Genetics, Vol. 151, 831-838, February 1999, Copyright © 1999

Meiotically and Mitotically Stable Inheritance of DNA Hypomethylation Induced by ddm1 Mutation of Arabidopsis thaliana

Tetsuji Kakutania, Kyoko Munakataa, Eric J. Richardsb, and Hirohiko Hirochikaa
a Department of Molecular Genetics, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-0856, Japan
b Department of Biology, Washington University, St. Louis, Missouri 63130

Corresponding author: Tetsuji Kakutani, Department of Molecular Genetics, National Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan., kakutani{at}abr.affrc.go.jp (E-mail)

Communicating editor: V. SUNDARESAN


*  ABSTRACT
*TOP
 *ABSTRACT
*MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

In contrast to mammalian epigenetic phenomena, where resetting of gene expression generally occurs in each generation, epigenetic states of plant genes are often stably transmitted through generations. The Arabidopsis mutation ddm1 causes a 70% reduction in genomic 5-methylcytosine level. We have previously shown that the ddm1 mutation results in an accumulation of a variety of developmental abnormalities by slowly inducing heritable changes in other loci. Each of the examined ddm1-induced developmental abnormalities is stably transmitted even when segregated from the potentiating ddm1 mutation. Here, the inheritance of DNA hypomethylation induced by ddm1 was examined in outcross progeny by HPLC and Southern analyses. The results indicate that (i) DDM1 gene function is not necessary during the gametophyte stage, (ii) ddm1 mutation is completely recessive, and (iii) remethylation of sequences hypomethylated by the ddm1 mutation is extremely slow or nonexistent even in wild-type DDM1 backgrounds. The stable transmission of DNA methylation status may be related to the meiotic heritability of the ddm1-induced developmental abnormalities.

IN both plants and mammals, epigenetic control of gene expression is often correlated with change in cytosine methylation of the affected locus. Mammalian epigenetic phenomena, such as parental imprinting and X-chromosome inactivation, are developmentally regulated, and "resetting" of the epigenetic status occurs in each generation. Similarly, methylation patterns in mammalian genome undergo reorganization (MONK et al. 1987 Down) by extensive demethylation and "de novo" methylation during gametogenesis and early development (YODER et al. 1997 Down). In contrast, the epigenetic states of plant genes such as the Arabidopsis SUPERMAN gene (JACOBSEN and MEYEROWITZ 1997 Down), PAI genes (BENDER and FINK 1995 Down), maize transposable elements (MCCLINTOCK 1967 Down; BRUTNELL and DELLAPORTA 1994 Down; MARTIENSSEN and BARON 1994 Down; SCHLAPPI et al. 1994 Down), and repeated transgenes of tobacco (PARK et al. 1996 Down) are often stably inherited through generations.

Eukaryotic mutants affecting genomic DNA methylation have been described in mouse (LI et al. 1992 Down), Neurospora (FOSS et al. 1993 Down), Ascobolus (MALAGNAC et al. 1997 Down), and Arabidopsis (VONGS et al. 1993 Down; FINNEGAN et al. 1996 Down; RONEMUS et al. 1996 Down; MITTELSTEN-SCHEID et al. 1998 Down). As in other eukaryotes (LI et al. 1992 Down; FOSS et al. 1993 Down; MALAGNAC et al. 1997 Down), developmental abnormalities were exhibited in the Arabidopsis DNA methylation mutants. In homozygous ddm1 mutants of Arabidopsis, genomic 5-methylcytosine (5mC) content in TaqI sites is reduced to ~30% of wild-type levels (VONGS et al. 1993 Down). The ddm1 mutations result in an accumulation of a variety of developmental abnormalities, by inducing heritable changes in other loci. Each of the ddm1-induced developmental abnormalities investigated was stably transmitted even when segregated from the potentiating ddm1 mutation (KAKUTANI et al. 1996 Down; KAKUTANI 1997 Down). A similar spectrum of developmental abnormalities was found in transgenic plants expressing a DNA methyltransferase gene MET1 (FINNEGAN and DENNIS 1993 Down) in an antisense orientation (FINNEGAN et al. 1996 Down; RONEMUS et al. 1996 Down).

In addition to revealing effects of altering DNA modification on development, DNA methylation mutants provide good systems with which to investigate de novo methylation in vivo. For example, disruption of a mouse DNA methyltransferase gene (Dnmt1) causes a reduction in overall DNA methylation levels (LI et al. 1992 Down). Expression of the wild-type Dnmt1 cDNA in mutant male embryonic stem (ES) cells causes an increase in methylation of bulk DNA to normal levels, while restoration of the methylation of the imprinted genes H19 and Igf2r occurs only after germline transmission (TUCKER et al. 1996 Down). These results suggest the existence of de novo methyltransferase activities specific during oogenesis and spermatogenesis.

We have previously proposed that remethylation of sequences hypomethylated by ddm1 mutations is slow, on the basis of the following observations using thin-layer chromatography (VONGS et al. 1993 Down): (i) Heterozygotes (DDM1/ddm1) produced by one outcross to wild-type plants contain 5mC in TaqI sites in amounts intermediate between those in the two parents; and (ii) repeated backcrossing of the heterozygotes to wild-type parents generates plants that contain an amount of 5mC at TaqI sites that approaches the amount found in wild-type plants. Given that 5mC levels of only TaqI sites were followed, however, it is possible that the wild-type DDM1 allele is incompletely dominant over the examined ddm1 mutant allele. More importantly, the extent of remethylation of genomic sequences hypomethylated by ddm1 mutation has only been examined in DDM1/ddm1 heterozygotes, not in DDM1/DDM1 wild-type backgrounds.

In this article, we extend the previous studies of TaqI sites to the whole genome using HPLC and examine methylation of specific loci and alleles in the outcross progeny including DDM1/DDM1 homozygotes by Southern analysis. The results substantiate our previous proposal and show that ddm1-induced hypomethylation in the majority of sequences in the Arabidopsis genome, both repeated and single-copy sequences, can be stably inherited both mitotically and meiotically. The stable transmission of DNA hypomethylation correlates with the stable property of the ddm1-induced developmental abnormalities.


*  MATERIALS AND METHODS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
*RESULTS
 *DISCUSSION
 *LITERATURE CITED

Plants and growth conditions:
Isolation of ddm1 mutants of Arabidopsis thaliana was reported by VONGS et al. 1993 Down. The ddm1-2 allele was used throughout. Plants were grown in a controlled environmental chamber under a long-day condition as described (KAKUTANI et al. 1996 Down).

Measurement of 5mC content by HPLC:
The levels of 5mC were measured by a modification of the reversed-phase HPLC method described by KUO et al. 1980 Down. Arabidopsis genomic DNA was isolated as described (KAKUTANI et al. 1995 Down). To remove RNA, the DNA solution was treated with ribonuclease A, precipitated by adding an equal volume of 13% PEG, 1.6 M NaCl, and centrifuged at 27,400 x g for 2 min at 4°. The precipitate was rinsed with 70% ethanol, air dried, and dissolved in TE (pH 8). The solution was extracted twice with an equal volume of phenol/chloroform and once with chloroform. After addition of 1/4 volume of 10 M ammonium acetate (pH 7.7) and 2 volumes of ethanol, the DNA was precipitated by centrifugation at 7790 x g for 3 min at room temperature. The precipitate was washed with 70% ethanol, air dried, and dissolved in TE. The digestion of DNA was performed as described by KUO et al. 1980 Down. Digested samples were filtered [UFC4 TGC, pore size 10,000 (Millipore, Bedford, MA)] and resolved on a Purasil C18 column (4.6 x 150 mm; Waters Associates, Inc., Milford, MA) with a 60-min isocratic gradient of 10 mM ammonium phosphate buffer with 2.5% methanol (pH 5.6). The position of each nucleoside was determined using commercially available standards (Sigma, St. Louis). The values were calculated by integration of peak areas of absorbance at 280 nm with DataModule, Waters 741. The 5mC content ([5mC])/([5mC]+[C]) was normalized for absorbance difference between cytosine and 5mC.

Southern analysis of genomic DNA:
Southern analysis of genomic DNA was performed as described by AUSUBEL et al. 1987 Down using the high-SDS hybridization buffer of CHURCH and GILBERT 1984 Down. Radiolabeled probes of 180-bp centromere repeat, rDNA repeat (VONGS et al. 1993 Down), retrotransposon Ta3 (KONIECZNY et al. 1991 Down), and m105 (PRUITT and MEYEROWITZ 1986 Down) were generated using the Megaprime DNA-labeling system (Amersham, Arlington Heights, IL).

Repeated backcrossing:
Figure 1 shows the lineage of the repeatedly backcrossed plants. DDM1/DDM1 and DDM1/ddm1 plants should segregate from the cross DDM1/ddm1 x DDM1/DDM1. Plants from such a segregating family were used as material for the next backcrossing without determining the genotype. DDM1/ddm1 plants were later identified by progeny tests, and progeny of the cross DDM1/ddm1 x DDM1/DDM1 was used for the next backcrossing generation.



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  		Figure 1. Lineage of BC-H2 (progeny of the repeatedly backcrossed heterozygote).


*  RESULTS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
*DISCUSSION
 *LITERATURE CITED

Cytosine methylation levels in F1 plants from a cross between a ddm1 mutant and wild-type plants are intermediate between those of the parents:
We have previously shown that heterozygotes (DDM1/ddm1) produced by crossing a ddm1/ddm1 homozygote to a wild-type plant contain 5mC at TaqI sites (TCGA) in amounts halfway between those of the two parents (VONGS et al. 1993 Down). Methylation in other sites, however, has not been examined. Here, we examined the 5mC content of the total genome as examined by HPLC analysis, which allows cytosine methylation at every site to be sampled. The 5mC content of the ddm1 mutant genome was reduced to ~30% of the wild-type level (Figure 2), a value consistent with previous reports (VONGS et al. 1993 Down; RONEMUS et al. 1996 Down). The F1 heterozygotes (DDM1/ddm1), produced by crossing a ddm1 homozygote to a wild-type plant, contain 5mC at levels halfway between those of the two parents (Figure 2), consistent with our previous study. At first glance, these results seem to suggest that the ddm1 mutation is semidominant. However, our previous observations (VONGS et al. 1993 Down) and the findings described below lead us to believe that this is not the case.



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  		Figure 2. 5-methylcytosine (5mC) level in wild-type Columbia (WT), homozygous ddm1 mutant, and the F1 measured by reversed-phase HPLC. The values under the magnified chart represent the averages and deviations of four individual runs.

Although the results of TLC and HPLC show the overall amount of 5mC in the genome, the distribution of the genomic 5mC in different loci and different alleles cannot be analyzed by these methods. This was investigated by Southern analysis using a methylation-sensitive restriction endonuclease. Figure 3 shows the results of Southern analysis using the methylation-sensitive restriction enzyme, HpaII, in the genome of ddm1/ddm1 mutant, wild-type, and the F1 plants. We examined three sequences: retrotransposon Ta3 (KONIECZNY et al. 1991 Down), rDNA, and 180-bp centromere repeats (VONGS et al. 1993 Down). Figure 3A illustrates the restriction map of Ta3. All four HpaII sites are demethylated in the ddm1 mutant, but methylated in wild-type plants. In DNA from the F1 plants, both the top band and the three bottom bands were observed, indicating that HpaII sites in the Ta3 locus are completely methylated in about half of the DNA, while all the HpaII sites are unmethylated in the rest. Consistent with this interpretation, a mixture of genomic DNA from DDM1/DDM1 and ddm1/ddm1 plants gave essentially the same banding pattern as that from a DDM1/ddm1 plant (Figure 3B). F1 plants from the cross DDM1/DDM1 x ddm1/ddm1 and the reciprocal cross gave the same banding pattern.



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  		Figure 3. Southern analysis of DNA methylation patterns of HpaII sites in ddm1 mutant, wild-type, and the F1 plants. (A) HpaII sites in Ta3 sequence. (B) The filter was probed with Ta3 (left), 180-bp centromere repeat (center), or rDNA (right). In each part, genomic DNA from the following plants was used: lane 1, wild-type Columbia (DDM1/DDM1); lane 2, ddm1/ddm1; lane 3, DDM1/DDM1 x ddm1/ddm1; lane 4, ddm1/ddm1 x DDM1/DDM1; lane 5, 1:1 mixture of DDM1/DDM1 and ddm1/ddm1 DNA.

Similar results were obtained using two repeated sequences, 180-bp repeats and rDNA, as hybridization probes. These probes recognize tandemly repeated sequences clustering in two (rDNA) or five (180-bp repeats) unlinked loci of the A. thaliana genome. Both types of repeats are hypomethylated in ddm1 mutants (VONGS et al. 1993 Down). HpaII sites in the rDNA sequences are completely demethylated in the ddm1 mutants, whereas most of the sites are methylated in the wild type. HpaII sites in the 180-bp repeats are demethylated in ddm1 mutants but completely methylated in the wild type. For both of these repeated sequences, the extent of the methylation of F1 appears to be intermediate between that of the two parents (Figure 3B). For the 180-bp probe, the ladder of bands did not shift upward, although the overall intensity was reduced. The absence of the shift in relative band intensities again suggests that about half of the DNA was hypomethylated as in the ddm1 mutant, and the rest was normally methylated as in the wild-type parent.

Methylation status was determined not only by DDM1 genotype but also by methylation status of the chromosome in the previous generation:
To explain why the methylation level of DNA in the F1 plants was intermediate between that of the two parents, three models were considered.

Model 1: The ddm1 mutation is semidominant and causes incomplete genome methylation in heterozygotes (DDM1/ddm1).

Model 2: When the genotype of a haploid gametophyte is ddm1, the mutation results in hypomethylated chromosomes, which remain hypomethylated after fertilization and during the development of the next sporophyte generation.

Model 3: Hypomethylated chromosome segments originating from a ddm1 mutant plant remain hypomethylated during meiosis and mitosis, resulting in hypomethylation of half of the chromosomes in F1.

To distinguish among these three models, methylation of 180-bp repeats was examined in the progeny resulting from backcrosses of F1 DDM1/ddm1 to DDM1/DDM1. If model 1 or model 2 is correct, methylation of each progeny plant should be determined only by the genotype of the DDM1 locus, plants with DDM1/DDM1 should have normally methylated chromosomes, and DDM1/ddm1 plants should have hypomethylated chromosomes. If model 3 is correct, most progeny should inherit hypomethylated 180-bp repeats (theoretically, 1 - ()5 = 31/32, because 180-bp repeats on five centromeres should segregate). All of the examined progeny from a F1 DDM1/ddm1 x DDM1/DDM1 cross (n = 7) and a reciprocal DDM1/DDM1 x F1 DDM1/ddm1 cross (n = 7) had the hypomethylated ladder of centromere repeat bands, although the intensity of the ladder differed from plant to plant (Figure 4). These results are consistent with model 3, but neither model 1 nor 2 can explain the results (possibility that all of the 14 plants are DDM1/ddm1, 2-14). Similarly, all 43 selfed F2 progeny from a F1 DDM1/ddm1 plant showed a hypomethylated ladder of bands (Figure 5), confirming the conclusion that the methylation status was not determined by the DDM1 genotype alone (possibility that none of the 43 F2 plants is DDM1/DDM1, 0.75-43). These results indicate that neither incomplete dominance (model 1) nor the effect of ddm1 mutant allele on the gamete (model 2) can explain the hypomethylated chromosomes in F1 plants and the progeny, whereas model 3 can explain all the results obtained.



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  		Figure 4. Southern analysis of DNA methylation patterns of the 180-bp repeat in wild-type (DDM1/DDM1), ddm1/ddm1, F1 (DDM1/DDM1 x ddm1/ddm1), and outcross progeny of the F1: (DDM1/DDM1 x ddm1/ddm1) x DDM1/DDM1 and DDM1/DDM1 x (DDM1/DDM1 x ddm1/ddm1).



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  		Figure 5. (A) Southern analysis of the DNA methylation pattern in the F2 family (from a cross ddm1/ddm1 x DDM1/DDM1) and BC-H2 family (progeny from a backcrossed DDM1/ddm1, see the lineage in Figure 1) using HpaII. (B) Summary of the methylation status of three genomic sequences in F2 and BC-H2. Generally, black and white boxes represent normal methylation and hypomethylation in the HpaII sites of the sequence examined, respectively. For Ta3, a gray box indicates one copy methylated and the other copy hypomethylated. For rDNA, a white box indicates no top band, in other words, all HpaII sites are unmethylated. A black box indicates that HpaII sites are methylated in at least one of the rDNA clusters. For 180-bp repeats (Cen.), a white box means a ladder of bands was observed, indicating that one or more copies of the repeats were hypomethylated in the HpaII sites. A black box means that no ladder was observed.

Is the ddm1 mutation completely recessive?
It is possible, however, that more than one mechanism is responsible for the hypomethylated chromosomes in F1 plants and their progeny. For example, inefficient de novo methylation and incomplete dominance of the DDM1 allele over the ddm1 allele together may result in hypomethylated chromosomes in DDM1 backgrounds. To examine whether ddm1 is completely recessive, in other words, whether a DDM1/ddm1 heterozygote plant can methylate genomic cytosine as efficiently as a DDM1/DDM1 homozygote, heterozygotes created by repeated backcrossing were used. Figure 1 illustrates the lineage of the materials used. We have previously suggested that the ddm1 mutations are recessive because repeated backcrossing of heterozygotes to wild-type parents generates plants that contain amounts of 5mC in TaqI sites that approach the amount found in wild-type plants (VONGS et al. 1993 Down). To see how complete the dominance of DDM1 allele over ddm1 allele is, the methylation of specific genomic sequences in the progeny from such a backcrossed DDM1/ddm1 was examined. Figure 5A shows the results obtained. In contrast to the F2 family (progeny of a heterozygote DDM1/ddm1 without backcrossing) in which all the plants had hypomethylated 180-bp repeats, only about one-quarter of the progeny from the backcrossed heterozygote (BC-H2) show the ladder of hypomethylated 180-bp repeats. Figure 5B summarizes the methylation status of the three genomic sequences in the F2 and the progeny of a backcrossed heterozygote. About one-quarter of the progeny of both types had hypomethylation in all of the sequences examined (i.e., 180-bp repeats, rDNA, and Ta3), suggesting that these individuals are ddm1/ddm1. The wild-type methylation pattern of the remaining three-quarters of the progeny of the backcrossed heterozygote indicates that DDM1/ddm1 plants are indistinguishable from DDM1/DDM1 plants in their ability to methylate all the sequences examined. The backcrossed heterozygote parent contained fully methylated chromosomes due to dilution of the hypomethylated chromosome by the normally methylated chromosomes during the repeated backcrossing. Furthermore, these results suggest that model 2 is not correct. The lack of detectable hypomethylation in DDM1/ddm1 plants demonstrates that hypomethylation does not occur in ddm1 gametophytes. The presence of hypomethylated chromosomes in all the F2 progeny indicates that hypomethylated chromosome segments can be inherited independently of the ddm1 mutation.

Stable inheritance of hypomethylation of rDNA and a retroelement in DDM1/DDM1 background:
The results shown in the previous sections indicate that one copy of wild-type DDM1 allele is sufficient for normal DDM1 function. Therefore, the observation that the methylation level of F1 is precisely intermediate between that of the two parents suggests that the rate of de novo methylation of unmethylated chromosome segments from a ddm1 mutant parent is extremely slow even in the wild-type DDM1 backgrounds. To test this interpretation, we estimate the rate of de novo methylation of hypomethylated sequences in DDM1/DDM1 background by Southern analysis. As hypomethylated sequences remain hypomethylated even when segregated from the potentiating ddm1 mutation, we could generate DDM1/DDM1 plants with unmethylated Ta3 or rDNA sequences from progeny of a cross between ddm1 mutants and wild-type plants.

We first investigated selfed progeny of a DDM1/DDM1 plant homozygous for hypomethylated nucleolus organizer regions (NORs) on chromosomes 2 and 4. From 12 F2 progeny of a cross: ddm1/ddm1 x DDM1/DDM1, 1 plant (95-89/10) with normally methylated Ta3 and partially hypomethylated rDNA was selected. Among 24 selfed progeny of 95-89/10, 5 plants had hypomethylated Ta3, indicating that 95-89/10 is DDM1/ddm1. All four copies of the rDNA loci (i.e., two copies each of nucleolus organizer regions NOR2 and NOR4) were hypomethylated in 7 plants of this family. One of these plants was determined to be DDM1/DDM1 by progeny tests. Forty-five progeny were examined in this family and no detectable remethylation of the rDNA sequences was detected, demonstrating stable inheritance of hypomethylation in a large number of rDNA repeat sequences (data not shown). All 45 progeny had normally methylated Ta3 sequences, confirming that the parent was DDM1/DDM1.

Similarly, hypomethylated Ta3 remained hypomethylated in DDM1/DDM1 background. From 12 F2 progeny of a cross ddm1/ddm1 x DDM1/DDM1, 4 plants with methylated rDNA and heterozygous for Ta3 methylation alleles (one copy of Ta3 was normally methylated and the other copy hypomethylated) were identified. One of them (95-89/6) was determined to be DDM1/DDM1 by progeny tests. The methylation status of the Ta3 locus was determined in 47 progeny of 95-89/6. Among the progeny, 11 plants were homozygous for the methylated Ta3 alleles, 27 plants were heterozygous, and 10 plants were homozygous for the hypomethylated Ta3 allele. Three plants homozygous for hypomethylated Ta3 alleles were used here to examine de novo methylation of hypomethylated Ta3 in a DDM1/DDM1 background. In all the examined progeny from these 3 plants (n = 24 + 24 + 23), the four HpaII sites (see Figure 3A) remained hypomethylated (data not shown). In conclusion, hypomethylation of Ta3 and rDNA induced by the ddm1 mutation was stably inherited even in DDM1/DDM1 background.

Stable inheritance of hypomethylation slowly induced by ddm1 mutation:
We have previously shown that ddm1 mutation induces a variety of developmental abnormalities by causing heritable changes on unlinked loci (KAKUTANI et al. 1996 Down; KAKUTANI 1997 Down). As this induction does not seem to be a random mutation event, we have proposed that it is due to ddm1-induced epigenetic change in other loci (KAKUTANI 1997 Down). Consistent with this interpretation, we found slowly accumulating hypomethylation in some of the single-copy sequences (KAKUTANI et al. 1996 Down), such as m105 and m118 (PRUITT and MEYEROWITZ 1986 Down). Most of the repeated sequences methylated in wild-type A. thaliana are hypomethylated in ddm1 mutants recovered in the segregating population. In contrast, although some of single-copy sequences such as Ta3 and telomere-associated sequence YpAtT1 are hypomethylated immediately as repeated sequences, most of the single-copy sequences are unaffected (VONGS et al. 1993 Down; RONEMUS et al. 1996 Down). These unaffected single-copy sequences generally become hypomethylated during the propagation by repeated selfing. To see if such slowly induced hypomethylation is also meiotically heritable, DNA of an F2 family from an interstrain cross between a plant with two hypomethylated m105 alleles (strain Columbia) and a wild-type plant (Landsberg) was analyzed. The origin of m105 allele could be detected by examining a BglII RFLP between the Landsberg and Columbia strain (CHANG et al. 1988 Down), as shown in the top of Figure 6A. The methylation status of the m105 sequence was detected using the methylation-sensitive restriction enzyme HpaII, as shown in the bottom of Figure 6A. Homozygous ddm1 mutants in each class were identified by hybridizing the filter with a cloned A. thaliana rDNA sequence (in parentheses in Figure 6B). The rDNA sequence becomes hypomethylated in ddm1 mutants before repeated selfing (VONGS et al. 1993 Down) and can be used for identifying ddm1 homozygotes immediately. As shown in Figure 6B, all of the 79 hypomethylated Columbia m105 alleles (38 from 19 Columbia m105 homozygotes and 41 from heterozygotes) remain hypomethylated through an outcross and a selfing. Out of these 79 hypomethylated alleles, 57 (30 from 15 Columbia m105 homozygotes and 27 from heterozygotes) were in a DDM1/- background. One demethylation event was observed in 1 of the 20 plants homozygous for the Landsberg m105 allele (Figure 6B). As this plant was ddm1/ddm1, the demethylation event is consistent with our previous observation that slow and stochastic hypomethylation of the m105 sequence occurs in ddm1 mutant backgrounds. In conclusion, hypomethylation of the m105 sequence was meiotically transmitted even upon segregation from the potentiating ddm1 mutation.



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  		Figure 6. Stable inheritance of hypomethylation of the m105 locus. DNA were prepared from F2 plants from an interstrain cross between a wild-type plant (Landsberg) and a ddm1 mutant plant (Columbia, line#6) with hypomethylation of the m105 locus. After Southern blotting, the membrane was probed with a 3.3-kb EcoRI subclone from the single-copy clone m105 (PRUITT and MEYEROWITZ 1986 Down). (A) Top: The origin of the m105 allele was detected by RFLP (CHANG et al. 1988 Down) after BglII cleavage. DDM1 and ddm1 plants in Columbia background showed the same band pattern (data not shown). Bottom: The methylation status of the m105 locus was detected by HpaII cleavage. The hypomethylation of the m105 allele had been generated during the self-pollination of the ddm1 mutant (KAKUTANI et al. 1996 Down) as shown in the two left lanes in the bottom. Methylated, ddm1 mutant plant before repeated selfing; Unmethylated, ddm1 mutant plant after six times of selfing. Both of them were in Columbia background; but DDM1 plants in Landsberg background also showed the top band (data not shown). (B) Methylation of m105 alleles in the F2 population. Number of plants is shown, with numbers of ddm1 plants in parentheses. Origin of m105: Col, Columbia homozygotes; Het, heterozygotes; La, Landsberg homozygotes. Methylation: -, signal at 2.1-kb position as plants 2 and 4; ±, signal at both 2.1 kb and 4.7 kb as plants 3, 6, and 7; +, signal at 4.7 kb as plants 1 and 5 of the F2.


*  DISCUSSION
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
*LITERATURE CITED

The results presented here indicate that (i) DDM1 gene function is not necessary during the gametophyte stage, (ii) the ddm1-2 mutation is completely recessive, and (iii) remethylation of sequences hypomethylated by the ddm1-2 mutation occurs extremely slowly, if at all, in wild-type DDM1 backgrounds.

A few rounds of DNA replication occur during the gametophyte stage of development: three for the female gametophyte to make egg cells and two for the male gametophyte to make sperm nuclei. If the maintenance methylation machinery does not function in a haploid ddm1 gamete, a substantial loss of DNA methylation should result, but this is not the case. One possible interpretation for the dispensability of DDM1 gene function in the gametes is that the function is developmental-stage-specific and not required in the gametophyte stage. An alternative interpretation is that sporophytic DDM1 gene product remaining in the gametes is sufficient for the normal DDM1 function.

A more important conclusion from the results presented here is that ddm1-induced hypomethylation in the majority of sequences in the Arabidopsis genome, both repeated and single-copy sequences, can be stably inherited through both mitotic and meiotic cell divisions. This indicates that epigenetic information, in the form of differential DNA methylation, can be transmitted between plant generations. Transgenic Arabidopsis plants expressing the MET1 gene in an antisense orientation (MET1as) exhibit a reduction in genomic methylation (FINNEGAN et al. 1996 Down; RONEMUS et al. 1996 Down). In progeny of the transgenic plants, hypomethylation of the 180-bp repeats is transmitted even to the plants losing the transgene (FINNEGAN et al. 1996 Down; RONEMUS et al. 1996 Down). The transmission was, however, not fully penetrant, and remethylation of at least the 180-bp repeats occasionally occurs in both outcross progeny (RONEMUS et al. 1996 Down) and selfed progeny from hemizygotes (J. FINNEGAN, personal communication) that do not inherit the transgene. Thus, DNA remethylation efficiency may differ between ddm1 and MET1as plants. Similarly, developmental abnormalities induced by MET1 antisense expression are often unstable compared to those induced by ddm1 mutation. For example, phenotypic revertants were occasionally found among outcross progeny of late-flowering MET1as plants without the transgene (RONEMUS et al. 1996 Down), in contrast to the stable inheritance of late-flowering traits in outcross progeny from ddm1 mutant (KAKUTANI 1997 Down).

The basis for these observed differences in stability is not clear. It may reflect a difference in the distribution of the hypomethylated sequences and the extent to which the sequences are hypomethylated. In ddm1 mutants, repeated sequences are more effectively hypomethylated than single-copy sequences (VONGS et al. 1993 Down), while both single-copy and repeated sequences are hypomethylated in MET1as lines (RONEMUS et al. 1996 Down). If there were positive cooperativity in de novo methylation of the endogenous genes, the extent of hypomethylation of particular genomic regions would affect the remethylation efficiency.

Alternatively, the effect of ddm1 mutation might be qualitatively different from that of MET1as. The DDM1 gene product is not likely to be DNA methyltransferase, because nuclear extracts of the ddm1 mutant have as much DNA methyltransferase activity as those of the wild type, and the ddm1 gene does not map to any known methyltransferase structural gene (KAKUTANI et al. 1995 Down). It is possible that the hypomethylation is a secondary effect of ddm1 mutation, and the primary effect is on another epigenetic state such as chromatin structure. The primary effect of ddm1 mutation could be more stably inherited than the hypomethylation itself. Meiotically stable inheritance of the epigenetic chromatin state has been found in fission yeast for both the centromere (EKWALL et al. 1997 Down) and mating-type locus (GREWAL and KLAR 1996 Down), despite a lack of detectable DNA methylation in its genome (ANTEQUERA et al. 1984 Down).

Mutation of ddm1 has recently been found to release silencing of repeated hygromycin phosphotransferase transgenes driven by the 35S promoter (MITTELSTEN-SCHEID et al. 1998 Down), repeated CHS transgenes (FURNER et al. 1998 Down), and the endogenous PAI2 gene (JEDDELOH et al. 1998 Down). Molecular and genetic characterization of ddm1, MET1as, and other recently identified Arabidopsis mutants affecting gene silencing (DEHIO and SCHELL 1994 Down; FURNER et al. 1998 Down; MITTELSTEN-SCHEID et al. 1998 Down) would be useful for further understanding the basis for the inheritance of epigenetic states in plant genes.


*  ACKNOWLEDGMENTS

We thank M. Saito for technical assistance. We give special thanks to L. Medrano, E. Meyerowitz, A. Konieczny, and D. Voytas for m105 and Ta3 clones, and to J. Finnegan, H. Higo, K. Higo, R. Martienssen, M. Tahir, and T. Toyama for comments on the manuscript. This work was supported by grants from Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation and the Science and Technology Agency of the government of Japan to T.K.

Manuscript received August 20, 1998; Accepted for publication October 12, 1998.


*  LITERATURE CITED
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

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