Spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells

Abstract

The establishment of de novo-generated patterns of DNA methylation is characterized by the gradual spreading of DNA methylation (I. Kuhlmann and W. Doerfler, J. Virol. 47:631-636, 1983; M. Toth, U. Lichtenberg, and W. Doerfler, Proc. Natl. Acad. Sci. USA 86:3728-3732, 1989; M. Toth, U. Müller, and W. Doerfler J. Mol. Biol. 214:673-683, 1990). We have used integrated adenovirus type 12 (Ad12) genomes in hamster tumor cells as a model system to study the mechanism of de novo DNA methylation. Ad12 induces tumors in neonate hamsters, and the viral DNA is integrated into the hamster genome, usually nearly intact and in an orientation that is colinear with that of the virion genome. The integrated Ad12 DNA in the tumor cells is weakly methylated at the 5'-CCGG-3' sequences. These sequences appear to be a reliable indicator for the state of methylation in mammalian DNA. Upon explantation of the tumor cells into culture medium, DNA methylation at 5'-CCGG-3' sequences gradually spreads across the integrated viral genomes with increasing passage numbers of cells in culture. Methylation is reproducibly initiated in the region between 30 and 75 map units on the integrated viral genome and progresses from there in either direction on the genome. Eventually, the genome is strongly methylated, except for the terminal 2 to 5% on either end, which remains hypomethylated. Similar observations have been made with tumor cell lines with different sites of Ad12 DNA integration. In contrast, the levels of DNA methylation do not seem to change after tumor cell explanation in several segments of hamster cell DNA of the unique or repetitive type. Restriction (HpaII) and Southern blot experiments were performed with selected cloned hamster cellular DNA probes. The data suggest that in the integrated foreign DNA, there exist nucleotide sequences or structures or chromatin arrangements that can be preferentially recognized by the system responsible for de novo DNA methylation in mammalian cells.

The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida

“…The previously observed occasional variation in CG methylation, detectable both in flowers and in leaves of line 17, which resulted in a reduction or inactivation of pigmentation (Linn et a/., 1990;Meyer et a/., 1992) also supports the assumption that a highly flexible methylation activity is specifically present within the integrated DNA. Similar observations have been made for integrated adenovirus copies where de-novo methylation within the central part of the transgene has been observed (Orend et a/., 1991) as well as a spread of methylation from the late E2A promoter of adenovirus into an environmental region which had been unmethylated previously (Toth et a/., 1989). A more detailed analysis of methylation variability within integrated DNA is required, however, to determine whether the observed fluctuation within the transgene represents a general mechanism reflecting a specific recognition for foreign or displaced DNA as it has been suggested for mammalian cells (Solter, 1988).…”

Identification of human papillomavirus (HPV) 16 DNA integration and the ensuing patterns of methylation in HPV‐associated head and neck squamous cell carcinoma cell lines

“…The methylation of viral DNA integrated into the human genome has been studied over the past decade 29 and in general it has been found that the local pattern of DNA methylation gradually spreads to involve the integrated viral genome. 30 Our study showed a statistically significant correlation between the average methylation level of the HPV16 DNA and that of the flanking host genome in 3 HPV-HNSCC cell lines. This correlation may be explained by our previous observation, indicating that the HBV genome often became significantly methylated when integrated into highly methylated host sites.…”

The structure of adenovirus type 12 DNA integration sites in the hamster cell genome

“…Ad12-transformed cells and Ad12-induced tumor cells. Origins and propagation of the Ad12-transformed hamster BHK21 cell line T637 (47), its morphological revertants TR1, TR2, TR3, TR4, TR7, TR11, TR12, TR14, and TR16 (9,15), cell line HA12/7 (51), the Ad12-induced tumor T191, and the cell line H191 (33,34) were described elsewhere.…”