A genome-wide transcriptional activity survey of rice transposable element-related genes
© Jiao and Deng; licensee BioMed Central Ltd. 2007
Received: 22 September 2006
Accepted: 27 February 2007
Published: 27 February 2007
Transposable element (TE)-related genes comprise a significant portion of the gene catalog of grasses, although their functions are insufficiently characterized. The recent availability of TE-related gene annotation from the complete genome sequence of rice (Oryza sativa) has created an opportunity to conduct a comprehensive evaluation of the transcriptional activities of these potentially mobile elements and their related genes.
We conducted a genome-wide survey of the transcriptional activity of TE-related genes associated with 15 developmental stages and stress conditions. This dataset was obtained using a microarray encompassing 2,191 unique TE-related rice genes, which were represented by oligonucleotide probes that were free from cross-hybridization. We found that TE-related genes exhibit much lower transcriptional activities than do non-TE-related genes, although representative transcripts were detected from all superfamilies of both type I and II TE-related genes. The strongest transcriptional activities were detected in TE-related genes from among the MULE and CACTA superfamilies. Phylogenetic analyses suggest that domesticated TE-related genes tend to form clades with active transcription. In addition, chromatin-level regulations through histone and DNA modifications, as well as enrichment of certain cis elements in the promoters, appear to contribute to the transcriptional activation of representative TE-related genes.
Our findings reveal clear, albeit low, general transcription of TE-related genes. In combination with phylogenetic analysis, transcriptional analysis has the potential to lead to the identification of domesticated TEs with adapted host functions.
The completion of the rice (Oryza sativa) genome sequence allowed further functional classification of the coding sequences of this important crop and model of grass species [1, 2]. Detailed annotation of the rice genome revealed that nearly a quarter of the rice open reading frame (ORF) coding capacity has features of transposable elements (TEs) and are therefore defined as TE-related genes . Like other genes, these TE-related genes have predicted normal gene structure with protein coding capacity. However, they share significant sequence similarity with known TEs in either or both of the following ways: they have TE signature sequences in The Institute for Genomic Research (TIGR) Oryza Repeat Database  or they contain TE-related protein domains . By this definition, TE-related genes can include potentially active TEs (based on the existence of a functional ORF) as well as cellular genes derived from TEs. Many of these TE-related genes encode reverse transcriptases, transposases, or other related proteins , and they can be further classified based on protein domain and other sequence features [3, 4]. Those TEs overwhelming in number that lack functional ORFs are not considered to be genes . Although there are many TE-related genes, the biologic functions of these genes remain elusive .
TEs are considered to be important for the maintenance and diversification of genomes. TEs are usually separated into two classes that differ in the mode of propagation: retrotransposons, or type I elements, which transpose by reverse transcription of an RNA intermediate; and type II elements, which only use a DNA intermediate in movement within the genome. Both classes can be further divided into several superfamilies, each with a unique evolutionary history. Representatives of virtually all superfamilies of TEs have been detected in grass genomes [7–9]. Accumulating evidence suggests that TE activities have profound impact on the genome , influencing genome size, genome rearrangement, chromatin transcription, and gene evolution [10–15]; many of these factors relying specifically on the transposition activity of TEs.
Although most TEs are considered inactive [16, 17], there have been isolated reports of TE transposition in rice and other grasses . A common condition promoting transposition is stress, including that which occurs in in vitro cell or tissue culture [19–22]. Developmental regulation of transposition has also been reported in intact plants [23, 24].
Transcription of TE-related genes is required for their own transposition and that of other related TEs, although transcription itself may not be sufficient for transposition [20, 25, 26]. Analysis of TE-related genes from certain subgroups of the type I class and the Mutator-like superfamily of the type II class suggests that their transcripts are widely present in grasses [27, 28]. Most of these transcribed TEs have coding capacity and are therefore considered TE-related genes. A recent study of expressed sequence tags (ESTs) in sugarcane identified 267 active TE-related transcripts . Transcription of TE-related genes was also reported in an unbiased survey of the transcriptional activity of a single rice chromosome using a tiling microarray .
Apart from the potentially active TEs among these TE-related genes, domesticated TE-related genes, which acquire new functions for the host, have also been found to exist. Although our current classification for distinguishing TE-related genes from non-TE-related genes is not definitive , two recent studies in Arabidopsis identified domesticated TE-related genes contributing to cellular processes [32, 33]. Similar examples were also found in animals [34, 35]. Such findings in part support the hypothesis that TE-related genes may influence the evolution of their host by providing a source of novel coding capacity.
The potential impact of domesticated TE-related genes on the evolution of genomes requires systematic investigation. One attempt to identify further domesticated TE-related genes is sequence mining . Because a change of position through transcription can be detrimental to the host, transposon-derived genes with known host function usually lack mobility. As a consequence, they may be devoid of transposon-specific terminal sequences [32, 36]. By employing this criterion in a search, one particular member of the MULE superfamily was identified as a domesticated gene candidate . Transcription is an important feature of domesticated TE-related genes, because it is generally required in cellular functions of the host [32, 33]. By surveying transcriptional activity and combining other approaches, we would be able to identify domesticated TE-derived gene candidates.
Another mechanism for the evolution of new genes from TEs is through their ability to acquire and fuse fragments of genes to new genomic locations, as seen in plant Pack-MULE and, more recently, in certain Helitron-like and CACTA elements [13, 14, 37, 38]. However, many of these Pack-MULEs have been suggested to possess pseudogene-like features . Pack-MULE, as a unique group of TE-related genes, is relatively well annotated and is a current focus of interest regarding the origin of genes .
Given the paucity of information on TE-related genes, a systematic study of their transcriptional activity in a well characterized genome is required to enhance our understanding of the activity of TE-related genes. That the sequence of the rice genome is now completely annotated makes it a good resource for such a genome-wide survey . Recent advances in microarray technology allow us to study the transcriptional activity of genes in a high-throughput manner. It is therefore possible to conduct a genome-wide survey of the transcriptional activity of rice TE-related genes, especially those more divergent ones for which unique oligomer probes can be designed. Different from simple TEs composing mostly repetitive sequences, many TE-related genes are diverged enough to have short oligomers representing their unique sequence regions. Such an approach has recently been utilized to analyze transcription of TE-related genes in plants and animals [11, 30, 40]. In addition to TE-related genes, TEs without protein-coding capacity and other tandem repeats may also exhibit transcriptional activity [26, 41]. Transcripts derived from tandem repeats in the heterochromatin can give rise to small RNAs, which in turn direct the modification of histones and DNA in TE-related sequences and nearby regions by means of RNA interference . Although transcripts from tandem repeats are important for the genome, their highly repetitive nature prohibits characterization of their unique identities in chromosomal organization on a genome-wide scale [42, 43].
We conducted an expression analysis for rice TE-related genes using 70-mer oligonucleotide microarrays. Expression profiles from 4,728 oligonucleotides covering organs from rice plants were analyzed under both normal conditions at various developmental stages as well as under stress conditions. Clear but restricted transcription of TE-related genes were found for all major superfamilies of TE-related genes. Mechanisms controlling representative TE transcription were further analyzed.
Representation of TE-related genes by an oligonucleotide microarray
A 70-mer oligonucleotide set was previously developed to span the rice genome . Many TE-related genes are included in this oligomer set design, allowing survey of a large number of rice TE-related genes. However, for the sake of simplicity, those oligonucleotide probes representing TE-related genes were removed from analysis in all prior genome profiling analyses [44–47]. Here, we collected all of our available datasets and systematically examined the transcriptional activities of TE-related genes in various tissues and growth conditions. In particular, we included datasets representing cell cultures and stress-exposed tissues.
Summary of annotated TE-related genes in rice and coverage by (cross-hybridization free) microarray probes
Number of TEs in TIGR
Number of TEs in TIGR and literature review
Covered by microarray
Transcriptional activity of TE-related genes
Summary of rice samples used in this study
Tillering stage shoot
Tillering stage root
Suspension cultured cells
Somatic root in culture
Somatic shoot in culture
Tillering stage shoot under drought stress
Tillering stage shoot under salt stress
Flag leaf under drought stress
Flag leaf under salt stress
Heading panicle under drought stress
Heading panicle under salt stress
By monitoring the expression of 2,191 TE-related genes using unique oligomer probes, we identified expression of 1,084 (61.7%) TE-related genes in at least one of our 15 samples. This is in contrast to findings in non-TE-related genes, 85.8% of which are expressed in at least one sample and 22.6% in all samples, using the same selection criteria. Expressed TE-related genes tend to exhibit transcription in a relatively small number of samples. The percentages of expressed TE-related genes in a wide range of samples are markedly lower than those of non-TE-related genes (Figure 1b). For those oligonucleotide probes that match multiple TE-related genes, 73.7% and 5.1% had hybridization signals in at least one sample or in all samples, respectively. Considering that those probes match multiple repetitive genes, a smaller portion of those TE-related genes that they represent is expected to be transcribed.
To gauge the reliability of our microarray data for TE-related genes, we first compared rice cDNA and EST collections with our data. We found 496 TE-related genes in the cDNA/EST collection in TIGR database . These cDNAs and ESTs were derived from six rice samples: callus, seed, shoot and stem, leaf, root, and flower (heading panicle). We have similar (although not identical) rice samples with microarray expression profiles for all of them except seed. A survey of these TE-related cDNAs/ESTs indicates that 80% of those covered by our microarray also had detectable transcription. We further used RT-PCR to verify the microarray data. An attempt to amplify a series of TE-related genes with different levels of microarray signals supported our choice of threshold used to determine expression. Of the 10 genes with expression level within 100 units above the threshold, seven were amplified by RT-PCR; in contrast, only two out of 10 with expression below the threshold were amplified. Moreover, 34 randomly selected TE-related genes identified through microarray analysis as being shoot expressed were tested with RT-PCR using seedling shoot RNA samples. Twenty-nine (85%) of them were clearly detected. An independent tiling microarray analysis of rice transcriptome also covered a significant portion of the TE-related genes . A preliminary survey of the transcriptional activities of TE-related genes in this dataset gives a similar portion of expression (about 30%) among tissues examined , although a different platform and hybridization detection procedure were used .
Transcription of type I TE-related genes
In addition to taking an inventory of transcribed TE-related genes in various tissues and under multiple growth conditions, the availability of high-quality complete genome sequence provided an opportunity to elucidate how transcriptional activities evolve following sequence divergence. To this end, phylogenic trees were generated for all major TE-related gene superfamilies and were integrated with their members' expression profiles.
LTR-type TE-related genes belong to two superfamilies, namely Ty1/copia and Ty3/gypsy, which are both ubiquitous throughout plants and believed to have contributed significantly to the evolution of genome structure and function . Both families are quite diverse in rice, with Ty3/gypsy elements outnumbering Ty1/copia elements . Our expression data indicate that both families are similarly transcribed at low levels at around 25% in most samples, but there are members in both families with strong transcription in widespread tissues. However, they are spread in different clades with only remote similarity (Additional data files 1 and 2). A few active LTR retrotransposons have been reported in rice. Among them, Tos17 is the best characterized and is known to exhibit active transposition in tissue culture . We found active transcription of Tos17 not only in cultured cells but also in a wide range of organs (Additional data file 1), suggesting that tissue culture may provide a way to propagate somatic transposition events to progeny. Sireviruses are a plant-specific lineage of the Ty1/copia retrotransposons that interact specifically with proteins related to dynein light chain 8 . We found one member of this lineage with ubiquitous strong transcription and several others with transcription in selected rice samples (Additional data file 1).
A large number of type I TE-related genes have not yet been further classified (Table 1). We detected transcription of a smaller proportion of this group of genes than for Ty1/copia and Ty3/gypsy superfamilies.
Transcription of type II TE-related genes
Type II TE-related genes are in general more actively transcribed than type I TE-related genes. Different from type I, type II TE-related genes are highly variable among major superfamilies with respect to transcriptional activity. Whereas CACTA and MULE superfamilies are actively transcribed, hAT-like, PIF/Pong-like, Mariner-like, and Helitron-like superfamilies have transcriptional activities similar to or lower than those of type I TE-related genes.
The CACTA superfamily is a diverse group of high-copy repetitive genes in grasses [53, 54]. CACTA transposons with active transcription or even transposition have been reported in rice and other grass genomes [54–57]. A total of 2,276 intact CACTA transposase-coding genes are identified in rice, making it the largest superfamily in type II TE-related genes (Table 1). The CACTA superfamily is also highly active, with more than 40% transcribed in each sample. Several clades with active transcription were identified (Additional data file 4). Among them, two clades include over 20 members. No members within these actively transcribed CACTA transposons have previously been characterized.
A recently identified unique type II TE superfamily, Helitron-like, is relatively under-characterized in the rice genome . Strikingly, Helitron-like transposons have the potential to move and shuffle genes or exons in maize [13, 14]. In rice, we found only one member with transcriptional activity in all the samples. There is no other Helitron-like transposon among the seven examined ones with transcriptional activity in any samples (Additional data file 5).
We were unable to further classify another 787 type II TE-related genes into any superfamilies (Table 1). Interestingly, a large percentage (>40% out of 128 with unique oligomer probes) was found to be transcribed.
Transcription of Pack-MULE
Association of transcription with DNA and histone modification
TEs, including TE-related ORF encoding genes, are under multiple levels of epigenetic control, including DNA methylation and histone modifications . In Arabidopsis, DNA methylation and histone H3 lysine-9 methylation (H3K9m) correlates with the silencing of TEs, and histone H3 lysine-4 methylation (H3K4m) is associated with transcribed genes . However, H3K4m is also found in silenced genes and therefore may not always be a marker for active transcription .
To explore these relationships further, we selected five TE-related genes with transcription in cultured cells but not in seedling shoots: the Ty1/copia retroelement Os10g22210; Ty3/gypsy retrotransposons Os09g11940 and Os10g06250; and CACTA DNA transposons Os07g23660 and Os08g32100 (Figure 8b). Three of these five genes were associated with higher levels of H3K9m in shoots (silenced) as compared with in cultured cells (transcribed), according to ChIP-PCR analysis. Levels of H3K4m did not exhibit a clear difference between shoots and cultured cells (Figure 8b). DNA methylation was reduced in three genes in cultured cells compared with shoots (Figure 8b). Thus, lower levels of DNA methylation and H3K4m tend to accompany TE-related gene transcription under developmental regulation.
It has been shown that small RNAs derived from repetitive genome sequences repress transcription by means of RNA interference in Arabidopsis . Small RNAs, both microRNAs (miRNAs) and small interfering RNAs (siRNAs), have also been identified in rice, albeit at a small scale [66, 67]. Sixteen out of a total of 44 predicted siRNAs have at least one TE-related gene as their target gene , whereas few miRNA have a TE-related gene target . For the five target TE-related genes covered by microarray, we found active transcription for only one. It is interesting to note that for siRNAs targeting multiple genes, the transcriptional profiles of these target genes may not be at all similar. For example, siRNA P96-E12 has two targets: Os07g10770 (a cellulose synthase) and Os01g05370 (a Ty1/copia family retrotransposon). The cellulose synthase gene has strong transcription in almost all samples we profiled. In contrast, the retrotransposon target does not exhibit transcription in any sample.
Upstream gene transcription affects TE-related gene transcription
Functions of cis-elements in transcription
Transcription profiles of TE-related genes in rice
TEs account for an overwhelming proportion of plant genomes. To ensure the viability of their host and hence their own survival, the transposition of TEs should be tightly controlled . Transcribed autonomous TEs among TE-related genes have the potential to self-activate or activate transcription of related nonautonomous TEs. Transcriptional regulation is therefore one major control step used by plants, but it remains insufficiently understood. The recently available rice genome sequence has enabled us to characterize TE-related gene transcription on a genome-wide scale.
Using 70-mer oligonucleotide microarrays covering more than 2,000 rice TE-related genes, we surveyed the transcription profiles under a wide range of organ samples under various conditions. Considering that TE-derived cellular genes are relatively rare, autonomous TEs probably contribute to most of these TE-related genes. Genome profiling revealed that 25% to 30% of the TE-related genes were transcribed in one sample, which was much lower than the corresponding percentage of non-TE-related genes (Figures 1 and 2). Moreover, TE-related genes differed from their non-TE-related counterparts in two additional aspects. First, TE-related genes tended to be transcribed in only a subset of organs or developmental stages, whereas non-TE-related genes had transcription in more samples on average (Figure 1 and Figure 2). Second, transcribed TE-related genes exhibited weaker transcription overall compared with non-TE-related genes in all of the samples we profiled (Figure 2). It worth noting that our estimation of TE-related gene transcription was biased toward low-copy elements, because it was difficult to distinguish transcripts among recently duplicated high-copy TE-related genes, which share high sequence similarity within clades. It has been reported in Arabidopsis and Drosophila that the activity of TE elements may reduce as the copy number increases [77, 78]. Therefore, we expect the transcriptional activity of those high-copy TE-related genes will be lower than for low-copy ones.
Among TE-related genes, a smaller proportion of type I than type II genes were transcribed (Figure 1a), a discrepancy that resulted primarily from the strong transcription of MULE and CACTA superfamilies as well as unclassified type II members. It is interesting to note that all TE-related gene superfamilies with potential to severely expand, including all type I TE-related genes and PIF/Pong-like, Mariner-like and Helitron-like type II TE-related genes, were more tightly controlled at the transcription level. Type I TE-related genes are amplified through a copy-and-paste mechanism . PIF/Pong-like and Mariner-like superfamilies regulate the activity of MITEs, which dominate the rice genome . Members of the Helitron-like superfamily go through a unique rolling cycle replication to rapidly amplify themselves .
Many TE-related genes exhibit organ-specific, growth stage-specific, and stress-specific expression profiles in our collection of samples. These genes exist in all superfamilies, as shown in Figures 3 to 7. A number of them, again from various superfamilies of both type I and type II TE-related genes, exhibit clear induction in cultured cells, in certain organs, or in certain stress challenged organs (Figure 2). The precise biologic significance for this observation remains to be elucidated.
It is important to note that transcriptional activity does not necessarily correspond to transpositional activity. Transcription is just the first of several steps required for the transposition of type I and type II TEs [79, 80]. Active transcription and even translation of TE-related genes has been reported in several isolated cases , but only in a few cases was transposition actually confirmed by observed copy number change . A two-step regulatory mechanism was therefore proposed for retrotransposons . In this model, some elements may have slipped the leash of transcriptional gene silencing . Nevertheless, they can be controlled by post-transcriptional gene silencing . We observed transcription of all major TE-related gene superfamilies in rice, but it is probable that most of them, if not all, are not actively transpositioned. It is therefore likely that such a two-step regulation exists not only for retrotransposons but also for other classes. Post-transcriptional regulation, which is still largely unexplored, is thought to repress transposition activity further .
Transcription of domesticated TE-related genes in the rice genome
It is well accepted that some TE-related genes have actually acquired host functions and play physiologic roles in the host. They can either be derived from TEs or include hijacked TEs or TE fragments by cellular genes. Not surprisingly, we have discovered active transcription of all potential domesticated TE genes previously described in Arabidopsis and rice. Interestingly, domesticated TE genes tend to be within actively transcribed TE gene clades. The rice homologs of the two reported cases of domesticated transposons in Arabidopsis, namely FAR1/FHY3 and DAYSLEEPER, were located in two actively transcribed clades. MUG1, a putative domesticated gene revealed by cross-species sequence comparison analysis, was shown to be transcribed from our data and located within an actively transcribed clade. These examples may suggest that actively transcribed clades of TE-related genes are a rich source for domesticated TE genes. In fact, several other actively transcribed clades have been observed, especially for the MULE and CACTA superfamilies, from our analysis (Figures 4 and 5). It is reasonable to suspect that those transcriptionally active clades may contain genes co-opted by hosts to serve adaptive functions. This notion will be worth testing in future research. Clearly, the combination of transcriptional analysis with phylogenetic analysis is instrumental in identifying those TE-derived genes with adapted host function.
A specific mechanism for the evolution of new genes by mobile DNA elements is through their ability to acquire and fuse fragments of genes to new genomic locations, as represented by Pack-MULE . By exploring the transcriptional activity of a subset of Pack-MULEs, we have shown that their transcriptional activity falls in between the levels of TE-related and non-TE-related gene models (Figure 7). This result suggests that many of them might not have biologic functions, and both pseudogenes and evolving new functional genes exist among these annotated Pack-MULEs. Alternatively, functional diversification of recently evolved genes may be another explanation, because newly formed genes usually have more specific expression profiles .
Mechanisms controlling TE-related genes transcription
The presence of such a diverse array of transcribed TE-related genes raises questions regarding the mechanisms that control the transcription. At the chromatin level, we found that actively transcribed TE-related genes have reduced levels of H3K9m and DNA methylation. This finding indicates that proper chromatin modification status is usually required for transcription of TE-related genes. However, histone and DNA modifications are unlikely to be efficient markers for distinguishing between autonomous TE genes and TE-derived cellular genes.
Consistent with the existence of chromatin-level control, we found that transcribed TE-related genes tend to be located near to transcribed neighboring genes. It is possible that the status of a chromatin domain is marked by histone and DNA modifications. Such chromatin status affects a few genes located in the same or neighboring chromatin domains. The orientation of upstream genes affects downstream TE-related gene transcription (Figure 9). If both genes are in the same orientation, then the downstream TE-related gene would have a greater chance of being transcribed. Readthrough of TE-related genes derived from upstream genes may account for this difference, besides possible chromatin effects.
Small RNA has been suggested to be a key regulator to silence TE elements transcriptionally and post-transcriptionally [18, 81]. However, only a few examples were found in our dataset. Small RNAs are known to be highly abundant in the Arabidopsis genome , whereas their counterparts in rice are yet to be discovered. A full catalog of small RNAs in rice will provide a better picture of their role in TE transcription.
Another possible mechanism controlling TE transcription is the existence of cis elements in their promoter regions. Examples have been found previously for LTR retrotransposons, which employ alternating cis elements present in their LTRs [29, 84–86]. Here, we identified nine cis elements that were clearly enriched in the promoter regions of transcribed TE-related genes. Among them, both basic transcription-related cis elements and elements that respond to developmental or environmental regulation are found to be enriched in the upstream regions of those transcribed TE-related genes (Figure 10). In addition, these enriched cis elements are probably not limited to a certain superfamily but rather widely spread in several superfamilies. Taken together, our data show that transcription of TE-related genes, mostly autonomous TE genes, in rice is a complex process, which is controlled, at least in part, by chromatin-level regulation and cis elements in promoters.
Materials and methods
The rice 70-mer oligonucleotide set was described previously . Briefly, 70-mer oligonucleotides were designed based on a combination of FGENESH predicted genes from an improved shotgun sequence  and the available full-length cDNAs and ESTs . Designed 70-mer oligonucleotides correspond to the sequence within the coding region of genes, and the design was corrected for such factors as oligo cross-hybridization, uniform TM value, GC content, and hairpin/stem nucleotide number. All oligonucleotides were remapped to TIGR rice genome annotation version 3.1 genes  using BLAST. We requested greater than 90% alignment of a 70-mer oligonucleotide probe to a gene during the remapping. Moreover, only those 70-mer probes without a greater than 80% second-best aligned gene were considered to be free from cross-hybridization. These criteria were selected because a mismatch of 20% removes more than 90% of the hybridization signals, whereas a 10% mismatch retains at least half of the hybridization signals .
TE-related genes were identified in accordance with TIGR annotation, with supplemental literature review of published TE-related genes. A total of 2,191 TE-related genes are represented by at least one oligonucleotide free from cross-hybridization. In addition, there are 1,966 70-mer oligonucleotides mapped to several but only TE-related genes. These oligonucleotides represent another 9,396 TE-related genes.
Oligonucleotides were custom synthesized by Operon Biotechnologies Inc. (Huntsville, AL, USA) and printed onto poly-L-lysin coated microscope slides using a contact microarrayer. The same recommended set of 12 unique negative control 70-mer oligonucleotides based on heterologous genes  were included in all slides. There were 240 negative control spots on each slide.
Microarray data and plant materials
Microarray experiments and detailed rice sample preparation were described previously [44–47]. Samples include organs harvested under normal growth conditions (seeding stage shoot, tillering stage shoot, tillering stage root, heading stage flag leaf, heading stage panicle, and filling stage panicle), organs under conditions of salinity or drought (tillering stage shoot, heading stage flag leaf, and heading stage panicle), and cultured cells (suspension-cultured cells, somatic root in culture, and somatic shoot in culture). A summary is provided in Table 2. The microarray data discussed in this publication have been deposited in NCBI Gene Expression Omnibus  and are accessible through GEO series numbers GSE2360, GSE2691, GSE6533, and GSE6552.
Microarray data processing
Microarray spot intensity signals were acquired using Axon GenePix Pro 3.0 software package (Molecular Devices, Sunnyvale, CA, USA). To identify and remove systematic sources of variation, including dye and spatial effects, spot intensities from the GenePix Pro output files of all repeats of a given sample pair were normalized using limma, a software package for the analysis of gene expression microarray . This normalization process identified and ameliorated spatial, intensity-based, and dye-specific artifacts using multiple step corrections. To determine objectively whether a gene exhibited significant expression in a given sample, we followed a method that relied on negative control spots and data reproducibility . To estimate nonspecific hybridization, a distribution of normalized intensities was obtained from the subset of negative control spots present on each array slide. From this distribution, we chose an intensity cutoff at which less than 10% of the distribution was greater than or equal to this threshold. Expression of a gene was only considered detectable if it was above the threshold in two or more repeats out of the three. These criteria had been demonstrated suitable for oligonucleotide arrays with an error rate range of 1% to 3% false negatives . RT-PCR results and independent analysis using different microarrays and statistical approaches  further supported this threshold.
TE family classification was according to TIGR annotation . Hand analysis led to the identification of another 208 TE-related genes according to published sequences and BLAST search. Multiple sequence alignments were conducted using Clustal W . The weighing matrix used was Gonnet Pam 250 with the penalty of gap opening 10 and gap extension 0.2. Phylogenetic trees were generated based on the neighbor-joining method, using PAUP* version 4.0b10 with default parameters .
Cluster analysis was applied to all TE-related genes and 1,353 randomly selected non-TE-related genes showing expression in at least one sample. Average normalized log-transformed expression intensities were subjected to cluster analysis. For hierarchical clustering, Pearson correlation was used to compute similarities, and a complete linkage clustering algorithm was used. Cluster analysis was performed using the software Cluster  and visualized using custom scripts.
Total RNA was extracted from independently prepared rice seedling shoots using Qiagen RNeasy kit (Qiagen, Valencia, CA, USA). After DNase I treatment, total RNA was used for cDNA synthesis using Superscript II (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's protocol. PCR primers were designed according to sequence using Primer3 . The amplification reaction was carried out for 35 cycles and at an annealing temperature of 55°C. Products were separated by 1% agarose gel electrophoresis. Negative controls using mock cDNA synthesis products without reverse transcriptase were included for all genes to detect potential genomic DNA contamination.
Histone and DNA methylation
ChIP was carried out as described elsewhere  using seedling shoots and cultured cells. Histone H3 anti-dimethyl lysine-4 or anti-dimethyl lysine-9 antibodies (Upstate, Avon, NY, USA) were used to precipitate genomic DNA, which was resuspended in water for PCR analysis. The same PCR and gel electrophoresis conditions were used as for RT-PCR analysis.
Methylation of DNA was assessed by McrBC digestion following a previously published protocol . Genomic DNA was isolated from seedling shoots and cultured cells using Qiagen DNeasy plant kit and divided into two equal samples. One sample was digested with McrBC, a methylation-dependent restriction enzyme that cuts the sequence A/G 5 mC (New England Biolabs, Beverly, MA, USA). Both digested and untreated samples were subject to PCR amplification as described previously. Successful amplification after digestion indicates lack of methylation.
The genome sequences 2 kilobases upstream of the annotated translation start site were retrieved from the TIGR database. Both DNA strands were searched for known plant motifs using the PLACE database . Enrichment levels were further calculated using custom scripts .
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 shows degrees of lineage-specific transcription in the Ty1/copia superfamily. Additional data file 2 shows degrees of lineage-specific transcription in the Ty3/gypsy superfamily. Additional data file 3 shows degrees of lineage-specific transcription in the CACTA superfamily. Additional data file 4 shows degrees of lineage-specific transcription in the Mariner superfamily. Additional data file 5 shows degrees of lineage-specific transcription in the Helitron superfamily.
We gratefully acknowledge Junli Zhou, Ning Su and Lei Li for sharing unpublished data, Junli Zhou and Xueyong Li for technical assistance in histone and DNA methylation experiments, and Valerie J Karplus and Yeqin Ma for critical reading of this manuscript. This work was supported by National Science Foundation Plant Genome Program Grant DBI-0421675 to XWD.
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