Roles of retrotransposons in benign and malignant hematologic disease

Abstract

Nearly half of our genomes are repetitive sequences derived from retrotransposons. These repeats have accumulated by a 'copy-and-paste' mechanism whereby: (i.) a genomic template sequence is transcribed to RNA, (ii.) the RNA is reverse-transcribed, and (iii.) the DNA copy is inserted at a new location in the host genome. As we remain susceptible to new retrotransposition events, many of these insertions are highly polymorphic. Transposons are of interest since insertions into both coding and non-coding gene regions have been associated with a wide variety of functional sequelae and because transposable elements can be involved in genomic rearrangements in transformed cells. In this review, we highlight how expression of retrotransposons, de novo and polymorphic transposon insertions, and genomic rearrangements that these repeats potentiate contribute to both benign and neoplastic hematopoietic diseases.

Figure 1. Structural features of HERVs, LINEs, SINEs and SVAs

(Modified from [101].) Endogenous retroviruses (ERVs) are examples of autonomous, LTR retroelements. These consist of partly overlapping coding regions bounded by long terminal repeats (LTRs). ERVs code for group-specific antigen (Gag), protease (PR), and polymerase (Pol). The LTR has promoter activity (arrow) on the 5′ as well as the 3′ end. There are also target site duplications (TSDs; thick black lines). The Pol gene contains domains for integrase (IN), reverse transcriptase (RT), and RnaseH (RH). While ERV templates are not excised in transposition, recombination between LTRs can result in proviral loss, leaving a ‘solo LTR’ at the integration site. LINE retrotransposons are a prototypical example of autonomous, non-LTR transposable elements. Human genomic L1 LINEs consist of an internal CpG-rich 5′UTR with a polymerase II promoter (arrow), two open reading frames (ORF1 and ORF2), and a poly(A) tail [An]. ORF1 encodes a 40 kDa protein involved in formation of the ribonuclear protein (RNP) complex; ORF2 encodes for reverse transcriptase (RT) and endonuclease (En) activities. Genomic L1 insertions are characteristically flanked by short target site duplications (TSD) reflecting fill in repair of staggered dsDNA breakage at the insertion site (thick black lines). Alu SINEs are examples of non-autonomous, non-LTR elements. Human Alus consist of two GC rich monomer sequences, termed the left (L Alu) and the right (R Alu), and an intervening A-rich linker. SVAs are another type of non-autonomous, non-LTR retrotransposon, and are composite structures named after constituent SINE-R (derived from HERV LTR sequence), VNTR and Alu repeats. Like LINEs and reflecting their dependence on LINE-encoded ORF2, Alus and SVAs have a poly (A) tail and are flanked by TSDs (thick black lines). Alus are transcribed by a RNA polymerase III promoter. Drawings are not to scale. Full-length HERVs approximate 10kb; L1 LINEs 6kb; Alus 300bp; SVAs 2kb.

Figure 2. Comparison of LTR (A) and non-LTR (B) retrotransposon mechanism

(Modified from [102].) A Retrotransposition of LTR retrotransposons starts with transcription of the TE from its genomic location (gray). This is followed by reverse transcription (RT) of the RNA template into cDNA, after which integrase (purple dots) acts to insert the cDNA into a new genomic location, generating a target site (TS) duplication at its flanks. B Reverse transcription and insertion of non-LTR retrotransposons is carried out in a coupled process involving intrachromosomal priming; this is referred to as target primed reverse transcription (TPRT). It involves ORF2p-mediated endonuclease activity at the DNA target site (TS) to expose a 3′ hydroxyl group, followed by ORF2p RT activity extension of the DNA strand to copy the retrotransposon sequence. Resolution of the resulting RNA-DNA hybrid is thought to be reliant on host DNA repair mechanisms.

Figure 3. L1 and Alu insertions in exon 14 of F8 causing hemophila A

Three examples of retrotransposition events causing loss-of-function of factor VIII are illustrated. All have occurred within exon 14, which is a 3.1 kb exon entirely protein coding (striped pattern). A The first is a 3.8 kb, 5′ truncated L1 LINE insertion in sense orientation that occurred with a 12 nucleotide target site duplication about 1 kb 3′ of the exon start. B The second retrotransposition event described resulted in insertion of 2.3 kb of rearranged 3′ L1 LINE sequence close to the midpoint of exon 14. It was flanked by a 13 bp target site duplication. These two L1 LINE insertions were reported by Kazazian and colleagues [1] and appeared to be new insertions in the affected probands. C The third insertion shown is an Alu insertion interrupting the same exon and oriented in antisense with respect to the F8 gene. It was reported by Sukarova et al. [24] who retrospectively identified the insertion in familial carriers, the affected patient’s mother and maternal grandmother.

Figure 4. Repeat-associated genetic recombination events

A Sister chromatids or homologous chromosomes are illustrated, with hypothetical repetitive sequences shown as blocks. Arrows indicate positions of a dsDNA break near or within one of the repeats. The first step in homologous repair pathways (open arrow) is a 5′ sequence resection, which prepares the 3′potential invader strands (black arrow). In this schematic, one correct (true allelic) pairing option is available to the invader (indicated by the gray arrow marked HR, homologous recombination). Erroneous pairing with a tandem repeat in the same orientation in cis, a type of single stranded annealing (SSA), results in deletion of one of the repeats and the intervening unique sequence. B Appropriate association of the invader strand with an allelic repeat is shown, with subsequent invader strand elongation. The other 3′ end left by the original dsDNA break is involved in a second strand capture that allows for elongation. After the Holiday junction (HJ) traverses sufficient distance during elongation, there is resolution of the complex (open arrow). Depending on whether there has been a cross-over event, there are one of two possible resulting products. True homologous recombination is non-mutagenic. C Misalignment of the homologs caused by repetitive sequence similarity is illustrated. The consequent non-allelic homologous recombination (NAHR) yields two products, one with deletion of one repeat and the sequence that occurred between the repeats, and one with interval duplication. If this occurs between homologous chromosomes, both deletion and duplication products can be expected to persist in the cell. If this occurs between sister chromatids, the abnormal copies are expected to separate in daughter cells, one persisting in a dominant neoplastic clone. Similar rearrangements between oppositely oriented repeats cause inversions of inter-repetitive sequence (not shown). D Intrastrand slip mispairing model for ssDNA break repair is expected to result in duplication after replication without incurrence of the corresponding deletion.