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11 all loss of function alleles of genes encoding

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Unformatted text preview: n and TGS requires DNA methyltransferases other than MET1, proper histone methylation3,23 and the chromatin-remodeling protein DDM1 (ref. 11). All loss-of-function alleles of genes encoding these proteins (several considered to be null alleles; refs. 24,25) affect DNA methylation and TGS only in homozygotes2–7,19. Their mutated alleles can be propagated through heterozygotes without epigenetic consequences2,26. This is consistent with a minor, or nonexistent, regulatory role for these a Fig. 3 Maintenance of methylation in somatic tissues of met1 heterozygotes and its loss during gametogenesis. a, Scheme of genetic crosses. Several independent met1-3 heterozygous T2 plants (MET1+/–) were crossed to tester line 6b5 (ref. 19) containing a silent homozygous GUS locus (GUS+/+). The met1 mutated allele was always inherited through heterozygotes. Three independent F1 plants (genotype MET1+/– GUS+/–; framed red) were reciprocally backcrossed (BC) to wild-type plants. b, F1 (left) and backcrossed (BC; right) plants of equivalent genotype (MET1+/– GUS+/–; framed red), the latter originating from reciprocal crosses (mat, maternal transmission or pat, paternal transmission of the met1 mutated allele) were examined for GUS expression (top) and methylation changes at the transgenic GUS locus analyzed by Southern blotting (bottom). Control DNA was from line 6b5, silent and hypermethylated GUS repeats; Col, Columbia wild-type; AsMET1:GUS, plants combining the transgenes of line 6b5 and AsMET1 expressing MET1 antisense RNA12,30; pl, parent line of backcross. DNA samples were digested with HpaΙΙ and hybridized with a GUS cDNA probe. Analyses used fully grown plants, thus some variations in methylation levels among met1+/–mat are probably due to the weak sporophytic de novo methylation. Therefore GUS mRNA expression levels were analyzed by northern blots and quantified by Phosphorimager (Molecular Dynamics). Relative values are shown above the lanes. c, Methylation analysis of endogenous loci. DNA from segregating for met1-4 T2 plants was digested with HpaΙΙ (upper panel) or CfoΙ (lower panel) and hybridized with Rap2.1 promoter probe, or FWA promoter probe18, respectively. d, Model of passive demethylation occurring in met1 mutants during gametogenesis. Black lines marked with lollipops represent methylated DNA. Vertical arrows mark mitoses during gametogenesis. Depletion of MET1 results in passive demethylation (gray line) and creates gametes with hemimethylated and completely demethylated DNA (75% female and 50% male gametes with complete demethylation). Hemimethylation provides a signal to restore methylation in a heterozygous zygote where MET1 is again available. DNA methylation status of a specific locus is presented and possible sister chromatid exchanges are not considered. b c nature genetics • volume 34 • may 2003 d 67 © 2003 Nature Publishing Group http://www.nature.com/naturegenetics letter epigenetic modifiers in the propagation of epigenetic states during gametogenesis and rest...
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