Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity

Abstract

The promoter homology-dependent inactivation of a 35Spro-hygromycin phosphotransferase (hpt) gene, which is present at the H2 locus, by the multipurpose 271 silencing locus has been studied. The 271 locus can silence any gene under the control of the 35Spro as well as endogenous nitrite reductase (NiR) genes of tobacco because of the presence of a chimeric antisense gene (35Spro-RiN). All F1 progeny of a cross between homozygous H2 and 271 lines were sensitive to hygromycin and were chlorotic (a symptom of nitrogen deficiency). These phenotypes were accompanied by a reduction in the steady-state levels of Hyg and NiR transcripts. Transcriptional run-on experiments indicated, however, that while NiR silencing occurred post-transcriptionally, the hpt gene was inactivated at the transcriptional level; this was associated with increased methylation of the 35Spro of the hpt gene. NiR gene expression recovered uniformly to wild-type levels in first generation backcross (BC1) progeny that did not inherit the 271 locus. In contrast, hygromycin resistance was only partially and non-uniformly regained among adult BC1 plants. Moreover, substantial silencing of the hpt gene could persist into the BC2 generation. Genomic sequencing demonstrated that the meiotic heritability of hpt silencing in the absence of the 271 locus was correlated with cytosine methylation primarily at CpG and CpNpG residues. Despite this residual methylation, H2 loci weakened by an association with 271 did not acquire the ability to silence a 'naive' H2 locus. Fluorescence in situ hybridization revealed that the 271 locus was located at a telomere. The results strengthen the distinction between silencing effects involving homology restricted to coding or promoter regions, respectively. The former is a post-transcriptional process that is meiotically reversible; the latter is due to transcriptional inactivation and is associated with increased promoter methylation, which can lead to meiotically heritable reductions in target gene activity. The relevance of these data for the meiotic heritability of silencing, the non-transferability of silencing activity, and the basis of 271 silencing effects is discussed.

A single transgene locus triggers both transcriptional and post-transcriptional silencing through double-stranded RNA production

“…Because the 271 locus is able to transcriptionally silence transgenes driven by the 35S promoter (Elmayan and Vaucheret 1996;Park et al 1996), we looked for 35S RNAs aberrantly expressed from the 271 locus. RNase protection assays revealed two RNAs, 220-nt and 180-nt in length, corresponding to the 35S promoter ( Fig.…”

“…The 271 locus also triggers PTGS of endogenous NIR genes (Elmayan and Vaucheret 1996;Park et al 1996). The 271 line does not accumulate mRNA from either of the four endogenous NIR genes or RNAs from the antisense transgene (35S-RIN) carried at the 123 271 locus (Vaucheret et al 1992).…”

“…The 271 transgenic tobacco line was regenerated from kanamycin-resistant callus that spontaneously became sensitive to kanamycin and was unable to grow on medium containing nitrate or nitrite as the sole source of nitrogen (Vaucheret et al 1992). The 271 locus was subsequently shown to silence, by TGS, any transgene driven by the 19S or 35S promoter and to silence, by PTGS, endogenous NIR genes as well as NIR transgenes (Elmayan and Vaucheret 1996;Park et al 1996;Thierry and Vaucheret 1996), indicating that the 271 locus is a universal silencer of 19S, 35S, and NIR sequences. The 271 locus, however, was unable to silence npt transgenes by PTGS, but only by TGS when they are driven by the 19S or 35S promoter.…”

RNA-Dependent RNA Polymerase 6 Is Required for Efficient hpRNA-Induced Gene Silencing in Plants

“…The efficiency and frequency of transitive silencing of an endogenous gene depends on the length of its sequence identity with the primary target (Bleys et al, 2006). Gene silencing mediated by promoter homology occurs at the level of transcription (Park et al, 1996). Mutants of C. elegance that are defective in transposon silencing and RNAi are also resistant to co-suppression, indicating that RNAi and co-suppression are mediated by the same molecular machinery, possibly through the formation of dsRNA/RNA duplex as a precursor to generate siRNA (Ketting and Plasterk, 2000).…”

“…Transcriptional gene silencing (TGS), which takes place through repression of transcription, is often associated with methylation of the corresponding homologous promoter (Neuhuber et al, 1994;Park et al, 1996). Post-transcriptional gene silencing (PTGS), occurring through sequencespecific degradation of target mRNAs, is characterized by accumulation of small interfering RNA (siRNA) and methylation of target gene sequences (Depicker and Montagu, 1997;Vaucheret et al, 2001).…”