Repetitive elements constitute over 50% of the human genomic sequence [1]. The most prevalent repeats are the Alu family of SINEs, which comprise approximately 10% of the human genome. A typical Alu element is approximately 300 bp long and contains two almost identical arms separated by an A-rich sequence. The ancestor of the Alu monomer is the 7 SL RNA gene, which encodes the RNA component of the signal recognition particle (SRP) that is involved in the translocation of newly synthesized proteins [2, 3]. Similar to the 7 SL gene, Alu elements with intact promoters - namely A and B boxes - may be transcribed by RNA polymerase III [2, 4]. With the aid of the LINE-encoded retrotransposition machinery, Alu transcripts gain mobility and expand in genomes through a process involving reverse transcription and integration [5].

Alu retrotransposition has been an important molecular evolutionary force reshaping the primate genomes [6]. The expansion of the Alu elements in the primate genomes is dated at least 60 million years ago [7]. Based on their evolutionary history, Alu elements can be classified in three major subfamilies: AluJ, AluS, and AluY [8]. Among them, the youngest Alu elements - AluY and its variants AluYa-g - remain very active, and exhibit the highest rate of retrotransposition in the human genome [9–12]. While several recent studies have shown that LINE-1 elements contribute substantially to the structural variations observed in the human genome [13–15], the retrotransposition rate of Alu elements is ten times higher than that of LINE-1, with an estimated new insertion at every 21 births [16].

Decades of research have demonstrated that Alu elements play important roles in the genome and transcriptome [17–20]. Alu elements may contribute a large number of transcription factor binding sites [21], some of which may serve as enhancers involved in tissue development [22, 23]. In addition, some Alu elements may be expressed and Alu transcription affects nearby gene expression, distal gene expression, and global translation. For instance, the expression of an Alu in the promoter of an epsilon-globin gene was found to negatively regulate globin gene expression by transcriptional interference [24]. Recently, Alu RNA was found to be a modular transacting repressor of mRNA transcription [25]. Interestingly, such transcriptional suppression was found to be specific and limited to certain genes. Alu RNAs also affect translational initiation and were found to form stable, discrete complexes with the double-stranded RNA-activated kinase PKR, and to antagonize PKR activation [26]. Transcription derepression of otherwise active Alu elements, which so often reside within genes, may lead to the formation of double-strand RNA - if in antisense orientation - and ultimately to heterochromatinization and silencing of the gene [27].

One of the key mechanisms controlling Alu expression is DNA methylation. The human genome has approximately 28 million CpG dinucleotides, 7 million of which are found within Alu elements [1]. In most somatic tissues, the CpG dinucleotides within the Alu sequence are heavily methylated to suppress Alu expression [28, 29]. It has been demonstrated that the A and B boxes (5-16 bp, and 75-84 bp from the 5' terminus, respectively) are critical cis-elements for Alu expression. In particular, methylation of the B box is thought to inhibit protein binding and hence block Alu transcription [30]. Albeit not sufficient, demethylation and consequently transcription of Alu elements is required for occurrence of de novo retrotransposition [28]. Methylated CpGs can undergo deamination and thereby lead to mutations that render them unable for retrotransposition [8, 9].

Although much effort has been made to identify structural variations resulting from Alu integration, much less is known with regard to the epigenetic status of newly inserted elements and of their flanking genomic sequences. Here we report the utilization of an Alu-anchored bisulfite PCR strategy to generate methylation maps for thousands of Alu elements in human cerebellum and in ependymomas [31, 32]. In this approach, most of the targeted Alu elements are members of the active AluY subfamilies. In this study, we analyzed the aforementioned datasets to identify newly integrated Alu elements, to investigate sequence characteristics and commonalities of their integration sites, to uncover their methylation statuses, and to determine whether the methylation patterns of the sequences surrounding their integration sites would be altered in the alleles harboring the insertion in individuals hemizygous for the Alu retrotransposon.

Recent studies demonstrated that major structural variants in the human genome are derived from retrotransposons, Alu elements in particular [16, 41]. Due to the extensive sequence homology that exists among young Alu repeats, the identification of such structural variants remains a challenging task. To date, a total of 2,558 polymorphic Alu retrotransposons have been reported to occur in human populations, 1,763 of which have been deposited in dbSNP and 795 in dbRIP. In this study we implemented a computational pipeline to identify recent Alu insertions, and examined the methylation status of the newly inserted Alu retrotransposons and their flanking sequences. At the time we developed this strategy the Genome Sequencer FLX System was the most suitable alternative available, given the greater length of the sequence reads that it generates, and the fact that sequences would encompass an Alu repeat and would be derived from bisulfite-converted genomic DNA. Altogether, the longer reads generated with the FLX System greatly facilitated their mapping back to the reference genome sequence. Notwithstanding this advantage, however, we anticipate that our approach may be adapted to take advantage of competing next generation sequencing platforms that have a higher throughput and that can now generate sufficiently long sequence reads.

Using this strategy a total of 327 putative Alu elements were identified. We found that 42.8% of their insertion sites fell within intronic regions, while one integration site mapped to the 3'-UTR of the TOMM40 gene. TOMM40 is a component of the preprotein translocase complex of the outer mitochondrial membrane, which consists of at least 7 different proteins (TOMM5, TOMM6, TOMM7, TOMM20, TOMM22, TOMM40, and TOMM70). These results are consistent with previous studies indicating that Alu retrotransposons tend to be inserted within intragenic regions [1, 42].

Out of the twenty-one insertion events that were randomly selected for validation analysis, nineteen were successfully verified. A limitation of the Alu-anchored bisulfite PCR approach that needs to be acknowledged is the fact that only 5' flanking sequences are obtained. The right arm of the Alu retrotransposons and their 3' flanking sequences are not represented in the sequence reads that are generated. Hence, in order to design primers for the validation experiments, we used the reference sequence of the human genome as source of putative 3'-flanking sequences for the Alu insertions. Accordingly, it is possible that the two cases that could not be verified may have been caused by the utilization of an incorrect 3' flanking sequence for primer design. Notwithstanding this limitation, the lowest estimated accuracy for the analysis pipeline that we have implemented in this study for the identification of de novo Alu retrotransposition events would be of 90.5% (19/21).

The sequence features (TSD, TT/AAAA cleavage sequence, and A-rich Alu tail) that are typically observed in newly inserted Alu elements constitute hallmarks of retrotransposition [5, 10, 36, 37]. Indeed, further analysis of the aforementioned nineteen PCR-cloned Alu elements and flanking sequences revealed the presence of both the TSD and TT/AAAA sites. Alu A-tails seem to be an important factor to enable Alu element retrotransposition [4, 35]. Roy-Engel et al. reported that the average A-tail length of active Alu elements is 26 [35]. Consistent to their finding, the Alu A-tail sizes of the Alu elements described in this study ranged from 11-45 with an average of 29 bp.

Most cancer genomes are characterized by localized hypermethylation as well as by global hypomethylation [43, 44]. This hypomethylation process may enable transcription and de novo retrotransposition of Alu elements which, in turn, may lead to genome instability [45]. Our previous study demonstrated that the methylation level of Alu elements decreased in ependymomas, and most significantly in recurrent tumors [31]. To examine whether some Alu insertions represented somatic events limited to recurrent ependymomas, which could have occurred in consequence of the loss of DNA methylation, we generated and compared PCR products from ten genomic loci in primary and in recurrent tumors derived from one individual. The same results were obtained in all ten genomic loci. In addition, five of the ten Alu insertions were also found in other individuals. These results suggested that such validated Alu insertions most likely represent germ-line rather than somatic events.

In this study, in addition to identifying structural variants in the genome of 6 individuals, we investigated epigenetic variations that might result from de novo retrotransponsition events. The Alu elements identified in this study were heavily methylated, as it was previously shown by high-throughput bisulfite sequencing and herein validated by cloning and sequencing analyses. The analysis of methylation throughout the mapped Alus and among the 19 loci verified revealed that there were no methylation differences among tissues (Additional File 4, Table S3). This result indicates that at least by the time these DNA samples were obtained most of the newly inserted Alu elements were already transcriptionally repressed. This finding is further supported by the fact that the promoters of the Alu elements, i.e. their A and B boxes, were found to be methylated. However, there was one exception. We found an Alu insertion that was partially unmethylated (chr9:37594172-37594310). Interestingly, the insertion of this Alu element occurred 1,576 bp downstream from a CpG island and 1,674 bp downstream from the transcription start site of TOMM5, a gene encoding the translocase of the outer mitochondrial membrane 5. With a completely unmethylated promoter (both the A box and the B box were unmethylated), it is conceivable that this Alu element may have remained transcriptionally active and hence have served as source for additional retrotransposition events. Another interesting finding was that a CpG island that is upstream of the element - i.e., that of TOMM5 - may be influencing the methylation pattern of this Alu repeat. Indeed, the methylation status of the CpG island was similar to that of the 5' end sequences of this Alu repeat, i.e. both were unmethylated. It would be interesting to explore the functional impact of this particular Alu on the nearby TOMM5 gene. Additionally, 3 Alu repeats and 1 MIR element that are localized between the newly inserted Alu repeat and the CpG island were found to exhibit very low methylation levels. Such striking pattern of DNA methylation may indeed be an indication of the influence exerted by the adjacent CpG island. It is also possible that other epigenetic factors might be affecting the methylation statuses of these Alu elements, such as nucleosome positioning. Two previous studies have reported the influence of nucleosome positioning, within and around Alu element, in Alu activity [46, 47]. Accordingly, it is noteworthy that an AluJo that is localized downstream of this newly inserted Alu exhibits a similar pattern of DNA methylation, i.e. its 5' half is unmethylated while its 3' half is methylated. In our previous study [32], we found that genomic localization has a profound impact on Alu methylation status. In this study, the identification of both methylated and unmethylated Alu elements provided additional support to there being a positional effect on Alu methylation. Last, but not least, it is noteworthy that two of the novel Alu insertions herein reported map within or near genes encoding members of the preprotein translocase complex of the outer mitochondrial membrane, namely TOMM40 and TOMM5, respectively. It is conceivable that given their housekeeping function and ubiquitous expression pattern, hence commonly open chromatin status, these genes may be more vulnerable to uptake de novo retrotransposition events.

To explore the epigenetic impact of Alu insertion on adjacent genomic sequences, we examined the methylation statuses for two loci harboring hemizygous insertions, and - in one case - obtained the methylation patterns of CpG dinucleotides flanking the Alu insertion sites. Both alleles - irrespective of the presence of an inserted element - were found to be heavily methylated, and no significant epigenetic variation was observed in association with the presence of the additional Alu element.

In this work we have identified a few novel Alu insertions sites. We used DNA samples from normal and from tumor tissues, but the data obtained did not show any tissue preference for these insertions. More studies are highly desired to further scrutinize the functional aspects of structural variants in the human genome, including epigenetic variations that might arise in consequence of a de novo retrotransposition event.