Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes

Abstract

Previous work has shown that two homologous, unlinked transgene loci can interact in plant nuclei, leading to non-reciprocal trans-inactivation and methylation of genes at one locus. Here, we report the structure and methylation of different transgene loci that contain the same construct but are variably able to inactivate and methylate a partially homologous, unlinked target locus. Silencing loci comprised multiple, methylated copies of the transgene construct, whereas a non-silencing locus contained a single, unmethylated copy. The correspondence between strength of silencing activity and copy number/degree of methylation was further demonstrated by producing novel alleles of a strong silencing locus: reducing the transgene copy number and methylation within this silencing locus decreased its ability to inactivate the target locus. The strong silencing locus, which was located close to a telomere, trans-inactivated various structural variants of the original target construct, regardless of their location in the genome. This suggests that the silencing locus can scan the entire genome for homologous regions, a process possibly aided by its telomeric location. Our data support the idea that epistatic trans-inactivation of unlinked, homologous transgenes in plants results from a pre-existing epigenetic difference between transgene loci, which is subsequently equalized by "epigene conversion" involving DNA-DNA pairing.

How and Why Do Plants Inactivate Homologous (Trans)genes?

“…Nevertheless, direct physical contact between alleles has been suggested to account for paramutation in each respective system. DNA-DNA pairing has also been suggested as a mechanism for two trans-inactivation systems comprising a silencing transgene locus and a target transgene locus that share homology only in promoter regions (Vaucheret, 1993;Matzke et al, 1994a). In the nopaline synthase promoter-based system, the extent of promoter homology was about 300 bp (Matzke and Matzke, 1993).…”

“…A transgene insert that has become methylated and inactivated in cis can act as a "silencer" by somehow imposing in trans a similar degree of methylation and inactivation on a homologous "target" gene on a separate DNA molecule. The target gene can be at either an allelic (Meyer et al, 1993) or nonallelic (ectopic) (Vaucheret, 1993;Matzke et al, 1994a) chromosomal location relative to the silencing transgene insert. Because the silencing allele/locus independently achieves a methylated, inactivated state, truns-inactivation is a nonreciprocal gene interaction, i.e.…”

“…Open and closed circles designate unmethylated and methylated cytosines, respectively. A locus that has become silenced in cis (A) can potentially trans-inactivate homologous, unlinked loci in a manner that might rely on DNA-DNA pairing (Matzke et al, 1994a). ds RNA, Double-stranded RNA; RNA-dep.…”

Suppression of Virus Accumulation in Transgenic Plants Exhibiting Silencing of Nuclear Genes.

“…Gene silencing in transgenic plants is manifested as decreased accumulation of specific mRNAs and occurs most often when there are multiple copies of a particular sequence present in the genome (Jorgensen, 1990;Napoli et al, 1990;Smith et al, 1990;van der Krol et al, 1990;Hobbs et al, 1993;Dehio andSchell, 1994: lngelbrecht et al, 1994;Matzke et al, 1994). The repeated sequences may consist of coding sequences, promoter regions, or both.…”

Direct gene transfer in potato: A comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts

“…Multiple transgene copies can contribute to transgene silencing and inhibit transgene expression (Matzke et al 1994). Additionally, very large transgenic loci can be meiotically unstable, leading to the loss of both loci and transgene expression in subsequent generations (Fu et al 2000; and references therein).…”