The epigenetic status of integrated adenovirus type 12 (Ad12) DNA in hamster cells cultivated for about 4 decades has been investigated. Cell line TR12, a fibroblastic revertant of the Ad12-transformed epitheloid hamster cell line T637 with 15 copies of integrated Ad12 DNA, carries one Ad12 DNA copy plus a 3.9-kbp fragment from a second copy. The cellular insertion site for the Ad12 integrate, identical in both cell lines, is a >5.2-kbp inverted DNA repeat. The Ad12 transgenome is packaged around nucleosomes. The cellular junction is more sensitive to micrococcal nuclease at Ad12-occupied sites than at unoccupied sites. Bisulfite sequencing reveals complete de novo methylation in most of the 1,634 CpGs of the integrated viral DNA, except for its termini. Isolated unmethylated CpGs extend over the entire Ad12 integrate. The fully methylated transgenome segments are characterized by promoter silencing and histone H3 and H4 hypoacetylation. Nevertheless, there is minimal transcriptional activity of the late viral genes controlled by the fully methylated major late promoter of Ad12 DNA.A major part of mammalian genomes is made up of repetitive DNA, and viral retrotransposons comprise a substantial part of it. Little is known about the stability or numerical plasticity of the repetitive sequences. Repetitive DNA in mammalian genomes is heavily methylated (for recent reviews, see references 6, 29, and 47), possibly as a defense against the potential activity of foreign genes in an established genome (5, 50). The degree of methylation, particularly of the repetitive cellular DNA sequences, is subject to alterations in cells with integrated foreign DNA in the genome (13,32). Transgenomes are frequently generated and exploited in experimental and applied molecular biology. Their structure and the effects of their insertion on the stability and functionality of the recipient genome require more detailed studies.In a model system, we have investigated the stability as well as the methylation and transcriptional profiles of integrated adenoviral genomes in hamster cells. The transformed cell line T637 has been generated by infecting baby hamster kidney cell line BHK21 (40) with human adenovirus type 12 (Ad12) (41). The genomes of T637 cells carry about 15 copies of Ad12 DNA integrated at a single chromosomal site (18,35,39). Upon continuous cultivation of cell line T637, morphological revertants, from epitheloid to more fibroblastic, arose spontaneously (10). In one of these revertants, TR12, only one copy and a fragment of a second copy of Ad12 DNA persist stably (7).The genomes of human adenoviruses are always chromosomally integrated and hypermethylated in Ad12-transformed cells and in Ad12-induced hamster tumor cells (26,42,43). Virion DNA or free intracellular adenoviral DNA is not methylated (11,17). The integrated Ad12 genomes in the revertant cell line TR12 are more extensively methylated than those in the parental cell line, T637 (27). Hence, hypermethylation of transgenic DNA might be related to its stability in t...