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Corresponding author: Reinhard Kunze, Botanisches Institut II, Universität zu Köln, Gyrhofstrasse 15, 50931 Cologne, Germany., reinhard.kunze{at}uni-koeln.de (E-mail)
Communicating editor: V. SUNDARESAN
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In maize the transposable elements Activator/Dissociation (Ac/Ds) transpose shortly after replication from one of the two resulting chromatids ("chromatid selectivity"). A model has been suggested that explains this phenomenon as a consequence of different affinity for Ac transposase binding to holo-, hemi-, and unmethylated transposon ends. Here we demonstrate that in petunia cells a holomethylated Ds is unable to excise from a nonreplicating vector and that replication restores excision. A Ds element hemi-methylated on one DNA strand transposes in the absence of replication, whereas hemi-methylation of the complementary strand causes a >6.3-fold inhibition of Ds excision. Consistently in the active hemi-methylated state, the Ds ends have a high binding affinity for the transposase, whereas binding to inactive ends is strongly reduced. These results provide strong evidence for the above-mentioned model. Moreover, in the absence of DNA methylation, replication enhances Ds transposition in petunia protoplasts >8-fold and promotes formation of a predominant excision footprint. Accordingly, replication also has a methylation-independent regulatory effect on transposition.
THE maize transposable element Activator (Ac) is the prototype element of the class of "hAT" eukaryotic transposons. These elements are characterized by common conserved transposase segments including a unique signature motif (
In the maize sporophyte Ac transposes predominantly during or shortly after replication, and only one of the two resulting daughter elements is transposition competent (chromatid selectivity;
However, DNA methylation-mediated replication dependence cannot completely explain the behavior of Ac/Ds transposition. In several studies it was found that in a transient assay Ac/Ds element excision from extrachromosomal geminivirus vectors in maize, barley, wheat, and rice cells is dependent on vector replication, although the transfected DNAs were not C-methylated (
In this article we studied the correlation between Ds transposition in petunia cells and DNA replication by use of a replicon sequence derived from the genome of the monopartite geminivirus tomato yellow leaf curl virus (TYLCV;
We show that transposition of a Ds element from an extrachromosomal vector in petunia cells is regulated by DNA replication in a methylation-dependent and independent mode. Holomethylation completely inhibits Ds excision from a nonreplicating plasmid, whereas Ds transposition is restored by replication. Moreover, Ds elements that are hemi-methylated on one DNA strand transpose in the absence of replication, whereas methylation on the complementary DNA strand results in at least 6.3-fold reduced excision frequencies. These data strongly support the transposition model of
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasmid constructs:
pMiDsf1 and pMiDs<f1 were derived from pNT150Ds (
Ds excision assay in petunia protoplasts and DNA extraction:
Transpositional activity of Ds reporter plasmids in petunia cells was assessed as described by
Preparation of holo- and hemi-methylated reporter plasmids:
To generate holomethylated plasmids, pMiDsf1 and pMiDsf1-RI were treated with M-SssI (New England Biolabs, Beverly, MA) for 16 hr at 37° as recommended by the manufacturer. After phenol extraction, the completeness of the reaction was confirmed by HpaII and BsiEI digestion. The mock-methylated plasmids were prepared under the same conditions except for the presence of M-SssI.
The preparation of locally hemi-methylated plasmid DNA is depicted in Fig 5. Phagemid single-stranded DNA was isolated from pMiDsf1, pMiDs<1f, and pMiDsf1-RI as described by
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Single-stranded methylated Ds element was annealed to single-stranded phagemid DNA at a molar ratio 1:1 in 50 mM NaCl, 10 mM Tris-HCl (pH 8), and 5 mM EDTA by heating for 3 min at 95° and allowing to cool down to 25° over 2 hr. The hybridization product was purified by gel filtration in Sephacryl micro spin columns (Amersham-Pharmacia, Freiburg, Germany).
Approximately 60 ng of hybridization product was filled in at 72° for 1 hr, in a 50-µl reaction mixture containing 20 mM Tris-HCl (pH 8.8), 2 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 0.1 mg/ml nuclease-free BSA, 0.05 mM of each dNTP, and 1.25 units of Pfu DNA polymerase (Stratagene). The product was purified by gel filtration, and the remaining nick was closed by treatment with 2 units T4 DNA Ligase (Roche Biochemicals, Basel, Switzerland) overnight at 16° in a final volume of 50 µl. Each ligation contained 
0.3 µg of DNA. Before transfection into petunia protoplasts the constructs were extracted with phenol and purified by ethanol precipitation.
Determination of Ds excision frequency and isolation of excision footprints by PCR:
Total DNA was prepared twice from four independent batches of protoplasts that were cotransfected with pNT600-10.ATG and pMiDsf1 or pMiDsf1-RI, respectively. On each DNA preparation two to four PCRs were performed, and the products were ligated into pCR2.1-Topo (Invitrogen, San Diego) and transformed into Escherichia coli. For each transformation, the plasmid inserts from 7 to 18 colonies were sequenced. To amplify the empty Ds donor site (300-bp excision product), PCRs were performed on 150 ng DNA using primers Pr_1 (GGATACTTACGTCACGTCTTGCGCACTGAT) and Pr_2 (CCACAGTTTTCGCGATCCAGACTGAA). The reaction mixture (50 µl) contained 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.4 µM of each primer, and 2.5 units Taq DNA polymerase.
Amplification was carried out by incubating for 5 min at 94°, followed by 25 cycles of 94°, 30 sec/60°, 20 sec, and one final step of 5 min at 72°. With the same primer pair a 250-bp fragment was coamplified from plasmid pNT150, which lacks the Ds insertion and which was added (1 pg) as a control to each reaction tube.
Sixty-five excision footprints each from pMiDsf1 and pMiDsf1-RI, originating from 20 independent PCR reactions and four transfection assays, were cloned into pCR2.1-TOPO (Invitrogen) and sequenced.
DNA analysis by gel blot hybridization:
For gel blot analyses DNA was size fractionated by 1% agarose gel electrophoresis and transferred to positively charged nylon membranes (Hybond-N+). Hybridization and washing were done using standard conditions (
-32P]dCTP.
Gel retardation assays:
Gel retardation assays were performed using renatured TPase103-465 essentially as described (
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Hemi-methylated Ds ends were prepared by hybridization of methylated and unmethylated single-stranded Ds DNAs, followed by DraI digestion of the hybridization products. Methylated and unmethylated single-stranded Ds DNAs were prepared as described in Fig 5, steps two to four, except that 5mdCTP or dCTP were included in the PCR reaction mixture. The fragments were radiolabeled by 5' phosphorylation using [
-32P]ATP and T4 kinase. The 5' end segments I, II, and III were generated by combinatorial hybridization of complementary methylated or unmethylated radiolabeled oligonucleotides.
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Replication promotes Ds excision:
In monocots it was found that excision of Ds elements from geminivirus vectors requires replication (
The replication activity of each construct in petunia cells was tested by cotransfecting protoplasts with a TPase expression plasmid and Dam-methylated reporter plasmid DNA. At different time points after transfection DNA was reisolated, subjected to MboI digestion, and analyzed by gel blotting. Plasmid DNA propagated in Dam+ bacteria is resistant to MboI, whereas replication in plant cells leads to MboI sensitivity due to the loss of Dam methylation. The reisolated pMiDsf1 DNA is resistant to MboI digestion, indicating that the plasmid does not replicate in the plant cells (Fig 1B, lanes 5–7). In contrast, MboI releases increasing amounts of the 1024-bp digestion fragment from pMiDsf1-RI DNA isolated 30 and 60 hr after transfection (Fig 1B, lanes 2–4). To ensure that the replication activity of pMiDsf1-RI depends on the functionality of the viral replicon, we tested two plasmids with defects in the cis- and trans-acting TYLCV replication sequences, respectively. In pMiDsf1-rI the C1 gene encoding the trans-acting Rep protein is destroyed, and in pMiDsf1-R the intergenic region, containing the promoter and the Rep protein binding sites (IR), is deleted. Both control plasmids are unable to replicate in the plant cells (Fig 1C, lanes 2–3).
Ds excision from the reporter plasmids is accompanied by reversion to GUS activity. Petunia protoplasts cotransfected with a TPase expression plasmid and pMiDsf1, pMiDsf1-RI, pMiDsf1-rI, or pMiDsf1-R, respectively, were spread on filters, stained, and inspected for frequency and staining intensity of GUS-positive (blue) cells. No Ds excision is detectable 5 min after transfection. Thirty hours after transfection Ds excision is observed with all four plasmids (Table 1). This confirms that in petunia Ds is able to transpose in the absence of replication. Remarkably, the frequency of blue staining cells with the replicating plasmid pMiDsf1-RI is two to three times higher compared to the three nonreplicating plasmids, and the staining intensity of the protoplasts is significantly higher (data not shown).
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The GUS assay allows no distinction regarding whether these two effects result from an increase in Ds excision frequency or from replicative amplification of plasmids before and after Ds excision. We therefore performed a quantitative gel blot hybridization analysis. DNA from protoplasts was digested with AseI, blotted, and hybridized with a probe that detects three fragments: a 2807-bp vector fragment whose abundance reflects the total amount of plasmid, a 1798-bp fragment from plasmids that still contain the Ds, and a 1089-bp fragment from plasmids where Ds has been excised (Fig 2A). The ratio of the 2807-bp band intensities between pMiDsf1- (lane 4) and pMiDsf1-RI-transfected cells (lane 5) reflects the overall difference in plasmid content due to replication ("copy number effect") times aliquot size. The ratio of the 1089-bp band intensities (ß) corresponds to the product of copy number effect times change in excision frequency times aliquot size. The ratio /ß indicates the factor by which the Ds excision frequency is enhanced (or reduced) by replication of the reporter plasmid pMiDsf1-RI. Quantification of the respective bands (Fig 2B; upon overexposure, the Ds excision band in lane 5 becomes visible as a faint signal) led to the conclusion that the Ds excision frequency in petunia cells is promoted at least eightfold by replication of the host plasmid.
To determine whether the reduced Ds excision frequency from the nonreplicating host plasmid correlates with de novo methylation in the petunia cells of the Ds and/or the whole plasmid, DNA was isolated from the cells 30 hr after transfection, digested with the methylation-sensitive enzyme BsiEI, and analyzed by gel blot hybridization. No de novo methylation of any BsiEI sites in the Ds or the flanking plasmid sequences was detected (data not shown).
Transposon footprint formation is influenced by replication:
Excision of Ac and Ds elements is associated with the formation of characteristic transposon "footprints" that are the products of DNA end joining and repair reactions. We wanted to investigate whether excision footprint formation is also influenced by replication. Using standard conditions, the Ds excision products from nonreplicating plasmids were not detectable by gel blot hybridization or by PCR. We therefore developed optimized reaction conditions that allow the selective amplification of rare excision site sequences among a large excess of "wild-type" plasmids (see MATERIALS AND METHODS).
Sixty-five footprints were amplified, cloned, and sequenced from the replicating and the nonreplicating reporter plasmids, respectively (Fig 3). The sampling strategy for the clones assures that these sequences are derived from independent plasmid molecules (see MATERIALS AND METHODS). However, we cannot exclude the possibility that individual excision products were preferentially amplified in the protoplasts by postexcisional plasmid replication. In the presence of replication, one predominant footprint ("gc") is formed in 84% of excision events, 8% contain a second type ("g"), and 8% of footprints have individual sequences (Fig 3A). The gc and g footprints dominate with frequencies of 51 and 15% also in the absence of replication; however, the frequency of deviating and individual footprints is almost fivefold higher (34%; Fig 3B). If we assume that most of the analyzed products are derived from independent excision reactions, these data suggest that replication not only facilitates transposon excision but also has an influence on excision site repair. Possibly, the efficiency and fidelity of the reactions leading to the formation of the predominant footprints are enhanced.
Methylation of Ds inhibits transposition:
Genetic and molecular experiments led to the hypothesis that in maize the DNA methylation status of Ac/Ds elements determines their transpositional competence (
We therefore directly tested the effects of Ds methylation in the presence and absence of replication. According to the proposed model, C-methylation of TPase binding sites on both DNA strands should inhibit transposition in the absence of replication. pMiDsf1 and pMiDsf1-RI plasmids, isolated from Dam+ bacteria, were treated with M-SssI that converts all cytosine residues in CpG motifs to 5mC. In Ac/Ds elements almost all CpG motifs are located in the cis-acting terminal regions that include the subterminal TPase binding sites (
To determine whether Ds is able to excise from methylated pMiDsf1 a PCR analysis was performed [the GUS reversion assay is not suitable here because methylation by M-SssI inactivates the GUS gene (data not shown)]. A PCR primer pair was used that coamplifies the empty Ds donor site (0.3-kb excision product; Fig 4B, x), the Ds-containing donor site (1-kb product; Fig 4B, s), and a 250-bp fragment from plasmid pNT150, which lacks the Ds insertion in the GUS gene and was added as a control in transfections with pMiDsf1 (Fig 4B, Fig C). Because Ds excises only in a small fraction of the transfected plasmids and thus in the reaction the rare empty donor sites have to compete against a large excess of Ds-containing donor sites, a PCR cycle was chosen that conditions underrepresentation of the donor site among the PCR products (see MATERIALS AND METHODS). PCR products were visualized by gel blot hybridization and quantified. In contrast to the unmethylated plasmid (Fig 4B, lane 2), with methylated pMiDsf1 no empty donor site is detectable (lane 1). With the replicating pMiDsf1-RI the empty donor site signals from methylated and unmethylated plasmids are similar (Fig 4B, lanes 3–4). In summary, these results prove that C-methylation of a Ds element severely inhibits transposition and that this inhibition is overcome by replication.
Hemi-methylation determines the transposition competence of Ds elements:
It has been suggested that the chromatid selectivity of Ac/Ds transposition is caused by differential transposition competence of the two transiently hemi-methylated daughter elements resulting from replication of a holomethylated element (
The results of three independent experiments are shown in Table 2. The Ds element that is hemi-methylated on the top strand (pMiDs<f1-hemi) achieves on average a 6.3-fold higher number of GUS-positive cells than the bottom-strand-methylated Ds (pMiDsf1-hemi). The apparent excision frequency of the top-strand-methylated Ds is as high as that of the mock-hemi-methylated Ds (pMiDsf1-mock). These results corroborate the proposed model, and they prove that the top-strand-hemi-methylated Ds is fully transposition competent, whereas C-methylation on the bottom strand severely inhibits transposition. Additional evidence that the C-methylation is responsible for the inactivity of pMiDsf1-hemi was gained by showing that replication completely restores the transposition competence of a bottom-strand-methylated transposon, pMiDsf1-RI-hemi (Table 1).
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TPase binding to transposition-competent and -incompetent Ds elements:
Ac/Ds elements contain multiple short TPase binding motifs in both ends that contain a 5'-CCG-3' sequence (Fig 6A;
To this end it remained an open question whether TPase binding affinity to these synthetic, hemi-methylated concatemers reflects the binding properties to hemi-methylated transposon ends (i.e., the TPase binding motifs in their native sequence environment). We therefore analyzed the in vitro TPase binding reaction to hemi-methylated, transposition-competent or -incompetent Ds ends. We separately synthesized the complete Ds 5' and 3' ends and the three TPase binding site clusters I, II, and III from the 5' end (Fig 6A) in the unmethylated ("u"), holomethylated ("M"), and both alternate hemi-methylated states ("tm" and "bm") and determined their in vitro binding affinities to a TPase103-465 protein fragment by gel shift assays (Fig 6B). TPase103-465 is an N- and C-terminally truncated Ac TPase protein containing the complete bipartite DNA-binding domain (
TPase binds efficiently to the unmethylated Ds 3' end (Fig 6B, lane 20). Remarkably, TPase affinity is even increased when the 3' end fragment is hemi-methylated on the top strand (Fig 6B, lane 19). This is the transposition-competent state (Table 2). In that hemi-methylation state, 12 out of 14 subterminal TPase binding site motifs are in the 5'-mCmCG-3'/5'-CGG-3' configuration and therefore presumably able to bind TPase protein (Fig 6C, tm). In the alternate hemi-methylation state, which correlates with very low transposition activity, the 3' end is only weakly bound (Fig 6B, lane 18). This is in accordance with the assumption that in this state TPase can recognize only two widely separated TPase binding site motifs (Fig 6C, bm). If the 3' end is methylated on both strands, it is bound by TPase only in traces (Fig 6B, lane 17).
At the 5' end the situation is different. As is the case at the 3' end, holomethylation prevents TPase binding (Fig 6B, lane 1). However, both alternatively hemi-methylated 5' ends are bound similarly well (lanes 2–3), a little less efficiently than the unmethylated 5' end (lane 4). The 5' end central cluster II has opposite orientations of TPase binding sites relative to clusters I and III. We therefore individually tested the occupation of each of these clusters with TPase. In the unmethylated state, clusters I and II are moderately well bound (lanes 8 and 12), and cluster III is more efficiently complexed (lane 16). Methylation on both DNA strands completely inhibits TPase binding to all three fragments (lanes 5, 9, and 13). The terminal cluster I is most efficiently bound in the transpositionally competent, top-strand-hemi-methylated state (lane 7). The innermost cluster III is also recognized in this state (lane 15), whereas the central cluster II is not bound at all (lane 11). In the transpositionally inactive, bottom-strand-hemi-methylated state the 5' terminal cluster I is not recognized by TPase (lane 6), whereas cluster II is efficiently bound (lane 10) and cluster III is weakly complexed (lane14).
These experiments demonstrate that hemi-methylation of the subterminal TPase binding sites is a cis-acting determinant of transposase binding that correlates with transposition activity of Ds elements. In the active state, both Ds ends have a high affinity for the Ac TPase protein, whereas the inactive state correlates with much reduced TPase binding to the 3' end and to the terminal 5' end TPase binding site cluster I. Fig 6C shows a model of the occupation of the Ds terminal fragments with TPase molecules dependent on the methylation state, according to the gel shift assays shown in Fig 6B and binding studies with synthetic target site oligomers (
| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The replication dependence of Ac/Ds transposition is more pronounced in monocots than dicots:
Using four different monocot species (maize, rice, wheat, and barley) it was found that excision of Ac/Ds elements from extrachromosomal, nonreplicating geminivirus vectors is extremely rare or even absent (
Replication facilitates Ds excision in the absence of DNA methylation:
In petunia cells replication of the reporter plasmid promotes Ds excision frequency at least eightfold. As no de novo methylation of the transfected plasmid DNA was detectable, this phenomenon does not depend on DNA (de)methylation. On the one hand, the Rep protein is required for reporter plasmid replication; on the other hand, it is known to be severely toxic for prokaryotic and eukaryotic cells (B. GRONENBORN, personal communication). This might explain the observation that only a fraction of the TYLCV-containing plasmids has replicated after 30 hr, indicated by the low amplification factor of 1.3. We therefore speculate that this toxicity is a limiting factor for plasmid replication and that without this effect the boost in Ds excision frequency would be even more pronounced.
At present we can only speculate about the molecular mechanism underlying the transposition activation by replication. The fact that a mutation in the Rep binding site (IR) abolishes the transposition boost excludes the possibilities of a stimulation by direct interaction of the replicase with the Ac TPase (because a Rep/TPase interaction should not be affected) and of an activation of a host cell accessory factor by Rep. It is conceivable that the access of the TPase to its binding sites is facilitated by the conformational change of the transposon ends in the replication fork and/or by the interaction with replication-specific components. However, such an effect cannot account for the different activation factors in monocots and dicots.
Transposon footprints are influenced by replication:
Replication not only promotes Ds excision from extrachromosomal vectors, but apparently also affects the formation of the transposon footprints. With and without replication the same two footprints dominate; however, the frequency of aberrant footprints is higher in the absence of replication. This indicates that replication has no influence on the mechanism but rather on the fidelity of the reactions leading to the formation of the predominant footprints. The predominant footprint we obtained is characterized by transversion of the nucleotides immediately flanking the Ds on both sides. The same type of footprint also dominates after Ac or Ds excision in maize and transgenic Arabidopsis plants (
C-methylation of Ac/Ds elements is responsible for the chromatid selectivity of transposition:
The model for the chromatid selectivity of Ac/Ds transposition suggested by Wang and coworkers rests on the assumption that holomethylated elements do not transpose because TPase cannot bind to their ends. In addition, it predicts that, following replication, only one of the two daughter transposons is transposition competent (
It is unclear whether the 6.3-fold difference in transposition activity between the two daughter elements in the petunia system truly reflects the situation of an element in the maize genome. The plasmid vectors supposedly differ in topology and association with proteins from the chromosomal chromatin structure. Also, in the synthetically methylated Ds elements all cytosines are modified to the same extent, whereas the Ac9 element in maize and its epigenetically inactivated derivative Ds-cy display distinct methylation patterns with an uneven distribution of 5mC residues (
| ACKNOWLEDGMENTS |
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We thank Bruno Gronenborn (CNRS, Gif sur Yvette, France) for the TYLCV plasmid and helpful discussions, and Ruth Adolphs for help with the mobility shift assays. This work was supported by Deutsche Forschungsgemeinschaft through a Heisenberg fellowship to R.K. and SFB 190.
Manuscript received October 23, 2000; Accepted for publication January 4, 2001.
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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