Integrated viral genomes
In a hamster cell line transgenomic for about twelve copies of chromosomally integrated human adenovirus type12 (Ad12) DNA, the levels of cellular CpG DNA methylation are altered genome-wide . Similar findings have been reported in hamster cells transgenomic for bacteriophage lambda DNA, although to a lesser extent . Moreover, the transcriptional profiles of cellular DNA in hamster cells transgenomic for Ad12 DNA or lambda DNA have changed . In a revertant of one of the Ad12 transformed cell lines, which has lost all of the Ad12 transgenomes, the increase of genome-wide DNA methylation in intracisternal A particle (transposon) DNA persisted even in the absence of the transgenomic Ad12 DNA. Hence, in this instance, altered DNA methylation profiles proved inheritable and were likely initiated by a hit-and-run mechanism. The guard for the stability of the methylation profile had been called upon by Ad12 DNA integration, remained alerted after the loss of foreign DNA, and was stably transmitted to the next cell generations [5, 8].
Small bacterial plasmid
A different experimental scenario was devised to challenge the stability of cellular transcriptional and DNA CpG methylation profiles in individual cell clones of human colon tumor (HCT116) cells upon the insertion of a 5.6 kbp bacterial plasmid. Single-cell clones were generated which contained either no transgenome or the 5.6 kbp plasmid DNA which carried the kanamycin resistance gene for clonal selection as sole transcribed element. In the subsequently expanded cell clones, differences in transcriptional and CpG methylation patterns between transgenomic and non-transgenomic cell clones were determined. Transcriptional and CpG methylation profiles of all transcripts investigated in five clonal cell populations of non-transgenomic controls were almost identical. This finding facilitated comparisons of the differential gene segment expression between non-transgenomic and transgenomic cell clones in 28,869 genomic DNA segments: 1343 showed differential expression with 907 regions up- and 436 regions downregulated . Thus, the integration of the 5.6 kbp bacterial plasmid into the human genome altered transcription profiles in 4.7% of the investigated gene segments. The functional details about the DNA segments differentially transcribed and further information on experimental procedures have been published earlier .
Furthermore, when CpG DNA methylation profiles in the same sets of non-transgenomic versus 5.6 kbp plasmid-transgenomic HCT116 cell clones were assessed in 361,983 CpG dinucleotide positions, 3791 CpGs were found differentially methylated in the transgenomic cell clones, and 1504 proved hyper- and 2287 hypo-methylated. Hence, both transcription and CpG methylation profiles were altered in human cells by genomically inserting a 5.6 kbp DNA plasmid . All the data in this experimental setting were statistically verified by Benjamini-Hochberg corrections [11, 12] to exclude false-positive values.
Episomal persistence of EBV genomes
In a different biological system a similar observation was made on changes in DNA methylation patterns in cells which carried foreign DNA. One of the most frequently occurring forms of mental retardation in humans is the Fragile X Syndrome (FXS) . It is molecularly characterized by the expansion of a naturally occurring CGG repeat in the untranslated first exon of the human fragile X mental retardation gene 1 (FMR1) and the CpG hyper-methylation in its promoter region. In the region upstream of this promoter, we have identified a distinct boundary between unmethylated genome segments, as they exist in unaffected individuals, and DNA sequences further upstream from the promoter which are strongly CpG methylated. In FXS individuals, the methylation boundary is obliterated; the promoter becomes CpG hyper-methylated and inactivated. As a consequence, the FMR1 gene product fails to be produced; its lack during an individual’s development leads to the FXS syndrome. The methylation boundary is thought to exhibit a specific chromatin structure which demarcates a hyper-methylated upstream sequence from the unmethylated downstream FMR1 promoter. The boundary sequence, as it were, protects the promoter from the spreading of DNA methylation and thus helps maintain its activity .
In normal human PBMCs, the far upstream genome region beyond the boundary is hyper-methylated. In PBMCs immortalized by transformation with EBV or with the human telomerase gene, the boundary is maintained. However, the region far upstream from the boundary becomes hypo-methylated by a factor of 4  in PBMCs both from non-FXS and from FXS individuals. These specific alterations in methylation levels likely are caused by the episomally persisting foreign EBV DNA genome or the integrated telomerase gene [8, 15]. Destabilization of cellular methylation patterns has also been reported in human lymphoblastoid cell lines after transformation by EBV .
Although many questions remain as to mechanisms involved and the generality of alterations in transcriptional and CpG DNA methylation profiles, the ensemble of data presented raises the question of to what extent the introduction of foreign DNA into mammalian cells destabilizes the epigenetic landscapes in mammalian cells. By the same token, due to the mentioned epigenetic sequelae of foreign DNA intrusion, functionally completely new cell types arise and likely impact on the mechanisms causing common diseases. The stochastic nature of disease incidence is paralleled by the stochasticity of the molecular events linked to foreign DNA intrusion and intranuclear fixation.