A repetitive DNA fragment carrying a hot spot for de novo DNA methylation enhances expression variegation in tobacco and petunia

Abstract

A 1.6 kb repetitive DNA sequence (RPS) from Petunia hybrida was identified that destabilizes expression of a GUS marker transgene. Following polyethylene glycol (PEG)-mediated tobacco and petunia protoplast transformations, GUS expression patterns analysed on callus and plant levels were clearly more variable when constructs contained the RPS sequence. The effect on transgene expression required chromosomal integration since the two different RPS constructs employed did not exhibit reduced levels of GUS activities in transient assays. DNA methylation analysis implies a hypermethylated default state of endogenous RPS copies present in the petunia genome. Analysis of the transgene DNA in different transgenic tobacco plants showed almost complete hypermethylation of a particular Hhal site of the RPS sequence. It is proposed that, due to the presence of specific signals within the RPS region or based on interaction of RPS with other endogenous homologous sequences, RPS functions as an initiation region for de novo methylation and induces expression variegation in adjacent sequences.

Epigenetic silencing of plant transgenes as a consequence of diverse cellular defence responses

“…In this study, a repetitive sequence from petunia was found to enhance variegated expression of a reporter transgene in petunia and tobacco [32]. Using the Cre-lox system for site-specific recombination to specifically remove a repeated sequence from a transgene locus, we have found recently that a tandem repeat could induce methylation and inactivation of a neighbouring NOSpro-driven gene ( fig.…”

Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas.

“…In higher plants, transcriptional inactivation of transgenes has been proposed to result from DNA methylation and/or a condensed chromatin structure, perhaps triggered in some cases by DNA-DNA or DNA-RNA interactions (Matzke and Matzke, 1995;Ten Lohuis et al, 1995;Meyer and Saedler, 1996;Park et al, 1996;Ye and Signer, 1996). In Chlamydomonas, the multiple expression states, temperature sensitivity of expression, chromosomal location, and lack of methylation or altered chromatin accessibility of the introduced RbcS2::aadA::RbcSP gene(s) all suggest a possible role for (hetero)chromatic chromosomal domains in this epigenetic phenomenon.…”

“…In some cases, this gene inactivation involves transcriptional repression, shows at least partia1 meiotic heritability, and has been correlated with cytosine methylation, particularly of the promoter regions (Meyer et al, 1993;Neuhuber et al, 1994;Park et al, 1996). In a few examples, transcriptional inactivation also seems to be accompanied by a condensed chromatin structure (Ten Lohuis et al, 1995;Ye and Signer, 1996). Transcriptional silencing of some transgenes is strongly reminiscent of paramutation, particularly at the maize b locus (encoding a transcriptional activator of the anthocyanin biosynthetic pathway) (Matzke and Matzke, 1995;Meyer and Saedler, 1996).…”

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

“…In the plant genome, methylation of transgene sequences might also affect chromatin structure, as previously suggested by lngelbrecht et al (1994). Ten Lohuis et al (1995) showed that in petunia, repetitive DNA that carries a hot spot for methylation induces variegated expression of adjacent sequences, which was suggested to be due to heterochromatinmediated gene inactivation. Genes in inactive or heterochromatin domains are not considered to be accessible to the regulation, either activation or repression, that is afforded by DNA elements in promoters (Lewin, 1994;Rivier and Pillus, 1994).…”