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Mobilization of transposons by a mutation abolishing full DNA methylation in
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Author: A. Miura
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"................................................................. Mobilization of transposons by a mutation abolishing full DNA methylation in Arabidopsis Asuka Miura*?, Shoji Yonebayashi??, Koichi Watanabe�, Tomoko Toyama�, Hiroaki Shimadak & Tetsuji Kakutani*?� * National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan ? National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan � CREST, Japan Science and Technology Corporation, Tokyo 101-0062, Japan kScience University of Tokyo, Noda, Chiba 278-8510, Japan ? These authors contributed equally to this work. .............................................................................................................................................. A major component of the large genomes of higher plants and vertebrates comprises transposable elements and their deriva- tives, which potentially reduce the stability of the genome 1 .It has been proposed that methylation of cytosine residues may suppress transposition, but experimental evidence for this has been limited 2?5 . Reduced methylation of repeat sequences results from mutations in the Arabidopsis gene DDM1 (decrease in DNA methylation) 6 , which encodes a protein similar to the chromatin- remodelling factor SWI2/SNF2 (ref. 7). In the ddm1-induced hypomethylation background, silent repeat sequences are often reactivated transcriptionally, but no transposition of endogenous elements has been observed 8?11 . A striking feature of the ddm1 mutation is that it induces developmental abnormalities by causing heritable changes in other loci 12,13 . Here we report that one of the ddm1-induced abnormalities is caused by insertion of CAC1, an endogenous CACTA family transposon. This class of Arabidopsis elements transposes and increases in copy number at high frequencies specifically in the ddm1 hypomethylation background. Thus the DDM1 gene not only epigenetically ensures proper gene expression 13?16 , but also stabilizes transposon behaviour, possibly through chromatin remodelling or DNA methylation. To study the molecular basis for ddm1-induced developmental abnormalities, we identified the mutated gene responsible for one of them, clam (clm), which is characterized by lack of elongation in shoots and petioles 12 (Fig. 1a). This phenotype was initially unstable, and phenotypically normal sectors were occasionally observed (Fig. 1b?f). In subsequent generations, the clm phenotype stabilized in some of the progeny families and was inherited as a recessive mendelian trait, which could be mapped genetically 12 .By genotyping 926 chromosomes from a mapping cross, we narrowed the clm locus to a 64-kilobase (kb) region in bacterial artificial chromosome (BAC) clone T3A5 (GenBank AL132979) on chromosome 3. This region contains the DWF4 gene, encoding 22-a-hydroxylase, a 513-amino-acid protein that mediates the biosynthesis of brassinosteroid, a regulator of cell elongation 17 . Complementation tests indicated that clm is allelic to the dwf4-4 mutation (see Supplementary Information). The sequence of the DWF4 gene in the stable clm plant revealed a 4-base-pair (bp) insertion mutation in the second exon at nucleotide +527 from the translation start site. This insertion converts TAG to TAGC- TAG, creating a stop codon after the 149th amino acid. Although this defect is probably the cause of the stable clm phenotype, it cannot account for the instability of the clm phenotype in initial generations. letters to nature 212 NATURE | VOL 411 | 10 MAY 2001 | www.nature.com Figure 1 Stable and unstable clm phenotypes. a, A wild-type Columbia plant showing expanded leaves (right) and a clm homozygous plant with a defect in leaf and stem elongation (left). Both are 35 days old. b?f, clm homozygous plants with (c, e, f )or without (b, d) reversion sectors. Plants were grown on soil (a) or nutrient agar plates 12 (b?f ). Scale bar, 10 mm. Wild type (Columbia) Original CAC1 locus DWF4 locus D1 + R1 D1 D2F1 A1 A2 R1 D1 + R1 A1 + R1 A1 + R1 D2 + F1 D2 + F1 A2 + F1 A2 + F1 Unstable clm Figure 2 Transposition of CAC1 element from the original donor site to the DWF4 locus. Arrows indicate primers used for PCR. The DNA length markers are 19.3, 7.74, 5.53, 3.14, 2.69 and 2.32 kb. The sizes of transposon and the starting position (but not the length) of arrows for primers reflect the physical length. � 2001 Macmillan Magazines Ltd In the unstable clm plant, we used genomic Southern analysis to identify a several-kilobase insertion in the DWF4 gene (data not shown). We amplified the inserted DNA from unstable clm plants using the suppression polymerase chain reaction (PCR) technique 18 . Sequences obtained from each side of the insertion (523 and 154 nucleotides) showed exact matches to the 39 and 59 ends of a 8,479-bp sequence in chromosome 2 (nucleotides 52,296 to 60,774 in GenBank AC005897). No other exact match was found in the Arabidopsis genome database (http://www.arabidopsis.org/ blast/). This 8,479 sequence has features typical of the CACTA family of transposons, which include maize Spm/En and snap- dragon Tam1 elements: the 59 and 39 ends have the conserved terminal inverted sequence CACTACAA, and the internal region includes a predicted gene (GenBank AAC97237) with similarity to the putative snapdragon transposase Tnp2 (ref. 19). To confirm the transposition of this sequence, we amplified two DNA fragments covering the entire element using internal sequences and the DWF4 gene sequence as primers (Fig. 2). The estimated length of the product and the restriction digestion pattern were identical for the original copy and the insert in the DWF4 gene, suggesting that the full-length element had transposed from chromosome 2 to the DWF4 gene on chromosome 3. This transposon, designated CACTA1 (CAC1), was responsible for the unstable clm phenotype; letters to nature NATURE | VOL 411 | 10 MAY 2001 | www.nature.com 213 a c b A H3 H3 H3 12 3 45 6 78 9 101 121314151617 1 kb WT clm H3 R5 R5R5 R5 R5 C CAC1 CAC2 CAC3 CAC4 ddm1 DDM1 BD Figure 3 Transposition of CAC elements in the ddm1 mutant background. a, The CAC transposon family. Open boxes in CAC2 and CAC4 show the internal deletions. Vertical bars show the restriction sites: H3, Hind III; R5, EcoRV; other bars above the elements, Hha I; below the elements, Hpa II. The transpositions of CAC elements were examined by Southern blot analysis using EcoRV and probe A. b, clm and wild-type Columbia (WT) plants. c, The ddm1 and wild-type DDM1 lines self-pollinated six to seven times in parallel 12 . Five out of 12 DDM1 lines are shown. The DNA length markers are 19.3, 7.74, 5.53 and 4.25 kb. 1 1H1 2K2 2K4 2M2 2K3 2K1 CAC4 CAC1 CAC3 CAC2 2M3 2F1 2F2 2G1 2L1 2I1 1D4 1D2 1K1 1K2 1F1 1D1 1D3 1C2 1C1 1C3 1C4 1E1 1E2 1L2 1L1 1G1 2345 Figure 4 Transposition of CAC1 and CAC2 elements to unlinked loci. Sequences flanking the transposed copies were determined by suppression PCR (for determined sequences and methods see Supplementary Information). The red and blue loci show CAC1 and CAC2 copies, respectively. The transposed copies with second character D, E, F, G, H, I, K, L, M and C are from the ddm1 lines shown in lanes 10, 9, 8, 7, 6, 5, 3, 2 and 1 of Fig. 3c and the clm line (Fig. 3b), respectively. abProbe B 12 34 56 78 9101112 EcoRV HindIII + Hhal HindIII + HpalI HindIII + Hhal HindIII + HpalI HindIII WT ddm1 Probe D Probe C Figure 5 The ddm1-specific hypomethylation and RNA accumulation of CAC elements. a, Methylation-sensitive restriction enzymes (HpaII or HhaI) and probes B, C, D (Fig. 3a) were used to compare the methylation status of CAC elements between ddm1 (even lanes) and Columbia wild-type (odd lanes) plants. The ddm1 plant is before the repeated self-pollination (four generations before the plant shown in lane 10 of Fig. 3c). It still keeps the donor copies of CAC elements (lane 2). The DNA length markers are 19.3, 7.74, 5.53, 3.14, 2.69 and 2.32 kb. b, RNA blot analysis. Probe A (Fig. 3a) was used to detect CAC transcript in wild-type and ddm1 (clm) plants. The RNA length markers are 6, 4 and 3 kb. Bottom panel, ribosomal RNA on the filter stained with methylene blue. � 2001 Macmillan Magazines Ltd in the sector without the clm phenotype, restoration of the insertion site to the normal structure was found by sequencing PCR-ampli- fied templates (see Supplementary Information). The Arabidopsis genome contains three additional sequences distinct from but significantly similar to CAC1; their terminal 200 bp are 90?98% identical (Fig. 3a). As they are likely to comprise a new transposon family with CAC1, we designated them CAC2 (GenBank AC069160, nucleotides 34,626?38,790), CAC3 (Gen- Bank AC006429, nucleotides 85,404?76,949) and CAC4 (GenBank AC027135, nucleotides 72,903?64,858). Genomic Southern analy- sis using the methylation-insensitive restriction enzyme EcoRV revealed that CAC elements transposed and increased in the copy number in the clm line (Fig. 3b). To see whether the mobilization of the CAC elements is a general phenomenon in the ddm1 background, we examined genomic DNA of twelve ddm1 lines that had independently and randomly self- pollinated six to seven times 12 (Fig. 3c). Eleven out of twelve ddm1 lines exhibited changes in the band pattern of CAC elements, and six showed several-fold increases in band number (up to . 20). Transposition of CAC1 and CAC2 was confirmed by sequencing the genomic regions flanking them. Both were found to transpose to unlinked loci throughout the genome (Fig. 4). In contrast, we never observed the change in EcoRV banding pattern for the CAC elements in any of the 12 control wild-type DDM1 lines that had independently self-pollinated seven times in parallel (Fig. 3c), indicating that the mobilization of CAC elements is a consequence of the ddm1 mutation. Mobilization of transposons is often associated with hypo- methylation and transcriptional activation 20?23 . Indeed, the CAC elements were hypomethylated and transcriptionally activated in ddm1 plants (Fig. 5), like other repeated sequences 8?11 . PCR with reverse transcriptase and sequencing confirmed that at least the CAC1 transcript was highly accumulated in ddm1 (unpublished data). However, it remains to be seen whether transcriptional activation alone is sufficient to trigger the ddm1-induced trans- positional activation of CAC elements; the ddm1 mutation might also affect the epigenetic chromatin state, as the gene encodes a protein similar to the chromatin-remodelling factor SWI2/SNF2. Maize transposons were the first plant genes found to be epigenetically regulated 24,25 , and the epigenetic control of the maize Spm by cis elements and transposon-encoded Tnp proteins have been studied extensively 21 . Little is known, however, about controlling host factors. We report here endogenous Arabidopsis elements that are mobilized by a host gene mutation affecting epigenetic states. In addition to ddm1, a variety of Arabidopsis mutants in gene silencing and methylation 26?32 provide excellent systems to dissect mechanistically the transpositional activation of CAC elements, which should further clarify the role of epigenetic controls on genome evolution. Note added in proof: It has been shown recently that Arabidopsis elements similar to Robertson?s Mutator in maize become active in the ddm1 mutant 33 . M Methods The growth conditions, preparation of plant genomic DNA and mapping of the CLM locus have been described 12 . The suppression PCR method was as described 18 . We used EcoRV-digested genomic DNA for adapter ligation and subsequent amplification. The sequences of primers used for mapping the CLM locus, sequencing the DWF4 gene, suppression PCR reactions, transposition monitoring (Fig. 2) and probe amplification (Fig. 3) are listed as Supplementary Information or on http://www.nig.ac.jp/labs/AgrGen/ cacta-Nature.html. The Supplementary Information also includes sequences flanking the transposed CAC copies (Fig. 4). Received 11 January; accepted 21 February 2001. 1. SanMiguel, P. et al. Nested retrotransposon in the intergenic region of maize genome. Science 274, 765?768 (1996). 2. Yoder, J. A., Walsh, C. P. & Bestor, T. H. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet. 13, 335?340 (1997). 3. Martienssen, R. Transposons, DNA methylation and gene control. Trends Genet. 14, 263?264 (1998). 4. Walsh, C. P., Chaillet, J. R. & Bestor, T. H. Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nature Genet. 20, 116?117 (1998). 5. Matzke, M. A., Mette, M. F., Aufsatz, W., Jakowitsch, J. & Matzke, A. J. Host defenses to parasitic sequences and the evolution of epigenetic control mechanisms. Genetica 107, 271?287 (1999). 6. Vongs, A., Kakutani, T., Martienssen, R. A. & Richards, E. J. Arabidopsis thaliana DNA methylation mutants. 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Modification of the 59 untranslated leader region of the maize Activator element leads to increased activity in Arabidopsis. Mol. Gen. Genet. 245, 608?615 (1994). 24. McClintock, B. Chromosome organization and genic expression. Cold Spring Harbor Symp. Quant. Biol. 16, 13?47 (1951). 25. McClintock, B. The suppressor-mutator system of control of gene action in maize. Carnegie Institution of Washington Year Book 57, 415?429 (1958). 26. Finnegan, E., Peacock, J. & Dennis, E. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proc. Natl Acad. Sci. USA 93, 8449?8454 (1996). 27. Ronemus, M., Galbiati, C., Ticknor, J., Chen, J. & Dellaporta, S. Demethylation-induced developmental pleiotropy in Arabidopsis. Science 273, 654?657 (1996). 28. Furner, I. J., Sheikh, M. A. & Collett, C. E. Gene silencing and homology-dependent gene silencing in Arabidopsis. Genetic modifiers and DNA methylation. Genetics 149, 651?662 (1998). 29. Amedeo, P., Habu, Y., Afsar, K., Scheid, O. M. & Paszkowski, J. Disruption of the plant gene MOM releases transcriptional silencing of methylated genes. Nature 405, 203?206 (2000). 30. Mourrain, P. et al. Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101, 533?542 (2000). 31. Dalmay, T, Hamilton, A., Rudd, S., Angell, S. & Baulcombe, D. C. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101, 543?553 (2000). 32. Fagard, M. et al. AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proc. Natl Acad. Sci. USA 97, 11650?11654 (2000). 33. Singer, T., Yordan, C. & Martienssen, R. A. Robertson?s Mutator transposons in A. thaliana are regulated by the chromatin-remodeling gene Decrease in DNA methylation (DDM1). Genes Dev. 15, 591?602 (2001). Supplementary information is available on Nature?s World-Wide Web site (http://www.nature.com) or as paper copy from the London editorial office of Nature. Acknowledgements We thank K. Munakata for technical assistance, A. Miyao for advice on the suppression PCR technique, and R. Martienssen and Y. Hiromi for comments on the manuscript. We acknowledge ABRC at the Ohio State University for the BAC clones. Correspondence and requests for material should be addressed to T.K. (e-mail: tkakutan@lab.nig.ac.jp). The CAC1, CAC2, CAC3 and CAC4 sequences are deposited in GenBank under accession numbers AB052792, AB052793, AB052794 and AB052795, respectively. letters to nature 214 NATURE | VOL 411 | 10 MAY 2001 | www.nature.com� 2001 Macmillan Magazines Ltd "
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