Christy R Hagan ^{1, 2}, Rebecca F Sheffield ¹, and Charles M Rudin ^{1,  2,  3}

¹ Biological Sciences Division, University of Chicago, Chicago, Illinois 60637, USA.
² Committee on Cancer Biology University of Chicago, Chicago, Illinois 60637, USA.
³ Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.

Correspondence should be addressed to CM Rudin. e-mail:  crudin@uchicago.edu

Alu elements are found exclusively in primate species and comprise over 10% of the human genome. To better
define the mechanisms responsible for Alu replication, we introduced a human Alu element into mouse cells. We
report that Alu retrotransposition can be induced in mouse cells by exposure to the topoisomerase II inhibitor etoposide and is mediated in trans by endogenous mouse long interspersed elements (LINEs).

The most common classes of retrotransposable elements in the human genome are short interspersed elements (SINEs) and LINEs. The Alu element, present in over 106 copies per haploid genome, is the most prevalent human SINE, but it is not found in mice and has a clearly distinct distribution even within closely related primates [1, 2]. Alu replication may have had an important role in primate speciation by promoting the generation of mutually incompatible genomic structures [3]. The mechanisms regulating SINE expression and retrotransposition in vivo, and the extent of evolutionary conservation of these mechanisms, have not been defined.

Alu elements are transcriptionally silent under most conditions and do not encode any of the proteins required for their retrotransposition [4]. Recent studies involving expression of modified SINE and LINE constructs derived from the same species (either an eel SINE and eel LINE, or a human Alu and human LINE) have shown that exogenously driven LINE expression can markedly enhance SINE retrotransposition [5, 6].

Our laboratory has found that exposure to a variety of DNA damaging agents (including the topoisomerase II inhibitor etoposide) results in transcriptional induction of endogenous SINE elements and concomitant activation of an endogenous reverse transcriptase [7]. We proposed that genotoxic stress may be a determinant of Alu element activation.

To test this idea, we stably transfected FL5.12 mouse hematopoietic cells with a human Alu element (pREP4-Alu; see Supplementary Methods and Supplementary Fig. 1 online) [7, 8]
http://www.nature.com/ng/journal/v35/n3/suppinfo/ng1259_S1.html
and, to facilitate survival after genotoxic exposure, a construct expressing the anti-apoptotic factor Bcl-xL. Alu transcription was detected by real-time RT-PCR in cells containing pREP4-Alu but not in control cells. Consistent with our previous data from human cells, Alu transcription in FL5.12 cells was increased over sixfold by etoposide exposure but was only minimally affected by vincristine (Fig. 1a).

Figure 1:

(a) Transcriptional  induction determined by real-time RT-PCR. Shown are relative Alu expression levels in FL5.x_L.Alu cells, untreated or after 96 h of exposure to vincristine or etoposide. Cells lacking pREP4-Alu (FL5.x_L) serve as a negative control. Error bars show s.d.

(b) Panhandle PCR clone frequency. The number of clones of each category is indicated.

(c) Insertion-site characteristics of panhandle PCR clones. Mouse chromosomal integration sites are indicated. Putative LINE ORF2p consensus sites are underlined and bold; Alu sequences is in italics.

Panhandle PCR (see Supplementary Fig. 2 online) was used to evaluate whether etoposide exposure was sufficient to induce Alu retrotransposition [9]. New retrotransposition events in the mouse genome could be readily distinguished from either random integration of the pREP4-Alu plasmid (highly unlikely to map to the Alu transcriptional start) or amplification of trace contaminating human DNA.

We analyzed a total of 101 PCR products from untreated cells and cells surviving after exposure to either vincristine or etoposide (Fig. 1b). Seven PCR products, derived exclusively from cells exposed to etoposide, showed characteristics consistent with retrotransposition (Fig. 1c). In each of these clones, Alu sequence initiated precisely at the Alu transcriptional start site. Contaminating human amplicons and clones derived from integration of the pREP4-Alu plasmid were evenly distributed among the three populations. The difference in detection of Alu retrotransposition between treatment groups was significant using Fisher's exact test, whether we considered only clones derived from mouse DNA (P = 0.019) or all PCR products isolated (P = 0.004). These data, representative of multiple independent experiments, support the possibility that both Alu transcription and retrotransposition can be induced by etoposide exposure.

Etoposide inhibits religation of topoisomerase II cleavage products, resulting in double-strand breaks. One hypothesis to explain the association of etoposide exposure and successful Alu retrotransposition is that etoposide facilitates re-entry by providing sites for Alu cDNA integration. Yet none of the genomic insertion sites have features characteristic of topoisomerase II cleavage sites [10].

Alternatively, etoposide may activate LINE-mediated Alu retrotransposition. Human L1 LINE retrotransposition occurs through target-primed reverse transcription and is dependent on the LINE-encoded ORF2p endonuclease–reverse transcriptase [11]. This mechanism results in generation of direct target-site duplications flanking the inserted element, terminating at the second nucleotide of the ORF2p cleavage consensus site 5'-TAAA(A). ORF2p consensus sites were identified within the first 24 bp flanking all new Alu
retrotranspositions identified by panhandle PCR. The distance from the Alu transcriptional start to these putative cleavage sites was consistent with that observed in recent LINE and Alu integrants in the human genome [12, 13].

To further characterize the integration of Alu elements in these cells, we carried out inverse PCR on genomic DNA from etoposide-treated cells (Supplementary Fig. 2). Nine new Alu integration events consistent with retrotransposition were isolated, all of which again showed a precise 5' junction between mouse DNA and the Alu transcriptional start site (Fig. 2). The DNA flanking each integrated Alu element represents a contiguous stretch of known mouse chromosomal DNA.

Figure 2: Insertion-site characteristics of retrotransposed Alu elements identified by inverse PCR.

Target-site duplications are boxed; bases hypothesized to represent consensus for ORF2p cleavage are in bold. Poly(A) tract lengths and putative endonuclease recognition sites are indicated. Black boxes indicate identity to the consensus, and gray boxes indicate A-->G substitutions.

All integrated Alu elements cloned by inverse PCR are flanked by target-site duplications of 12–17 bp, terminating at the second nucleotide of a consensus TAAAA site, strongly suggestive of LINE-mediated endonucleolytic cleavage (Fig. 2). These insertion-site characteristics are indistinguishable from those of retrotransposition driven by an exogenously expressed L1 LINE in human cells [5]. Taken together, these results strongly support the notion that Alu retrotransposition in mouse cells is mediated by an endogenous mouse LINE.

Both Alu and LINE sequences terminate in adenosine-rich sequences of variable length. The parental pREP4-Alu clone contained a poly(A) sequence of 21 nt, whereas the retrotransposed derivatives had an average of 53 consecutive adenosine residues (Fig. 2). These data are consistent with reports that recent naturally occurring Alu insertions have atypically long poly(A) tracts [12]. It has been suggested that poly(A) tract length could be a determinant of competency for retrotransposition [12]. Our data support an alternative
interpretation, that recent Alu insertions have long poly(A) tracts as the result of incorporation of additional adenosine nucleotides in the process of retrotransposition.

The upregulation of substrate (Alu RNA) for retrotransposition may be sufficient to explain the observed association of Alu retrotransposition with etoposide exposure. Alternatively, concomitant induction of endogenous retrotransposition machinery may be required. Several stressors other than genotoxic exposure, including adenoviral infection and cycloheximide treatment, have been associated with concomitant transcriptional induction of both Alu and LINE elements [14]. It will be of interest to evaluate whether these other cellular stresses induce Alu retrotransposition.

These data provide the first definitive demonstration of Alu retrotransposition in the absence of an exogenously expressed LINE and the first demonstration of Alu retrotransposition in nonhuman cells. Both transcriptional induction and retrotransposition of Alu elements were associated with etoposide exposure. The insertion-site characteristics noted here are identical to those observed in naturally occurring Alu insertion events, indicating that the requisite machinery for Alu retrotransposition is conserved in mouse cells. Taken together, our results imply that an endogenous mouse LINE, with cleavage-site specificity similar to the human L1 LINE, is active
in mouse cells after etoposide exposure and is able to recognize and retrotranspose human Alu transcripts.

Note: Supplementary information is available on the Nature Genetics website.

ACKNOWLEDGMENTS

The Alu clone used in construction of pREP4 was a generous gift of P. Deininger. This work was supported by grants from the National Cancer Institute and the National Institute of General Medical Sciences of the US National Institutes of Health and from the Howard Hughes Undergraduate Research Fellowship Program.

Competing interests statement: The authors declare that they have no competing financial interests.

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