5-azacytidine induces transgene silencing by DNA methylation in Chinese hamster cells

Abstract

The cytosine analog 5-azacytidine (5-AzaC) is a demethylating agent that is also known to induce mutagenesis in mammalian cells. In this study, the mutagenic potential of this drug was tested in the G10 and G12 transgenic Chinese hamster cell lines, which have a single bacterial gpt gene integrated into the genome at different sites, with its expression driven by a simian virus 40 (SV40) promoter. We show that the mutation frequencies following a 48-h exposure to different concentrations of 5-AzaC were 10 to 20 times higher than those of any of the other numerous mutagens that have been tested in the G10-G12 system. Moreover, the mutation frequencies were much higher in the G10 cell line than in the G12 cells. Detailed molecular analysis of the 6-thioguanine (6-TG)-resistant variants demonstrated that transgene silencing by de novo DNA methylation and increased chromatin condensation in the SV40 promoter was the major factor responsible for this high level of 6-TG resistance. As would be expected, exposure to 5-AzaC lowered the overall genomic DNA methylation levels, but it unexpectedly caused hypermethylation and increased chromatin condensation of the transgene in both the G10 and G12 cell lines. These results provide the first evidence that 5-AzaC may also induce transgene-specific DNA methylation, a phenomenon that can further be used for the elucidation of the mechanism that controls silencing of foreign DNA.

FIG. 1

Mutation frequency and cytotoxicity of 5-AzaC in G10 and G12 cells. The cells were exposed to various concentrations of 5-AzaC for 48 h and then incubated for a 7-day expression period. The selection for gpt⁻ cells was then done in a medium containing 6-TG (10 μg/ml) for 10 days. The data represent the median values (for mutation frequencies) and the means ± standard deviations (for survivals) of three to eight determinations. Filled symbols and open symbols represent the mutation frequency and percent survival, respectively. The spontaneous mutation frequencies of the G10 (▴) and G12 (●) cells were 100 and 30 per 10⁶ surviving cells, respectively.

FIG. 2

Analysis of gpt transcription in several 6-TG-resistant G10 and G12 variants. (A) Northern blot analysis of gpt transcription. Fourteen micrograms of total RNA per cell line was fractionated on 1% agarose gels and transferred to a nylon membrane. The membrane was hybridized with a ³²P-labeled gpt probe that was generated by random primer labeling of a 561-bp PCR product of the gpt coding region. The arrows indicate the expected size of the gpt transcript in the G10 (top arrow) and G12 (bottom arrow) parental lines. (B) To control for gel-loading differences, the membranes were stripped and rehybridized with a GAPDH probe.

FIG. 3

Deletion screen by PCR amplification of the gpt coding sequence in G12-derived (A) and G10-derived (B) 6-TG-resistant cell lines. PCR products were separated on 1.2% agarose gels and stained with EtBr. The expected 561-bp PCR product is clearly visible in the control G10 and G12 cell lines and most of the examined variants. No products are shown in the negative control reaction (no template DNA) (lane N). M, molecular weight markers.

FIG. 4

Methylation of the gpt gene and its flanking sequence in G12 (A) and G10 (B and C) 5-AzaC-induced 6-TG-resistant cell lines. Ten micrograms of EcoRV-digested DNA was further digested with the methylation-sensitive restriction enzyme HaeII (A) or HpaII (B). Lane C contains EcoRV-digested DNA not subjected to digestion with the other enzymes. (C) Digestion of G10 and the A2, A3, and A7 5-AzaC variants with the methylation-sensitive enzyme HpaII and the methylation-insensitive isoschizomer MspI. To better resolve the bands, the DNA was cut with both EcoRV and HindIII, and fragments were separated in a 1.7% agarose gel (6 h at 55 V). The DNA was transferred to a membrane in 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). (D) Restriction of the gpt gene on the genomic map of G12 and G10 cell lines. The fragments were separated on agarose gels (1% [A], 1.7% [B], and 1.7% [C]), transferred to nylon membranes, and then hybridized with a ³²P-labeled gpt probe. The variants are identified as in Fig. 2. H1, H2, H3, and H4 are the first, second, third, and fourth HpaII sites, respectively.

FIG. 5

Map of 408 bp from the SV40 early promoter region flanking the gpt coding region in G10 and G12 transgenic cell lines. CpG dinucleotides are underlined. The numbers above the CpG sites correspond to the methylation map data in Table 3. Bold numbers represent the sites that were methylated or partially methylated in the genomes of five out of the eight variants that were examined. The arrows indicate the sequences used for the primer design. (The primers were constructed after the bisulfite conversion reaction had been taken into account.)

FIG. 6

Resistance of 5-AzaC-induced 6TG-resistant clones to DNase I. Nuclei isolated from G12, N37, A13, A14, and A15 cells (A) and G10, A2, A6, and A7 cells (B) were treated with 0, 0.5, 1, 2, and 10 U of DNase I. PCRs were performed on 50 ng of DNase I-digested DNA. PCR products were separated on 1.2% agarose gels and stained with EtBr. The expected gpt product is 561 bp. M, molecular weight markers.