Genetic and epigenetic variations contributed by Alu retrotransposition

Abstract

Background: De novo retrotransposition of Alu elements has been recognized as a major driver for insertion polymorphisms in human populations. In this study, we exploited Alu-anchored bisulfite PCR libraries to identify evolutionarily recent Alu element insertions, and to investigate their genetic and epigenetic variation.

Results: A total of 327 putatively recent Alu insertions were identified, altogether represented by 1,762 sequence reads. Nearly all such de novo retrotransposition events (316/327) were novel. Forty-seven out of forty-nine randomly selected events, corresponding to nineteen genomic loci, were sequence-verified. Alu element insertions remained hemizygous in one or more individuals in sixteen of the nineteen genomic loci. The Alu elements were found to be enriched for young Alu families with characteristic sequence features, such as the presence of a longer poly(A) tail. In addition, we documented the occurrence of a duplication of the AT-rich target site in their immediate flanking sequences, a hallmark of retrotransposition. Furthermore, we found the sequence motif (TT/AAAA) that is recognized by the ORF2P protein encoded by LINE-1 in their 5'-flanking regions, consistent with the fact that Alu retrotransposition is facilitated by LINE-1 elements. While most of these Alu elements were heavily methylated, we identified an Alu localized 1.5 kb downstream of TOMM5 that exhibited a completely unmethylated left arm. Interestingly, we observed differential methylation of its immediate 5' and 3' flanking CpG dinucleotides, in concordance with the unmethylated and methylated statuses of its internal 5' and 3' sequences, respectively. Importantly, TOMM5's CpG island and the 3 Alu repeats and 1 MIR element localized upstream of this newly inserted Alu were also found to be unmethylated. Methylation analyses of two additional genomic loci revealed no methylation differences in CpG dinucleotides flanking the Alu insertion sites in the two homologous chromosomes, irrespective of the presence or absence of the insertion.

Conclusions: We anticipate that the combination of methodologies utilized in this study, which included repeat-anchored bisulfite PCR sequencing and the computational analysis pipeline herein reported, will prove invaluable for the generation of genetic and epigenetic variation maps.

Figure 1

Computational pipeline developed to identify putative Alu insertions from Alu anchored bisulfite PCR libraries.

Figure 2

PCR validation of putative Alu insertions (a-g). The Alu insertions were sorted based on genomic coordinates. The Alu insertions were named AI1 through AI21. N-normal brain tissue DNA; E1, E2, and E3-brain tumor tissue (ependymoma) DNA from different individuals; P and R- ependymoma DNA, P is primary and R is relapsed tumor from the same individual.

Figure 3

Bisulfite PCR cloning and sequencing to validate methylation status of an unmethylated Alu insertion (chr9:37594172-37594310). Asterisk indicates the CpG dinucleotides that are flanking the Alu element; the scheme shows the relative location of TOMM5 and the CpG island in relation to the Alu AI19 insertion (USCS Genome Bioinformatics). a) methylation status of a downstream AluJo (sequence coordinates: chr9:37594745-37595002) near the newly inserted Alu element; b) newly inserted Alu element and its methylation status; c) methylation status of 2 CpGs upstream of the newly inserted Alu; d), e), f), and g) methylation statuses of 3 Alu repeats and 1 MIR element localized between the newly inserted Alu and the CpG island, respectively, AluSx, AluJo, MIRb, and AluSx; h) methylation status of the 5'end of a CpG island located 1,576 bp (sequence coordinates: chr9:37592324-37592701) upstream from the newly inserted Alu element. Note that the TOMM5 transcription unit is in opposite orientation to that of the newly inserted Alu element. The methylation levels for a, b, c, d, e, f, g, and h were 40%, 33.7%, 79.1%, 4.2%, 0%, 3%, 32%, and 0.6%, respectively.

Figure 4

Methylation statuses of two pairs of hemizygous alleles, i.e. before and after Alu insertions. a) chr10:72605338-72605440 locus; b) chr2:48276482-48276601 locus. Schemes on the left side represent the allele not containing the Alu insertion, while the figure on the right side represents the allele in which the Alu element inserted. The blue arrow indicates the probable site of Alu integration; CG and asterisk indicate the CpG dinucleotides that are flanking the Alu element; red bar indicates the Alu element that was inserted.