Epigenetic Transcriptional Silencing and 5-Azacytidine-Mediated Reactivation of a Complex Transgene in Rice

Abstract

Despite a growing number of reports indicating non-Mendelian inheritance of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants generated by direct DNA-transfer techniques, making the cause for these observations difficult to determine. In this paper we describe the complex organization of Btf crylllA and bar transgenes in rice (Oryza safiva 1.) that displayed aberrant segregation in R, progeny. Silencing rather than rearrangement of the bar gene was implicated because the herbicide-sensitive R, plants had a DNA hybridization profile identical to that of the resistant R, parent and R, siblings. Cenomic DNA analysis revealed substantial methylation of the Ubil/bar sequences in silenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuclease protection and nuclear run-on assays confirmed that silencing was due to transcriptional inactivation. Treatment of R, progeny of silenced plants with 5-azacytidine resulted in demethylation of the Ubil promoter and reactivation of bar gene expression, demonstrating a functional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals transformed by direct DNA protocols that insert multiple, often rearranged sequences.

Epigenetic memory of DNAi associated with cytosine methylation and histone modification in fern

“…3). [22][23][24] These studies suggest that DNA methylation and histone deacetylation are synergistic in the gene silencing of RNAi in Arabidopsis. However, our results showed that histone modification is independent of DNA methylation in Adiantum (Fig.…”

Gene silencing from plant DNA carried by a Geminivirus

“…Some examples of pathogen-derived host resistance to RNA viruses have been attributed to a posttranscriptional gene silencing mechanism (Covey et al, 1997;Mueller et al, 1995;Ratcliff et al, 1997;Tanzer et al, 1997). Transcriptional gene silencing has been hypothesized to involve DNA/DNA pairing, DNA methylation or heterochromatinization (Kumpatla et al, 1997;Neuhuber, 1995;Park et al, 1996). Repeated DNA has a tendency to undergo transcriptional silencing, which may be associated with changes in chromatin structure (Meyer, 1996;Ye and Signer, 1996), as well as to induce certain types of posttranscriptional silencing (Stam et al, 1997).…”

Genetic transformation of Sorghum bicolor

“…One possible reason for this may be that the transgenic lines obtained in their investigation had a large number of uidA gene copies integrated with possible concatameric arrangements. There are several reports demonstrating transgene inactivation as a result of high copy number integration (Rathore et al, 1993;and Kumpatla et al, 1997). However, results from a number of other studies suggested that transgenes with even low copy number integration could become silenced (Casas et al, 1993;Casas et al, 1997).…”