The initiation of de novo methylation of foreign DNA integrated into a mammalian genome is not exclusively targeted by nucleotide sequence (original) (raw)
Related papers
Journal of Virology, 1991
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 gradual...
Expression of a cloned adenovirus gene is inhibited by in vitro methylation
Proceedings of the National Academy of Sciences, 1982
In many viral and nonviral eukaryotic systems, an inverse correlation has been observed between the extent of DNA methylation at 5'-C-C-G-G-3' sites and the extent of expression of specific genes as mRNA. The E2a region of adenovirus serotype 2 (Ad2) DNA encodes the Ad2-specific DNA binding protein required for viral DNA replication. In three lines of Ad2 transformed hamster cells (HE1, HE2, and HE3), multiple copies of the major part of the Ad2 genome persist in an integrated state. Cell lines HE2 and HE3 do not express the DNA-binding protein whereas line HE1 does so. It has been shown that, in cell line HE1, all 5'-C-C-G-G-3' (Hpa II/MspI) sites in the E2a region remain unmethylated. Conversely, in lines HE2 and HE3 lacking expression of the E2a region all Hpa II sites are methylated. The cloned E2a region of Ad2 DNA, the HindIII A fragment in pBR322, was methylated in vitro by using Hpa II DNA methyltransferase (5'-C-C*G-G-3') or was left unmethylated. In...
Epigenetic Status of an Adenovirus Type 12 Transgenome upon Long-Term Cultivation in Hamster Cells
Journal of Virology, 2007
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 transcripti...
Biochimica Et Biophysica Acta Gene Structure and Expression, 1989
Integrated adenovims type 12 (Adl2} genes in Adl2-transformed cell lines were investigated for chromatin structure, expression levels and states of DNA methylation. The E3 region in the Adl2-transformed cell line HAl2/7 is hypemtethylated and not expressed. The same region in the Adl2-tra~Mormed hamster cell lines T637 and A2497.3 is ~bed and undormethylated (Kruczek, !. and Dnerfler, W. (1982) EMBO J. 1, 409--414). There was no significant difference In the DNase I sensitivity of the E3 region when nuclei of the aforementioned cell lines were incubated with this nuclesse. In contrast, incubation of these nuclei with the restriction endo~uclease Pstl and subsequent cleavage of the DNA with BamHl generated an additional 0.9 kbp fragment in T637 and A2497-3 DNA which was not observed after treating HAI2/7 nuclei and DNA in the same way. This finding was interpreted as indicative of differences in the chromatin structure of the E3 region depending on its state of transcriptional activity and its level of methyintion. The E1 and major late promoter regions, which were transcriptionally active and inactive, respectively, in all three cell lines investigated, did not exhibit differences in sensitivity towards DNase I or Pat| treatment of nuclei. More refined technology will be required to compare the chromatin structure of active venus inactive genes.
Journal of Virology, 1994
We investigated whether, upon the integration of multiple copies of adenovirus type 12 (Ad12) DNA into an established mammalian (hamster) genome, the pattern of foreign DNA insertion would remain stable or change with consecutive passages of cells in culture. By the injection of purified Ad12 into newborn hamsters, tumors were induced, cells from these tumors were cultivated, and five independent cell lines, HT5, H201/2, H201/3, H271, and H281, were established. These cell lines carried different copy numbers of Ad12 DNA per cell in an integrated form and differed in morphology. Cell line HT5 had been passed twice through hamsters as tumor cells and was subsequently passaged in culture. Patterns of Ad12 DNA integration were determined by restriction cleavage of the nuclear DNA with BamHI, EcoRI, HindIII, MspI, or PstI followed by Southern blot hybridization using 32P-labeled Ad12 DNA or its cloned terminal DNA fragments as hybridization probes. In this way, the off-size fragments, w...
Integration sites of adenovirus type 12 DNA in transformed hamster cells and hamster tumor cells
Journal of virology, 1980
The patterns and sites of integration of adenovirus type 12 (Ad12) DNA were determined in three lines of Ad12-transformed hamster cells and in two lines of Ad12-induced hamster tumor cells. The results of a detailed analysis can be summarized as follows. (i) All cell lines investigated contained multiple copies (3 to 22 genome equivalents per cell in different lines) of the entire Ad12 genome. In addition, fragments of Ad12 DNA also persisted separately in non-stoichiometric amounts. (ii) All Ad12 DNA copies were integrated into cellular DNA. Free viral DNA molecules did not occur. The terminal regions of Ad12 DNA were linked to cellular DNA. The internal parts of the integrated viral genomes, and perhaps the entire viral genome, remained colinear with virion DNA. (iii) Except for line HA12/7, there were fewer sites of integration than Ad12 DNA molecules persisting. This finding suggested either that viral DNA was integrated at identical sites in repetitive DNA or, more likely, that...
Virus Research, 1999
In adenovirus type 12 (Ad12)-induced tumor cells, in Ad12-transformed cells and in continuously passaged cell lines from these sources, the viral DNA is integrated in multiple copies, usually at a single chromosomal location. In different tumors or cell lines, the sites of integration of Ad12 DNA are all different. Rare exceptions exist. In most instances, the integrated viral DNA resides very stably in the host cell genomes. However, upon continuous serial passage of such cell lines, the integrated viral DNA can be destabilized and lost. In two instances, i.e. in the Ad12-induced hamster tumor cell lines H1111(1) and CLAC1, we have investigated the loss of integrated viral DNA in detail. After extended serial passage, these two cell lines seemed to be devoid of Ad12 DNA sequences, as detectable by Southern blot hybridization, but continued to induce tumors after reinjection into hamsters. Cells from these two cell lines were now recloned three times, and DNAs from cultures derived from several individual clones were reinvestigated for the presence of several parts of the viral genome by the polymerase chain reaction (PCR). Some of the clones still carried parts of the Ad12 genome. However, several clones were isolated that proved free of all parts of the viral genome, except for minute segments from the right terminus of the Ad12 genome. Apparently, the loss of integrated viral DNA from these cell lines proceeded as a continuous, gradual, multistep process whose pattern could differ from cell clone to cell clone, once destabilization had been initiated. The mechanism of destabilization is not understood. Cell populations of 2 × 10 6 to 3× 10 7 , and as low as 10 2 , cells from the clones, that contained only minimal remnants from the right viral DNA terminus, were reinjected into newborn or 13 -20 day-old weanling Syrian hamsters (Mesocricetus auratus). Tumors developed within 5 -17 days after injection. Tumor cell clones also grew in soft agar. The injection of primary hamster skin fibroblasts never elicited tumor formation. The : S 0 1 6 8 -1 7 0 2 ( 9 8 ) 0 0 1 3 1 -2 A. Pfeffer et al. / Virus Research 59 (1999) 113-127 114 tumor cells induced by this reinjection proved repeatedly free of Ad12 DNA both by Southern blot hybridization and by PCR, except for those cell and tumor clones that contained small segments of the right terminal E4 region of the Ad12 genome. The tumor cells, however, retained their oncogenic phenotype. The results raise questions about the cell clone-specific excision patterns of integrated foreign DNA from the recipient genome and the possibility of a hit-and-run mechanism of adenoviral oncogenesis.
Molecular and cellular biology, 1983
The transforming activity of cloned Moloney sarcoma virus (MSV) proviral DNA was inhibited by in vitro methylation of the DNA at cytosine residues, using HpaII and HhaI methylases before transfection into NIH 3T3 cells. The inhibition of transforming activity due to HpaII methylation was reversed by treatment of the transfected cells with 5-azacytidine, a specific inhibitor of methylation. Analysis of the genomic DNA from the transformed cells which resulted from the transfection of methylated MSV DNA revealed that the integrated MSV proviral DNA was sensitive to HpaII digestion in all cell lines examined, suggesting that loss of methyl groups was necessary for transformation. When cells were infected with Moloney murine leukemia virus at various times after transfection with methylated MSV DNA, the amount of transforming virus produced indicated that the loss of methyl groups occurred within 24 h. Methylation of MSV DNA at HhaI sites was as inhibitory to transforming activity as me...
Revertants of adenovirus type-12-transformed hamster cells have lost part of the viral genomes
International Journal of Cancer, 1979
Spontaneously arising morphological revertants of the adenovirus type 12 (Adl2)-transformed hamster cell line T637 had been previously isolated, and it had been demonstrated that in these revertants varying amounts of the integrated Ad12 genome were eliminated from the host genome. In this report, the patterns of persistence of the viral genome in the revertants were analyzed in detail. In some of the revertant cell lines, F10, TR3, and TR7, all copies of Adl2 DNA integrated in line T637 were lost. In lines TR1, -2, -4 to -6, -8 to -10, and -13 to -16, only the right-hand portion of one Ad12 genome was preserved; it consisted of the intact right segment of Adl2 DNA and was integrated at the same site as in line T637. In revertant lines G12, TR1l, and TR12, one Ad12 DNA and varying parts of a second viral DNA molecule persisted in the host genome. These patterns of persistence of Adl2 DNA molecules in different revertants supported a model for a mode of integration of Adl2 DNA in T637 hamster cells in which multiple (20 to 22) copies of the entire Ad12 DNA were serially arranged, separated from each other by stretches of cellular DNA. The occurrence of such revertants demonstrated that foreign DNA sequences could not only be acquired but could also be lost from eucaryotic genomes. There was very little, if any, expression of Adl2-specific DNA sequences in the revertant lines TR7 and TR12. Moreover, Adl2 DNA sequences which were found to be undermethylated in line T637 were completely methylated in the revertant cell lines G12, TR1l, TR12, and TR2. These findings were consistent with the absence of T antigen from the revertant lines reported earlier. Hence it was conceivable that the expression of integrated viral DNA sequences was somehow dependent on their positions in the cellular genome. In cell line TR637, the early segments of Adl2 DNA were expressed and undermethylated; conversely, in the revertant lines G12, TRll, TR12, and TR2, the same segments appeared to be expressed to a limited extent and were strongly methylated.