Mobilization of Stowaway-like MITEs in newly formed allohexaploid wheat species

Transposable elements (TEs) dominate the genetic capacity of most eukaryotes, especially plants, where they can account for up to 90?% of the genome, such as in wheat. The relationship between TEs and their hosts and the role of TEs in organismal biology are poorly understood. In this study, we have applied next generation sequencing, together with a transposon display technique in order to test whether a Stowaway-like MITE, termed Minos, transposes following allopolyploidization events in wheat. We have generated a 454-pyrosequencing database of Minos-specific amplicons (transposon display products) from a newly formed wheat allohexaploid and its parental lines and retrieved hundreds of novel MITE insertions in the allohexaploid. Clear mobilization of Minos was also seen by site-specific PCR analysis and sequence validation. In addition, using real-time qPCR analysis we observed an insignificant change in the relative quantity of Minos from the expected value of merging the two parental genomes, indicating that, despite its activation, no significant burst in Minos copy number can be seen in the newly formed allohexaploid. Interestingly, we found that CCGG sites surrounding Minos underwent massive hypermethylation following the allohexaploidization process. Our data suggest that MITEs have maintained their capacity for activity throughout the evolution of wheat and might be epigenetically deregulated in the first generations following allopolyploidization.     

Mobilized retrotransposon Tos17 of rice by alien DNA introgression transposes into genes and causes structural and methylation alterations of a flanking genomic region

Tos17 is a copia-like endogenous retrotransposon of rice, which can be activated by various stresses such as tissue culture and alien DNA introgression. To confirm element mobilization by introgression and to study possible structural and epigenetic effects of Tos17 insertion on its target sequences, we isolated all flanking regions of Tos17 in an introgressed rice line (Tong35) that contains minute amount of genomic DNA from wild rice (Zizania latifolia). It was found that there has been apparent but limited mobilization of Tos17 in this introgression line, as being reflected by increased but stable copy number of the element in progeny of the line. Three of the five activated copies of the element have transposed into genes. Based on sequence analysis and Southern blot hybridization with several double-enzyme digests, no structural change in Tos17 could be inferred in the introgression line. Cytosine methylation status at all seven CCGG sites within Tos17 was also identical between the introgression line and its rice parent (Matsumae)-all sites being heavily methylated. In contrast, changes in structure and cytosine methylation patterns were detected in one of the three low-copy genomic regions that flank newly transposed Tos17, and all changes are stably inherited through selfed generations.     

Sleeping Beauty transposition in the mouse genome is associated with changes in DNA methylation at the site of insertion

The Sleeping Beauty (SB) transposon (Tn) system is a nonviral gene delivery tool that has widespread application for transfer of therapeutic genes into the mammalian genome. To determine its utility as a gene delivery system, it was important to assess the epigenetic modifications associated with SB insertion into the genome and potential inactivation of the transgene. This study investigated the DNA methylation pattern of an SB Tn as well as the flanking genomic region at insertion sites in the mouse genome. The ubiquitous ROSA26 promoter and an initial part of the eGFP coding sequence in the SB Tn exhibited high levels of CpG methylation in transgenic mouse lines, irrespective of the chromosomal loci of the insertion sites. In contrast, no detectable CpG methylation in the endogenous mouse ROSA26 counterpart was observed in the same animals. Furthermore, significant hypomethylation was detected in neighboring chromosomal sequences of two unique SB Tn insertions compared to wild-type patterns. Taken together, these results suggest that SB Tn insertions into the mouse genome can be discriminated by DNA methylation machinery from an identical endogenous DNA sequence and can profoundly alter the DNA methylation status of the transgene cargo as well as flanking host genomic regions.     

Extensive and heritable epigenetic remodeling and genetic stability accompany allohexaploidization of wheat

Allopolyploidy has played a prominent role in organismal evolution, particularly in angiosperms. Allohexaploidization is a critical step leading to the formation of common wheat as a new species, Triticum aestivum, as well as for bestowing its remarkable adaptability. A recent study documented that the initial stages of wheat allohexaploidization was associated with rampant genetic and epigenetic instabilities at genomic regions flanking a retrotransposon family named Veju. Although this finding is in line with the prevailing opinion of rapid genomic instability associated with nascent plant allopolyploidy, its relevance to speciation of T. aestivum remains unclear. Here, we show that genetic instability at genomic regions flanking the Veju, flanking a more abundant retroelement BARE-1, as well as at a large number of randomly sampled genomic loci, is all extremely rare or nonexistent in preselected individuals representing three sets of independently formed nascent allohexaploid wheat lines, which had a transgenerationally stable genomic constitution analogous to that of T. aestivum. In contrast, extensive and transgenerationally heritable repatterning of DNA methylation at all three kinds of genomic loci were reproducibly detected. Thus, our results suggest that rampant genetic instability associated with nascent allohexaploidization in wheat likely represents incidental and anomalous phenomena that are confined to by-product individuals inconsequential to the establishment of the newly formed plants toward speciation of T. aestivum; instead, extensive and heritable epigenetic remodeling coupled with preponderant genetic stability is generally associated with nascent wheat allohexaploidy, and therefore, more likely a contributory factor to the speciation event(s).     

Allopolyploidy in wheat induces rapid and heritable alterations in DNA methylation patterns of cellular genes and mobile elements

Whereas accumulating recent evidences indicate that allopolyploid formation in plants is accompanied by rapid and non-Mendelian genomic changes, some other works showed genomic stasis in both nascent and natural allopolyploids. To further study the issue, we performed global DNA fingerprinting of a newly synthesized allohexaploid wheat and its natural counterpart, the common wheat, by AFLP analysis. It was found that ca. 20% bands showed deviation from parental additivity in both synthetic and the natural common wheat. Sequence analysis indicates that a majority of the changed bands represent known-function genes and transposable elements. DNA gel blot analysis showed that the main type of changes in the amphiploid is epigenetic in nature, i.e., alteration in DNA methylation patterns. Two types of alterations in methylation, random and non-random, were detected, and both types were stably inherited. Possible causes and implications of the epigenetic changes in allopolyploid genome evolution and speciation are discussed.     

Cytosine Methylation Changes in Rice Centromeric and Telomeric Sequences Induced by Foreign DNA Introgression

Cytosine methylation changes (hyper- or hypomethylation) in centromeric and telomeric sequences were observed in all three studied rice introgression lines containing DNA from wild rice, Zizania latifolia Griseb. The changed genomic Southern hybridization patterns were complex and non-concordant between a pair of isoschizomers (HpaII/MspI) digests, indicating methylation modifications at both the inner and outer cytosines of the CCGG sites. The changed patterns were inherited through generations. Possible mechanism for the methylation changes and their potential implications for the phenotypic variation and genome organization are discussed.     

Nonadditive changes to cytosine methylation as a consequence of hybridization and genome duplication in Senecio (Asteraceae)

The merger of two or more divergent genomes within an allopolyploid nucleus can facilitate speciation and adaptive evolution in flowering plants. Widespread changes to gene expression have been shown to result from interspecific hybridisation and polyploidy in a number of plant species, and attention has now shifted to determining the epigenetic processes that drive these changes. We present here an analysis of cytosine methylation patterns in triploid F(1) Senecio (ragwort) hybrids and their allohexaploid derivatives. We observe that, in common with similar studies in Arabidopsis, Spartina and Triticum, a small but significant proportion of loci display nonadditive methylation in the hybrids, largely resulting from interspecific hybridisation. Despite this, genome duplication results in a secondary effect on methylation, with reversion to additivity at some loci and novel methylation status at others. We also observe differences in methylation state between different allopolyploid generations, predominantly in cases of additive methylation with regard to which parental methylation state is dominant. These changes to methylation state in both F(1) triploids and their allohexaploid derivatives largely mirror the overall patterns of nonadditive gene expression observed in our previous microarray analyses and may play a causative role in generating those expression changes. These similar global changes to DNA methylation resulting from hybridisation and genome duplication may serve as a source of epigenetic variation in natural populations, facilitating adaptive evolution. Our observations that methylation state can also vary between different generations of polyploid hybrids suggests that newly formed allopolyploid species may display a high degree of epigenetic diversity upon which natural selection can act.     

Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer.

Cytosine methylation at CpG dinucleotides is thought to cause more than one-third of all transition mutations responsible for human genetic diseases and cancer. We investigated the methylation status of the CpG dinucleotide at codon 248 in exon 7 of the p53 gene because this codon is a hot spot for inactivating mutations in the germ line and in most human somatic tissues examined. Codon 248 is contained within an HpaII site (CCGG), and the methylation status of this and flanking CpG sites was analyzed by using the methylation-sensitive enzymes CfoI (GCGC) and HpaII. Codon 248 and the CfoI and HpaII sites in the flanking introns were methylated in every tissue and cell line examined, indicating extensive methylation of this region in the p53 gene. Exhaustive treatment of an osteogenic sarcoma cell line, TE85, with the hypomethylating drug 5-aza-2'-deoxycytidine did not demethylate codon 248 or the CfoI sites in intron 6, although considerable global demethylation of the p53 gene was induced. Constructs containing either exon 7 alone or exon 7 and the flanking introns were transfected into TE85 cells to determine whether de novo methylation would occur. The presence of exon 7 alone caused some de novo methylation to occur at codon 248. More extensive de novo methylation of the CfoI sites in intron 6, which contains an Alu sequence, occurred in cells transfected with a vector containing exon 7 and flanking introns. With longer time in culture, there was increased methylation at the CfoI sites, and de novo methylation of codon 248 and its flanking HpaII sites was observed. These de novo-methylated sites were also resistant to 5-aza-2'-deoxycytidine-induced demethylation. The frequent methylation of codon 248 and adjacent Alu sequence may explain the enhanced mutability of this site as a result of the deamination of the 5-methylcytosine.     

Accuracy of DNA methylation pattern preservation by the Dnmt1 methyltransferase

DNA methyltransferase 1 (Dnmt1) has a central role in copying the pattern of DNA methylation after replication which is one manifestation of epigenetic inheritance. With oligonculeotide substrates we show that mouse Dnmt1 has a 30- to 40-fold preference for hemimethylated DNA that is almost lost after addition of fully methylated oligonucleotides. Using long hemimethylated DNA substrates that carry defined methylation patterns and bisulfite analysis of the methylation reaction products, we show a 15-fold preference for hemimethylated CG sites. Dnmt1 moves along the DNA in a random walk methylating hemimethylated substrates with high processivity (>50 sites are visited on average which corresponds to linear diffusion over 6000 bp). The frequency of skipping sites is very low (<0.3%) and there is no detectable flanking sequence preference. CGCTC sites tend to terminate the processive methylation of DNA by Dnmt1. Unmethylated DNA is modified non-processively with a preference for methylation at CCGG sites. We simulate the propagation of methylation patterns using a stochastic model with the specificity of Dnmt1 observed here and conclude that either methylation of several sites is required to propagate the methylation information over several cellular generations or additional epigenetic information must be used.     

Extent and pattern of DNA methylation alteration in rice lines derived from introgressive hybridization of rice and Zizania latifolia Griseb

We have reported previously that introgression by Zizania latifolia resulted in extensive DNA methylation changes in the recipient rice genome, as detected by a set of pre-selected DNA segments. In this study, using the methylation-sensitive amplified polymorphism (MSAP) method, we globally assessed the extent and pattern of cytosine methylation alterations in three typical introgression lines relative to their rice parent at ∼2,700 unbiased genomic loci each representing a recognition site cleaved by one or both of the isoschizomers, HpaII/MspI. Based on differential digestion by the isoschizomers, it is estimated that 15.9% of CCGG sites are either fully methylated at the internal Cs and/or hemi-methylated at the external Cs in the rice parental cultivar Matsumae. In comparison, a statistically significant increase in the overall level of both methylation types was detected in all three studied introgression lines (19.2, 18.6, 19.6%, respectively). Based on comparisons of MSAP profiles between the isoschizomers within the rice parent and between parent and the introgression lines, four major groups of MSAP banding patterns are recognized, which can be further divided into various subgroups as a result of inheritance of, or variation in, parental methylation patterns. The altered methylation patterns include hyper- and hypomethylation changes, as well as inter-conversion of hemi- to full-methylation, or vice versa, at the relevant CCGG site(s). Most alterations revealed by MSAP in low-copy loci can be validated by DNA gel blot analysis. The changed methylation patterns are uniform among randomly selected individuals for a given introgression line within or among selfed generations. Sequencing on 31 isolated fragments that showed different changing patterns in the introgression line(s) allowed their mapping onto variable regions on one or more of the 12 rice chromosomes. These segments include protein-coding genes, transposon/retrotransposons and sequences with no homology. Possible causes for the introgression-induced methylation changes and their implications for genome evolution and crop breeding are discussed.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Z. Y. Dong and Y. M. Wang have equally contributed to the work.     

Allopolyploidy in Wheat Induces Rapid and Heritable Alterations in DNA Methylation Patterns of Cellular Genes and Mobile Elements

Whereas accumulating recent evidences indicate that allopolyploid formation in plants is accompanied by rapid and non-Mendelian genomic changes, some other works showed genomic stasis in both nascent and natural allopolyploids. To further study the issue, we performed global DNA fingerprinting of a newly synthesized allohexaploid wheat and its natural counterpart, the common wheat, by AFLP analysis. It was found that ca. 20% bands showed deviation from parental additivity in both synthetic and natural common wheat. Sequence analysis indicates that a majority of the changed bands represent known-function genes and transposable elements. DNA gel blot analysis showed that the main type of changes in the amphiploid is epigenetic in nature, i.e., alteration in DNA methylation patterns. Two types of alterations in methylation, random and non-random, were detected, and both types were stably inherited. Possible causes and implications of the epigenetic changes in allopolyploid genome evolution and speciation are discussed.__________From Genetika, Vol. 41, No. 8, 2005, pp. 1089–1095.Original English Text Copyright © 2005 by Dong, Liu, Shan, Qiu, He, Liu.This text was submitted by the authors in English.     

Detection of changes in DNA methylation induced by low-energy ion implantation in Arabidopsis thaliana

This study evaluated changes in DNA methylation in Arabidopsis thaliana plants grown from seeds implanted with low-energy N(+) and Ar(+) ions. Methylation-sensitive amplified polymorphism (MSAP) testing revealed altered DNA methylation patterns after ion implantation at doses of 1 × 10(14) to 1 × 10(16) ions/cm(2). Comparison of the MSAP electrophoretic profiles revealed nine types of polymorphisms in ion-implanted seedlings relative to control seedlings, among which four represented methylation events, three represented demethylation events, and the methylation status of two was uncertain. The diversity of plant DNA methylation was increased by low-energy ion implantation. At the same time, total genomic DNA methylation levels at CCGG sites were unchanged by ion implantation. Moreover, a comparison of polymorphisms seen in N(+) ion-implanted, Ar(+) ion-implanted, and control DNA demonstrated that the species of incident ion influenced the resulting DNA methylation pattern. Sequencing of eight isolated fragments that showed different changing patterns in implanted plants allowed their mapping onto variable regions on one or more of the five Arabidopsis chromosomes; these segments included protein-coding genes, transposon and repeat DNA sequence. A further sodium bisulfite sequencing of three fragments also displayed alterations in methylation among either different types or doses of incident ions. Possible causes for the changes in methylation are discussed.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

Frequencies and variation in cytosine methylation patterns in diploid and tetraploid cytotypes of Paspalum notatum

Paspalum notatum Flügge is a grass species organized as an agamic complex. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in diploid and tetraploid genotypes, and to determine the occurrence of methylation changes associated with tetraploidization by using methylation-sensitive amplification polymorphism (MSAP) markers. No differences were found in the average proportions of methylated CCGG sites between cytotypes, but methylation patterns were significantly more variable in tetraploids. In both groups of plants, epigenetic and non-epigenetic variation correlated significantly when compared by Mantel tests. The evaluation of 159 common MSAP markers showed that 18.86 % of them differed in their methylation status in the different ploidies. Dendrogram analysis, reflecting epigenetic distances, showed that the four diploids and one experimentally-obtained sexually-reproducing tetraploid, grouped together. MSAP analysis performed on a diploid plant and its autotetraploid derivative showed that new epialleles emerged after tetraploidization. Sequencing of several MASP markers showed homologies with low copy genes, non-coding sequences and transposon/retrotransposon elements.     

Absence of global genomic cytosine methylation pattern erasure during medaka (Oryzias latipes) early embryo development

Two techniques were used to analyze global genomic 5-methyl cytosine methylation at CCGG sites of medaka embryo DNA. DNA was labeled by incorporation of microinjected radiolabeled deoxynucleotide into one-cell embryos. After Hpa II or Msp I digestion the radiolabeled DNA was fractionated in agarose gels and the distribution of label quantified throughout each sample lane to detect differences in fragment distribution. Alternately isolated DNA was digested with Hpa II or Msp I and the resulting generated termini end-labeled. The end-labeled digestion products were then analyzed for fragment distribution after gel fractionation. These techniques proved to be extremely sensitive, allowing comparison of genomic DNA methylation values from as few as 640 fish cells. The data suggest that in medaka embryos the vast majority (>90%) of genomic DNA is methylated at CCGG sites. Furthermore, these data support the conclusion that the extent of methylation at these sites does not change or changes very little during embryogenesis (from 16 cells to the hatchling). These data argue against active demethylation, or loss of methylation patterns by dilution, during the developmental stages between the one cell zygote and gastrulation. From a comparative viewpoint, these data may indicate that mammals and fishes methylate and demethylate their genomes in very different manners during development.     

Contrasting evolutionary patterns and target specificities among three Tourist-like MITE families in the maize genome

Miniature inverted-repeat transposable elements (MITEs) are short, non autonomous DNA elements that are widespread and abundant in plant genomes. The high sequence and size conservation observed in many MITE families suggest that they have spread recently throughout their respective host genomes. Here we present a maize genome wide analysis of three Tourist-like MITE families, mPIF, and two previously uncharacterized families, ZmV1 and Zead8. We undertook a bioinformatic analysis of MITE insertion sites, developed methyl-sensitive transposon display (M-STD) assays to estimate the associated level of CpG methylation at MITE flanking regions, and conducted a population genetics approach to investigate MITE patterns of expansion. Our results reveal that the three MITE families insert into genomic regions that present specific molecular features: they are preferentially AT rich, present low level of cytosine methylation as compared to the LTR retrotransposon Grande, and target site duplications are flanked by large and conserved palindromic sequences. Moreover, the analysis of MITE distances from predicted genes shows that 73% of 263 copies are inserted at less than 5 kb from the nearest predicted gene, and copies from Zead8 family are significantly more abundant upstream of genes. By employing a population genetic approach we identified contrasting patterns of expansion among the three MITE families. All elements seem to have inserted roughly 1 million years ago but ZmV1 and Zead8 families present evidences for activity of several master copies within the last 0.4 Mya.     

A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction.

We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation(Ds) transposon lines with insertions on all 5 Arabidopsischromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsisgenome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsislines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

Dynamics of <Emphasis Type="Italic">Vulmar/VulMITE</Emphasis> group of transposable elements in <Emphasis Type="Italic">Chenopodiaceae</Emphasis> subfamily <Emphasis Type="Italic">Betoideae</Emphasis>

Transposable elements are important factors driving plant genome evolution. Upon their mobilization, novel insertion polymorphisms are being created. We investigated differences in copy number and insertion polymorphism of a group of Mariner-like transposable elements Vulmar and related VulMITE miniature inverted-repeat transposable elements (MITEs) in species representing subfamily Betoideae. Insertion sites of these elements were identified using a modified transposon display protocol, allowing amplification of longer fragments representing regions flanking insertion sites. Subsequently, a subset of TD fragments was converted into insertion site-based polymorphism (ISBP) markers. The investigated group of transposable elements was the most abundant in accessions representing the section Beta, showing intraspecific insertion polymorphisms likely resulting from their recent activity. In contrast, no unique insertions were observed for species of the genus Beta section Corollinae, while a set of section-specific insertions was observed in the genus Patellifolia, however, only two of them were polymorphic between P. procumbens and P. webbiana. We hypothesize that Vulmar and VulMITE elements were inactivated in the section Corollinae, while they remained active in the section Beta and the genus Patellifolia. The ISBP markers generally confirmed the insertion patterns observed with TD markers, including presence of distinct subsets of TE insertions specific to Beta and Patellifolia.