Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing

Abstract

The chromatin structures of two epigenetic alleles of a transgene were investigated by measuring the local accessibility of transgene chromatin to endonucleases. The two epialleles represented the active, hypomethylated state of a transgene in line 17-I of Petunia hybrida, and a transcriptionally inactive, hypermethylated derivative of the same transgene in line 17-IV. In nuclear preparations the inactive epiallele was significantly less sensitive to DNasel digestion and nuclease S7 digestion than the transcriptionally active epiallele, whereas no significant differences in accessibility were observed between naked DNA samples of the two epialleles. Our data suggest that a condensed chromatin structure is specifically imposed on transcribed regions of the construct in line 17-IV. In contrast, in both epialleles the plasmid region of the transgene, which is not transcriptionally active in plants, retains the same accessibility to endonucleases as the chromosomal integration site. These data suggest that transcriptional inactivation is linked to the process of transcription, and imply that control of transgene expression via the use of inducible or tissue-specific promoters might prevent transgene silencing and conserve the active state of transgenes during sexual propagation.

The Arabidopsis Class II Sirtuin Is a Lysine Deacetylase and Interacts with Mitochondrial Energy Metabolism

“…Subfractionation of mitochondria was performed as described in detail in Finkemeier et al (2005). Nuclei were isolated from 4-week-old Arabidopsis leaves as described previously (van Blokland et al, 1997).…”

Cell Culture-Induced Gradual and Frequent Epigenetic Reprogramming of Invertedly Repeated Tobacco Transgene Epialleles

“…The reduced methylation in Lo1E may reflect a more general trend toward genome hypomethylation in plant cell cultures (Kaeppler and Phillips, 1993;Kubis et al, 2003). On the other hand, the proximity of inverted repeats occupying distinct chromatin structure (Ebbs et al, 2005) and intensive transcription over the methylated coding region (van Blokland et al, 1997) could promote methylation spreading from the coding region into the promoter. Inverted repeat structure could potentially generate read-through transcripts, giving rise to methylation-inducing smRNA molecules (Mette et al, 2000).…”

Chromatin conversations: mechanisms and implications of paramutation

“…Trans-interactions were described between two homologous transgenes 57,83,84 , and between transgenes and endogenous genes 60,62 .Where tested, transcription was shown to be altered 57,80,84,85 and the alleles or homologous sequences often showed differences in chromatin, such as DNA methylation and/or NUCLEASE HYPERSENSITIVITY 3,24,83,86 .We now know that paramutation involves trans-interactions between homologous sequences that establish distinct heritable chromatin changes and that paramutation is not limited to plants; similar phenomena have been described in organisms from several other kingdoms (TABLE 1) pl1 loci occurs with 100% penetrance. Rare, darkly pigmented F 1 plants potentially carry dominant mutations that prevent the establishment of paramutation; rare F 2 families segregating darkly pigmented plants might carry recessive mutations that fail to maintain the low expression levels that are associated with paramutation and could affect maintenance of the paramutagenic state.…”