DNA Methylation Is Involved in Maintenance of an Unusual Expression Pattern of an Introduced Gene

Abstract

In one of 30 transgenic tobacco (Nicotiana tabacum) plants, the expression of an introduced ,B-glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S promoter, was found to be repressed as the plant matured, whereas the endogenous GUS activity was unaffected. Plants grown from seeds or regenerated from leaf discs derived from this plant showed a similar temporal pattern of expression. Suspension-cultured cells established from nonexpressing leaves did not express the introduced gene. In these cells, the silent gene could be reactivated by treatment for 5 or 10 days with 5-azacytidine. Overall, demethylation of the genome preceded recovery of the enzyme activity. The increase in the fraction of reactivated cells progressed in two phases. Up to 8 weeks after starting the 5-azacytidine treatment, approximately 2 to 4% of the cells were expressing GUS, followed by a dramatic increase of GUS-expressing cells. Thirteen weeks after starting the 5-azacytidine treatment, the fraction of GUS-expressing cells amounted to 80%. At this time, the original overall level of DNA methylation was reestablished. The degree of DNA demethylation, as well as the magnitude of reactivation, was dependent on the duration of the 5-azacytidine treatment. These results demonstrate that DNA methylation appears to be involved in the regulation of the introduced GUS gene and that this development-dependent pattern of expression can be inherited.Among different transformants, a variation in the expression of introduced genes has often been detected (2,25). Such intertransformant variability has been explained by "positional effects" or a variety of epigenetic and genetic causes (11,18), such as DNA methylation.Methylation of cytosine residues in DNA is involved in the regulation of gene expression, and an inverse correlation between the transcriptional activity of certain genes and the level of methylation of these genes has been found (1, 5, 23). Tissue-specific patterns of methylation have been observed in gene sequences that encode tissue-specific functions (5,8,23 azaC is a cytidine analog, which, when incorporated into DNA, inhibits DNA methyltransferase activity (6,24). Apparently, azaC is able to activate genes in a selective manner rather than causing a global increase in gene expression, even though it is thought to act as a nonspecific DNA-demethylating agent (15). Here, we report the azaC-induced reactivation of a silent, introduced GUS gene. Because azaC is a specific inhibitor of DNA methylation, the demonstration of the azaC-induced reactivation of the gene indicates that DNA methylation should be involved in repression of the gene in cells grown in suspension and in the leaves from which these cells are derived. MATERIALS AND METHODS Plant Material and Growth ConditionsMature (flowering stage) primary transgenic plants, R1, of tobacco (Nicotiana tabacum L. cv Wisconsin 38) transformed with the Ti plasmid pMON9749 (10) were used. The transfer DNA of pMON9749 contains the NPTII gene (encoding NPTII) driven by ...

Treatment of Agrobacterium or leaf disks with 5-azacytidine increases transgene expression in tobacco

“…However, treatment with azaC resulted in a substantial increase in expression of GUS after about 3 weeks, indicating that the temporally decreased expression was not only due to loss of the transgene but to its inactivation as well. Increase in transgene expression after azaC treatment of plant cells is a well known effect [3,18,29,31], which assumes the transgene to be methylated and consequently inactivated. Indeed, such de novo methylation has been shown to occur in tobacco protoplasts after direct gene transfer [29].…”

“…This list of genes includes endogenous genes, such as those encoding storage proteins [2,7], photosynthetic proteins [22] and rRNA [8,27], as well as transposable elements [4,19] and the tumour inducing genes on Agrobacterium tumefaciens wild-type T-DNA [1,9]. DNA methylation also seems to be a mechanism for inactivation of chimeric transgenes [ 10,20] and the demethylating agent 5-azacytidine (azaC) has been used to reactivate silent transgenes [3,29,31].…”

Transgenic sorghum plants via microprojectile bombardment.

“…GUS activity, which was high in transient assays, could not be detected in callus that had been maintained for prolonged periods on bialaphos selection pressure, despite the fact that Southern analysis indicated the presence of the uidA gene. This suggests that DNA methylation occurs in sorghum cells that inactivates the expression of transferred genes (39,40).…”

RNA-Mediated Virus Resistance: Role of Repeated Transgenes and Delineation of Targeted Regions.

“…We have tried to determine whether methylation at transcribed sequences of the transgene has a direct role in establishing RNA-mediated resistance. We germinated and cultivated seedlings of resistant TR lines on 5-azacytidine (Weber et al, 1990;Bochardt et al, 1992;Palmgren et al, 1993). However, at concentrations of 5-azacytidine (20 to 60 wM), at which plants just survived, only very limited demethylation had occurred (DNA gel blot analyses not shown) and the plants remained resistant.…”