed phenotype. We have now isolated clonal BHK21 hamster cell lines t hat carry in their genomes bacteriophage lambda and plasmid pSV2neo DNAs in an integrated state. Most of these cell lines contain one or multiple copi es of integrated lambda DNA, which often colocalize with the pSV2neo DNA, u sually in a single chromosomal site as determined by the fluorescent in sit u hybridization technique. In different cell lines, the loci of foreign DNA insertion are different. The inserted bacteriophage lambda DNA frequently becomes de novo methylated. In some of the thus-generated hamster cell line s, the levels of DNA methylation in the retrotransposon genomes of the endo genous intracisternal A particles (IAP) are increased in comparison to thos e in the non-lambda-DNA-transgenic BNK21 cell lines. These changes in the m ethylation patterns of the LAP subclone I (IAPI) segment have been document ed by restriction analyses with methylation-sensitive restriction endonucle ases followed by Southern transfer hybridization and phosphorimager quantit ation. The results of genomic sequencing experiments using the bisulfite pr otocol yielded additional evidence for alterations in the patterns of DNA m ethylation in selected segments of the IAPI sequences. In these experiments , the nucleotide sequences in >330 PCR-generated cloned DNA molecules were determined. Upon prolonged cultivation of cell lines with altered cellular methylation patterns, these differences became less apparent, perhaps due t o counterselection of the transgenic cells. The possibility existed that th e hamster BHK21 cell genomes represent mosaics with respect to DNA methylat ion in the IAPI segment. Hence, some of the cells with the patterns observe d after lambda DNA integration might have existed prior to lambda DNA integ ration and been selected by chance, A total of 66 individual BHK21 cell clo nes from the BHK21 cell stock have been recloned up to three times, and the DNAs of these cell populations have been analyzed for differences in IAPI methylation patterns. None have been found. These patterns are identical am ong the individual BHK21 cell clones and identical to the patterns of the o riginally used BHK21 cell line. Similar results have been obtained with nin e clones isolated from BHK21 cells mock transfected by the Ca2+-phosphate p recipitation procedure with DNA omitted from the transfection mixture, In f our clonal sublines of nontransgenic control BHK21 cells, genomic sequencin g of 335 PCR-generated clones by the bisulfite protocol revealed 5'-CG-3' m ethylation levels in the IAPI segment that were comparable to those in the uncloned BHK21 cell line. We conclude that the observed changes in the DNA methylation patterns in BNK21 cells with integrated lambda DNA are unlikely to preexist or to be caused by the transfection procedure. Our data suppor t the interpretation that the insertion of foreign DNA into a preexisting m ammalian genome can alter the cellular patterns of DNA methylation, perhaps via changes in chromatin structure. The cellular sites affected by and the extent of these changes could depend on the site and size of foreign DNA i nsertion.