The unmethylated state of the promoter/leader and 5'-regions of integrated adenovirus genes correlates with gene expression.

An inverse correlation has been established between the levels of DNA methylation at 5'-CCGG-3' (MspI/HpaII) sites in specific genes of integrated viral DNA in adenovirus type 12 (Ad12)-transformed hamster cell lines and the extent to which these genes are expressed ( Sutter and Doerfler , 1979, 1980). In general, early genes are transcribed into mRNA, while late genes are permanently switched off in these cell lines. Adenovirus type 2 genes methylated in vitro at 5'-CCGG-3' sites are not transcribed upon microinjection into nuclei of Xenopus laevis oocytes - unmethylated genes are expressed ( Vardimon et al., 1982a ). The MspI sites in the early and in some of the late Ad12 genes in cell lines HA12 /7, T637 , and A2497 -3 have now been precisely mapped. The data presented here reveal that the promoter/leader and 5'-regions of the early genes are unmethylated both at MspI sites and at 5'-GCGC-3' (HhaI) sites. In some instances, e.g., in the E2a regions in all three lines, the main parts of the early genes are partly methylated, even though the genes are expressed. In cell line HA12 /7, the early region E3 is not expressed, and the promoter/leader and 5'-regions of this segment are fully methylated. All late regions are completely methylated. The results suggest that the state of methylation in the promoter/leader and 5'-regions of integrated adenovirus genes is important in the control of gene expression.     

Integrated adenovirus type 12 DNA in the transformed hamster cell line T637: sequence arrangements at the termini of viral DNA and mode of amplification.

Approximately 20 to 22 copies of adenovirus type 12 (Ad12) DNA per cell were integrated into the genome of the cell line T637. Only a few of these copies seemed to remain intact and colinear with virion DNA. All other persisting viral genomes exhibited deletions or inversions or both in the right-hand part of Ad12 DNA. Spontaneously arising morphological revertants of T637 cells has lost viral DNA. In most of the revertant cell lines only the intact or a part of the intact viral genome was preserved; other revertant cell lines has lost all viral DNA. In three other Ad12-transformed hamster cell lines, HA12/7, A2497-3, and CLAC3 (Stabel et al., J. Virol. 36:22-40, 1980), major rearrangements at the right end of the integrated Ad12 DNA were not found. These studies were performed to investigate the phenomena of amplification, rearrangements, and deletions of Ad12 DNA in hamster cells.     

Integration Sites of Adenovirus Type 12 DNA in Transformed Hamster Cells and Hamster Tumor Cells

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 one or a few viral DNA molecules were amplified upon integration together with the adjacent cellular DNA sequences, leading to a serial arrangement of viral DNA molecules separated by cellular DNA sequences. Likewise, in the Ad12-induced hamster tumor lines (CLAC1 and CLAC3), viral DNA was linked to repetitive cellular sequences. Serial arrangement of Ad12 DNA molecules in these lines was not likely. (iv) In general, true tandem integration with integrated viral DNA molecules directly abutting each other was not found. Instead, the data suggested that the integrated viral DNA molecules were separated by cellular or rearranged viral DNA sequences. (v) The results of hybridization experiments, in which a highly specific probe (143-base pair DNA fragment) derived from the termini of Ad12 DNA was used, were not consistent with models of integration involving true tandem integration of Ad12 DNA or covalent circularization of Ad12 DNA before insertion into the cellular genome. (vi) Evidence was presented that a small segment at the termini of the integrated Ad12 DNA in cell lines HA12/7, T637, and A2497-3 was repeated several times. The exact structures of these repeat units remained to be determined. The occurrence of these units might reflect the mechanism of amplification of viral and cellular sequences in transformed cell lines.     

Excision of amplified viral DNA at palindromic sequences from the adenovirus type 12-transformed hamster cell line T637.

In the DNA of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 approximately 20-22 viral DNA molecules per cell are covalently linked to cellular DNA. Spontaneously arising morphological revertants of T637 cells have lost the bulk of the viral DNA. We have been able to mimic the excision event of viral DNA, as it occurs during reversion, by autoincubation of isolated nuclei from T637 cells. The same Ad12 DNA sequences, which had been deleted in morphological revertants, proved highly sensitive to endogenous nucleases in isolated nuclei of T637 cells. Viral DNA sequences, which persisted in the revertants, are resistant to endogenous nucleases in isolated T637 nuclei. All attempts to clone the nuclease-sensitive sites of Ad12 DNA in cell line T637 have so far failed. After denaturation and renaturation of T637 DNA followed by treatment with S1 nuclease, large fold-back structures of DNA have been found. These snap-back structures were derived from precisely those viral DNA restriction fragments which were uncloneable. The fragments containing palindromic sequences were both highly sensitive to endogenous nucleases in isolated T637 nuclei and were absent from the DNA of all revertant cell lines. Moreover, the palindromic sequences are susceptible to the phage T4-specific endonuclease VII which specifically attacks cruciform structures in DNA. The peculiar structures at the termini of integrated Ad12 DNA molecules are highly sensitive to endogenous nucleases in isolated nuclei. These nucleases may be related to the reversion event.     

DNA methylation and viral gene expression in adenovirus-transformed and -infected cells.

The level of DNA methylation in adenovirus type 2 (Ad2) and type 12 (Ad12) DNA was determined by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI. As previously reported virion DNA of Ad2 and Ad12 is not methylated. Parental or newly synthesized Ad2 DNA in productively infected human KB or HEK cells is not methylated either, nor is the integrated form of Ad2 DNA in productively infected cells. Hamster cells and Muntiacus muntjak cells are abortively infected by Ad12. We have not detected methylation of Ad12 DNA in hamster or Muntiacus muntjak cells. An inverse correlation between the level of methylation and the extent of expression of viral DNA in Ad12-transformed hamster cells has been described earlier. A similar relation has been found for the EcoRI fragment B of Ad2 DNA which is not methylated but is expressed as the Ad2 DNA-binding (72K) protein in the Ad2-transformed hamster line HE1. Conversely, the same segment is completely methylated in lines HE2 and HE3, and there is apparently no evidence for the expression of the 72K protein in these cell lines.     

Isolation and characterization of four adenovirus type 12-transformed human embryo kidney cell lines.

Four transformed cell lines were established from cultures of human embryo kidney (HEK) cells microinjected or transfected with cloned adenovirus 12 (Ad12) EcoRI-C DNA (0 through 16.5 map units of the left-hand end of the viral genome). Each cell line showed a different growth pattern. Southern blotting demonstrated that all of the cell lines contained Ad12-specific DNA sequences, but in the microinjected isolates these were at a much lower copy number than in the transfected isolate. Two cell lines (Ad12 HEK 1 and 3) appeared to contain tandemly repeated Ad12 EcoRI-C DNA fragments. Immunoprecipitation and Western blotting confirmed that Ad12 early region 1 (E1) proteins were being expressed by all four of the transformed cell lines, but indicated that E1A polypeptide expression was considerably less than E1B polypeptide expression. All of the Ad12-transformed HEK cell lines were tumorigenic when inoculated intracranially into athymic nude mice.     

Patterns of integration of viral dna sequences in the genomes of adenovirus type 12-transformed hamster cells

The patterns of integration of the viral genome have been analyzed in four hamster cell lines transformed by adenovirus type 12 (Ad12). It has previously been shown that in each of the cell lines HA12/7, T637, A2497-2 and A2497-3, the viral genome persists in multiple copies, and that different parts of the viral DNA are represented non-stoichiometrically (Fanning and Doerfler, 1976). All four cell lines are oncogenic when injected into hamsters.The DNA from each of the cell lines was extracted and cleaved in different experiments with restriction endonucleases Bam HI, Bgl II, Eco RI, Hind III, Hpa II or Sma I. The DNA fragments were separated on 1% agarose slab gels and transferred to nitrocellulose filters by the Southern technique. Ad12 DNA sequences were detected by hybridization to Ad12 DNA, which was ³²P-labeled by nick translation, and by subsequent autoradiography. In some experiments, the ³²P-labeled Eco RI restriction endonuclease fragments of Ad12 DNA were used to investigate the distribution of specific segments of the viral genome in the cellular DNA.For each cell line, a distinct and specific pattern of integrated viral DNA sequences is observed for each of the restriction endonucleases used. Moreover, viral sequences complementary to the isolated Eco RI restriction endonuclease fragments are also distributed in patterns specific for each cell line. There are striking differences in integration patterns among the four different lines; there are also similarities. Because the organization of cellular genes in virus-transformed as compared to normal cells has not yet been determined, conclusions about the existence or absence of specific integration sites for adenovirus DNA appear premature. Analysis of the integration patterns of Ad12 DNA in the four hamster lines investigated reveals that some of the viral DNA molecules are fragmented prior to or during integration. Analysis with specific restriction endonuclease fragments demonstrates that the Eco RI B, D and E fragments, comprising a contiguous segment from 0.17–0.62 fractional length units of the viral DNA, remain intact during integration in a portion of the viral DNA molecules. Although each cell line carries multiple copies of Ad12 DNA, the viral DNA sequences are concentrated in a small number of distinct size classes of fragments. This finding is compatible with, but does not prove, the notion that at least a portion of the viral DNA sequences is integrated into repetitive sequences, or else that the integrated viral sequences have been amplified after integration.In the three cell lines which were tested, the integration pattern is stable over many generations, with continuous passage-twice weekly-of cells for 6–7 months. In the three cell lines which were examined, the integration pattern is identical in a number of randomly isolated clones. Hence it can be concluded that the patterns of integration are identical among all cells in a population of a given line of transformed cells.     

Revertants of Adenovirus Type 12-Transformed Hamster Cell Line T637 as Tools in the Analysis of Integration Patterns

Spontaneously arising morphological revertants of the adenovirus type 12 (Ad12)-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 Ad12 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 Ad12 DNA and was integrated at the same site as in line T637. In revertant lines G12, TR11, and TR12, one Ad12 DNA and varying parts of a second viral DNA molecule persisted in the host genome. These patterns of persistence of Ad12 DNA molecules in different revertants supported a model for a mode of integration of Ad12 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 Ad12-specific DNA sequences in the revertant lines TR7 and TR12. Moreover, Ad12 DNA sequences which were found to be undermethylated in line T637 were completely methylated in the revertant cell lines G12, TR11, 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 Ad12 DNA were expressed and undermethylated; conversely, in the revertant lines G12, TR11, TR12, and TR2, the same segments appeared to be expressed to a limited extent and were strongly methylated.     

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5'' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.     

Chromatin structure of active and inactive human X chromosomes

Nuclei from a variety of human cell lines and tissues were digested with gradually increasing levels of DNase I. The DNA was then purified, treated with restriction enzymes and subjected to Southern blot hybridization using a cloned cDNA probe to 3-phosphoglycerate kinase (PGK) a housekeeping enzyme. At relatively high levels of DNase I, a specific, slightly sensitive site in chromatin sequences encoding PGK was observed in all of the cell types examined. This slightly sensitive site resides on the active X-chromosome since cell lines with increased numbers of inactive X-chromosomes do not show an increase in the region of chromatin which is sensitive. Except for this restricted region of enhanced sensitivity on the active X-chromosome, the data suggest that, for PGK encoding sequences, chromatin configurations on the active and inactive X-chromosomes are similar.     

Selective sites of adenovirus (foreign) DNA integration into the hamster genome: changes in integration patterns.

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, which represented the sites of linkage between Ad12 and cellular DNAs, were determined. At early passage levels in culture, the integration sites of Ad12 DNA in the hamster genome, as characterized by the positions of off-size fragments in agarose or polyacrylamide gel electrophoresis, were different in the five different tumor cell lines. Upon repeated passage, however, the off-size fragment patterns generated by the five restriction endonucleases became very similar in the five tumor cell lines. This surprising result indicates that under cell culture conditions, Ad12-transformed tumor cell lines that carry the foreign (Ad12) genome in selective, probably very similar sites of the cellular genome evolve.     

Methylation state and DNase I sensitivity of chromatin containing Moloney murine leukemia virus DNA in exogenously infected mouse cells

The nature of Moloney murine leukemia virus (M-MuLV)-specific proviral DNA in exogenously infected mouse cells was studied. M-MuLV clone A9 cells, NIH-3T3 fibroblasts productively infected with M-MuLV, were used. These cells contain 10 to 15 copies of M-MuLV proviral DNA. The state of methylation of M-MuLV proviral DNA was examined by cleaving A9 cell DNA with restriction endonucleases which have the dinucleotide CpG in their cleavage sequences. Analysis with such enzymes, which recognized nine different sites in M-MuLV DNA, indicated that most if not all of the M-MuLV proviruses in A9 cells were completely unmethylated. An individual proviral integration was examined, using as probe adjacent single-copy cellular sequences. These sequences were obtained from a lambda phage recombinant clone containing an M-MuLV provirus from the A9 cells. This individual integration also showed no detectable methylation. In contrast, endogenous MuLV-related sequences present in NIH-3T3 cells before infection were largely methylated. The configuration chromatin containing M-MuLV proviruses was also investigated by digesting A9 nuclei with DNase I, followed by restriction analysis of the remaining DNA. Endogenous MuLV-related DNA was in chromatin relatively resistant to DNase I digestion, whereas the majority of M-MuLV-specific proviruses were in domains of intermediate DNase I sensitivity. Two proviral copies hypersensitive to DNase I digestion were identified. Analogy to the DNase I sensitivity of expressed and nonexpressed globin genes suggested that the proviral copies containing DNase I-hypersensitive sites were transcribed.     

Adenovirus E1A-mediated regulation of class I MHC expression.

Expression of class I MHC transplantation antigens has been shown to be reduced in baby rat kidney (BRK) cells transformed by highly oncogenic adenovirus type 12 (Ad12), as compared with untransformed cells and cells transformed by non-oncogenic Ad5. Here we show that this reduction of class I expression also occurs in a variety of other primary cell cultures transformed by Ad12, and that reduction of class I gene expression occurs for all class I loci. Transfection of Ad5E1 into class I-negative Ad12-transformed BRK cells leads to complete restoration of class I expression. Introduction of Ad12E1 into most class I-positive established cell lines does not result in suppression of class I expression. However, transfection of the Ad12E1A region into a class I-positive cell line which was immortalized by a mutant Ad12E1A region resulted in suppression of class I gene expression, implying that the suppression of class I activity in Ad12-transformed cells is due to an active switching-off process.     

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

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.