Gene silencing mediated by promoter homology occurs at the level of transcription and results in meiotically heritable alterations in methylation and gene activity.
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.
Park
Y D
YD
Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg, Austria.
Papp
I
I
Moscone
E A
EA
Iglesias
V A
VA
Vaucheret
H
H
Matzke
A J
AJ
Matzke
M A
MA
D016375:Antisense Elements (Genetics); D001483:Base Sequence; D017796:Caulimovirus / Q000235:genetics; D003433:Crosses, Genetic; D018506:Gene Expression Regulation, Plant; D018392:Genomic Imprinting; D008540:Meiosis; D008745:Methylation; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000235:genetics; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000235:genetics; D014158:Transcription, Genetic
D016375:Antisense Elements (Genetics); D009572:Nitrite Reductases; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase