Insertion of foreign DNA into an established mammalian genome can alter the methylation of cellular DNA sequences

Abstract

The insertion of adenovirus type 12 (Ad12) DNA into the hamster genome and the transformation of these cells by Ad12 can lead to marked alterations in the levels of DNA methylation in several cellular genes and DNA segments. Since such alterations in DNA methylation patterns are likely to affect the transcription patterns of cellular genes, it is conceivable that these changes have played a role in the generation or the maintenance of the Ad12-transformed phenotype. We have now isolated clonal BHK21 hamster cell lines that carry in their genomes bacteriophage lambda and plasmid pSV2neo DNAs in an integrated state. Most of these cell lines contain one or multiple copies of integrated lambda DNA, which often colocalize with the pSV2neo DNA, usually in a single chromosomal site as determined by the fluorescent in situ hybridization technique. In different cell lines, the loci of foreign DNA insertion are different. The inserted bacteriophage lambda DNA frequently becomes de novo methylated. In some of the thus-generated hamster cell lines, the levels of DNA methylation in the retrotransposon genomes of the endogenous intracisternal A particles (IAP) are increased in comparison to those in the non-lambda-DNA-transgenic BHK21 cell lines. These changes in the methylation patterns of the IAP subclone I (IAPI) segment have been documented by restriction analyses with methylation-sensitive restriction endonucleases followed by Southern transfer hybridization and phosphorimager quantitation. The results of genomic sequencing experiments using the bisulfite protocol yielded additional evidence for alterations in the patterns of DNA methylation in selected segments of the IAPI sequences. In these experiments, the nucleotide sequences in >330 PCR-generated cloned DNA molecules were determined. Upon prolonged cultivation of cell lines with altered cellular methylation patterns, these differences became less apparent, perhaps due to counterselection of the transgenic cells. The possibility existed that the hamster BHK21 cell genomes represent mosaics with respect to DNA methylation in the IAPI segment. Hence, some of the cells with the patterns observed after lambda DNA integration might have existed prior to lambda DNA integration and been selected by chance. A total of 66 individual BHK21 cell clones from the BHK21 cell stock have been recloned up to three times, and the DNAs of these cell populations have been analyzed for differences in IAPI methylation patterns. None have been found. These patterns are identical among the individual BHK21 cell clones and identical to the patterns of the originally used BHK21 cell line. Similar results have been obtained with nine clones isolated from BHK21 cells mock transfected by the Ca2+-phosphate precipitation procedure with DNA omitted from the transfection mixture. In four clonal sublines of nontransgenic control BHK21 cells, genomic sequencing of 335 PCR-generated clones by the bisulfite protocol revealed 5'-CG-3' methylation levels in the IAPI segment that were comparable to those in the uncloned BHK21 cell line. We conclude that the observed changes in the DNA methylation patterns in BHK21 cells with integrated lambda DNA are unlikely to preexist or to be caused by the transfection procedure. Our data support the interpretation that the insertion of foreign DNA into a preexisting mammalian genome can alter the cellular patterns of DNA methylation, perhaps via changes in chromatin structure. The cellular sites affected by and the extent of these changes could depend on the site and size of foreign DNA insertion.

FIG. 1

Clonal cell lines of BHK21 cells that carry integrated λ DNA and pSV2neo DNA. (a) Integration patterns of λ DNA in some of the clonal, exemplarily selected λ DNA-transgenic BHK21 cell lines. The DNA extracted from BHK21-λ clones as indicated was cleaved with EcoRI (E) or PstI (P), and the fragments were separated by electrophoresis on a 0.6 or 0.8% agarose gel. The DNA was then transferred by Southern blotting to a Qiagen Nylon-Plus membrane, and the λ DNA-specific fragments were visualized by hybridization to ³²P-labeled λ DNA followed by autoradiography. As size and quantity markers, λ DNA cut with EcoRI or PstI was coelectrophoresed. Amounts of 1, 5, or 10 genome equivalents (ge) of λ DNA were used. (b) Integration patterns of the pSV2neo plasmid used in cotransfection experiments. Experimental conditions were identical to those described for panel a except that ³²P-labeled pSV2neo DNA was used as the hybridization probe. The same Qiagen Nylon-Plus filter as shown in panel a was used in these experiments, after removing the λ DNA probe by boiling in 0.1% sodium dodecyl sulfate–0.1 × SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The BHK21-λ18 and -λ18* clones are two distinct λ DNA-carrying BHK21 cell clones.

FIG. 2

FISH analyses of the chromosomal locations of the integrated λ genomes and the integrated pSV2neo plasmids in a series of BHK21 cell lines rendered transgenic for λ DNA and pSV2neo DNA. Experimental details are described in the text. (a) Control BHK21 cell devoid of foreign DNA; (b) BHK21-λ7; (c) BHK21-λ17; (d) BHK21-λ18*; (e and f) BHK21-λ15. (a and f) Biotinylated λ DNA alone was used as hybridization probe; (b to e) a mixture of biotinylated λ and pSV2neo DNAs was used for hybridization. The finding of a single signal demonstrated that both transgene DNAs were located at one chromosomal site, which was different for each cell line.

FIG. 3

De novo methylation of the integrated λ DNA (a) or pSV2neo DNA (b) in several clonal BHK21 cell lines transgenic for λ and pSV2neo DNAs. The DNAs from the λ and pSV2neo DNA-transgenic cell lines were isolated and cleaved with HpaII (H), HhaI (Hh), or MspI (M). Subsequently, the DNA fragments were separated by electrophoresis in 1.0% agarose gels and analyzed as described in the legend to Fig. 1. ³²P-labeled λ DNA (a) or ³²P-labeled pSV2neo DNA (b) was used as the hybridization probe.

FIG. 4

BHK21 cells transgenic for bacteriophage λ DNA do not detectably transcribe this DNA. An autoradiogram of an RNA (Northern) blotting experiment in which 30 μg of total RNA was electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde is shown. Upon transfer of the RNA to a Qiagen Nylon-Plus membrane, the RNA was hybridized to ³²P-labeled λ DNA (a) or to the ³²P-labeled pBluescript-cloned serine proteinase gene from Syrian hamster (b). In this autoradiogram, RNA samples from only eight of the BHK21 cell lines transgenic for λ DNA were analyzed. When ³²P-labeled pSV2neo DNA was used as the hybridization probe, results similar to those in panel a were obtained (not shown).

FIG. 5

Increases in DNA methylation in the 5′-CCGG-3′ (HpaII [H]) and 5′-GCGC-3′ (HhaI [Hh]) sequences in the IAPI segments of five cloned BHK21 cell lines with integrated λ DNA in comparison to DNA from the non-λ-DNA-transgenic BHK21 cell lines. Experimental procedures were similar to those described in the legend to Fig. 3, except that ³²P-labeled IAPI DNA was used as the hybridization probe. The data for the BHK21-λ7 clone have been shown previously (15). The results of phosphorimager analyses are presented in Table 1.

FIG. 6

Methylation analyses of the IAPI segments in the BHK21 and T637 cell lines and in the λ DNA-transgenic clonal BHK21 cell lines BHK21-λ7 and BHK21-λ10 by using the bisulfite protocol of the genomic sequencing procedure. A map of subclone I in the IAP retrotransposon in hamster cells (25) is shown. The locations of the primers used in the amplification step of the genomic sequencing procedure following the bisulfite reaction with clonal DNAs are designated with horizontal arrows. Several segments were genomically sequenced. The data shown in Tables 2 and 3 were derived from the primer p3–p4-flanked subsegment of the IAPI region, which contains 28 5′-CG-3′ dinucleotide sequences (vertical lines), as published previously (25). For experimental details see the text and the footnotes to Tables 2, 3, and 4. HpaII (stars) and HhaI (circles) sites are indicated. Numbers refer to the published nucleotide sequence (25).

FIG. 7

Alterations in DNA methylation at 28 5′-CG-3′ sites in the p3–p4 segment (Fig. 6) of the IAPI retrotransposon sequence in the Ad12-transformed hamster cell line T637 and in two λ DNA-transgenic BHK21 hamster cell lines, BHK21-λ7 and -λ10. Experimental details are described in the text. The numbering of the 5′-CG-3′ dinucleotides corresponds to that in Fig. 6. During the genomic sequencing experiments, 123 DNA clones from the reference BHK21 cell line, 79 DNA clones from the Ad12-transformed T637 cell line, 58 DNA clones from the BHK21-λ7 cell line, and 73 DNA clones from the BHK21-λ10 cell line were sequenced. The percentage values represent the average of methylated 5′-CG-3′ dinucleotides at each site for DNA from each of the cell lines. In many of the clones, 5′-CG-3′ positions 23 to 27 were in a deleted segment as part of a naturally occurring polymorphism in this region compared to the published nucleotide sequence (25). Furthermore, 5′-CG-3′ positions 19 and 35 were altered to 5′-TG-3′ in many clones and were therefore omitted from this analysis.

FIG. 8

The IAPI segment methylation patterns obtained upon HpaII (5′-CCGG-3′) cleavage were identical in 66 nontransfected and 9 mock-transfected individual BHK21 cell clones and did not differ from those in DNA preparations from unselected, uncloned BHK21 cells. As examples, the IAPI DNA hybridization results with HpaII (H)-cleaved DNAs from unselected BHK21 cells and from 16 individual BHK21 cell clones are shown. M, MspI control pattern.