Release of epigenetic gene silencing by trans-acting mutations in Arabidopsis - PubMed (original) (raw)

Release of epigenetic gene silencing by trans-acting mutations in Arabidopsis

O Mittelsten Scheid et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Gene silencing in plants inactivates trans-genes introduced into plants and/or endogenous homologous genes. This stable but potentially reversible loss of gene activity resembles epigenetic changes that occur in normal development. The stability of silencing implies the involvement of trans-acting components, although none of them have been identified so far. Here we report the finding of second-site mutations interfering with maintenance of the silent state. We mutagenized Arabidopsis thaliana plants carrying a silent transgene encoding hygromycin phosphotransferase (hpt) and therefore show a heritable hygromycin-sensitive phenotype. The M2 generation was screened for hygromycin resistance. Eight putative mutants (som1 to 8) were found that expressed the transgene and transmitted the expressed state to their progeny. All mutations were shown to reactivate a silent transgenic test locus in trans. The level of DNA methylation at the hpt locus and at centromeric repeats was found to be reduced in the som mutants. Complementation crosses indicated complex epigenetic interactions among the som mutant alleles and with the previously described ddm1 allele, which elicits DNA hypomethylation [Vongs, A., Kakutani, T, Martiensen, R.A. & Richards, E.J. (1993) Science 260, 1926-1928]. Som mutants can be classified into three groups: (i) allelic or interacting with ddm1 and with each other (som 1,4, and 5), (ii) nonallelic with ddm1 and som mutants of group A (som2), and (iii) mutants with slow resilencing after out-crosses, which hinders their classification (som 3, 6, 7 and 8).

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Figures

Figure 1

Figure 1

Selection of mutants. Primary selection of mutant candidates: hygromycin-resistant seedling after 8 days selection on 15 mg/l hygromycin (a), Northern (b), and Southern (c) blot analysis of the parental line A with the silent hpt gene and the mutants _som1_-som8. Both membranes were probed with the hpt coding region. Genomic DNA was digested with _Bam_HI.

Figure 2

Figure 2

Reactivation of a silent gene in test crosses with the mutants. (a) Scheme of the crosses for mutants with hemizygous transgene. (b) Scheme of the crosses for mutants with homozygous transgene. (c) F2 populations after 2 weeks on 10 mg/l hygromycin. som, mutant locus; SOM, corresponding wild-type allele; _A_s, hpt gene in silent state; _A_r, hpt gene in reactivated state; −, transgene-free locus allelic to A.

Figure 3

Figure 3

Methylation analysis. The same Southern blot of DNA cut with _Hpa_II was probed with the hpt coding region (a), or with a fragment containing the centromeric 180-bp repeat (b; ref. 20). Lanes: 1, pGl2 plasmid used to generate the parental line A; 2, wt; 3, parental line A; 4–11, som 1–8, 12, mutant ddm1/ddm1 with reduced methylation (20); 13, _ddm1/_A; 14, METIas T3 #10.5 transgenic for a methyltransferase antisense construct (21); 15, METIas T3 #10.5/A.

Figure 4

Figure 4

Complementation analysis. F1 hybrids of som2, som5, and som7 with ddm1 or wt, after 2 weeks on 10 mg/l hygromycin.

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