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Seminars in Cancer Biology 20 (2010) 211–221

Contents lists available at ScienceDirect

Seminars in Cancer Biology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / s e m c a n c e r

eview

mobile threat to genome stability: The impact of non-LTR retrotransposons pon the human genome

iriam K. Konkel, Mark A. Batzer ∗

epartment of Biological Sciences, Biological Computation and Visualization Center, Louisiana State University, 202 Life Sciences Building, Baton Rouge, LA 70803, USA

r t i c l e i n f o

eywords: etrotransposon on-allelic homologous recombination ransposable element enome instability ancer

a b s t r a c t

It is now commonly agreed that the human genome is not the stable entity originally presumed. Deletions, duplications, inversions, and insertions are common, and contribute significantly to genomic struc- tural variations (SVs). Their collective impact generates much of the inter-individual genomic diversity observed among humans. Not only do these variations change the structure of the genome; they may also have functional implications, e.g. altered gene expression. Some SVs have been identified as the cause of genetic disorders, including cancer predisposition. Cancer cells are notorious for their genomic instability, and often show genomic rearrangements at the microscopic and submicroscopic level to which transposable elements (TEs) contribute. Here, we review the role of TEs in genome instability, with particular focus on non-LTR retrotransposons. Currently, three non-LTR retrotransposon families – long interspersed element 1 (L1), SVA (short interspersed element (SINE-R), variable number of tan- dem repeats (VNTR), and Alu), and Alu (a SINE) elements – mobilize in the human genome, and cause

genomic instability through both insertion- and post-insertion-based mutagenesis. Due to the abundance and high sequence identity of TEs, they frequently mislead the homologous recombination repair path- way into non-allelic homologous recombination, causing deletions, duplications, and inversions. While less comprehensively studied, non-LTR retrotransposon insertions and TE-mediated rearrangements are probably more common in cancer cells than in healthy tissue. This may be at least partially attributed to the commonly seen global hypomethylation as well as general epigenetic dysfunction of cancer cells.

ide ex

Where possible, we prov

. Introduction

Less than a decade ago, with the availability of the first human raft genome sequence, the human genome was considered to be very stable entity [1]. However, with the identification of struc-

ural variations (SVs) as a major cause of inter-individual variation,

t is now evident that the human genome is distinguished by a igh inter-individual variability [2–6]. SVs are usually at the sub- icroscopic level and include insertions, deletions, duplications,

ranslocations, and inversions. It is now commonly believed that

Abbreviations: TE, Transposable element; SV, structural variation; ERV, endoge- ous retrovirus; ORF, open reading frame; UTR, untranslated region; LINE 1, L1, long

nterspersed element 1; SINE, short interspersed element; VNTR, variable number f tandem repeats; SVA, SINE-R/VNTR/Alu; TSD, target site duplication; NHEJ, non- omologous end joining; SSA, single strand annealing; DSB, double strand break; HR, omologous recombination; NAHR, non-allelic homologous recombination; SNP, ingle nucleotide polymorphism; HDGC, hereditary diffuse gastric cancer; AML, cute myeloid leukemia; T-ALL, T-cell acute lymphoblastic leukemia; Dnmt3L, DNA ethltransferase 3-like. ∗ Corresponding author. Tel.: +1 225 578 7102; fax: +1 225 578 7113.

E-mail address: [email protected] (M.A. Batzer).

044-579X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. oi:10.1016/j.semcancer.2010.03.001

amples that impact cancer predisposition and/or development. © 2010 Elsevier Ltd. All rights reserved.

SVs comprise more nucleotides than single nucleotide polymor- phisms (SNPs) in the human genome [3]. Cancer cells are notorious for their genome instability. It is generally accepted that structural rearrangements at the microscopic level are common features of the genome of most human cancers. Recently the full impact of SVs, including those at the submicroscopic level in cancer cells, has become more apparent. Inherited rearrangements have also been associated with cancer predisposition and will be discussed in more detail in this review.

Transposable elements (TEs) are often involved in the genesis of SVs due to their inherent ability to mobilize, their abundance, and their high sequence identity. About half of the human genome is comprised of repetitive sequences, with TEs being the largest contributors [1,7]. The repeat content of the human genome is likely even higher, given that the decay of TEs over time makes the identification and characterization of ancestral TEs difficult if not impossible, and sequencing and assembly is less than perfect in

repeat (and transposon)-rich regions [8,9]. Altogether, the impact of TEs onto the human genome has been underappreciated for some time; only recently are we beginning to comprehend the impact of TEs upon genome architecture and, consequently, onto the evolu- tion of the human genome [10].

2 rs in Cancer Biology 20 (2010) 211–221

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Fig. 1. Structure of non-LTR retrotransposons. Shown is structure of actively mobilizing retrotransposons in the human genome: an Alu element (blue), a full-length L1 (purple), and a full-length SVA (dark green). The non-LTR retrotransposons are not drawn to scale. All full-length non-LTR retrotrans- posons end in a homopolymeric tract of Adenosines (polyA-tail, yellow). SVA and L1 contain a polyadenylation signal (pA) immediately before the polyA-tail. Insertions are flanked by target site duplications (TSDs, green). Alu (blue): The A and B stand for the A and B boxes of the internal promoter. The left and right monomers are linked by a spacer sequence A5 TACA5 . L1 (purple): Pro stands for the internal Polymerase II promoter within the 5′ untranslated region (UTR). A full-length L1 element contains two open reading frames (ORF1, ORF2). SVA (dark green): A full-length composite element contains from 5′ to 3′ a hexamer (CCCTCT), an Alu-homologous region of

12 M.K. Konkel, M.A. Batzer / Semina

A recent comparative genomics study of two human genomes ound that TEs are associated with ∼10% of all SVs larger than 100 bp 11]. TEs impact genome integrity in several ways, including TE nsertions and rearrangements. TEs are now commonly recognized n genetic diseases (reviewed in [7,12–16]). They have also been ssociated with cancer genesis; this is not unexpected, as a typical haracteristic of cancer cells is their genomic instability. Here, we iscuss the impact of TEs – in particular, non-LTR retrotransposons upon the architecture of the genome; and review how and to what xtent TEs have been associated with the genesis of cancer.

. TE background

.1. TE classification and activity

To understand the role of TEs within genome instability, t is important to review several key aspects of TE biology. or more detailed information we refer to other reviews (e.g. 7,12,15,17,18]). TEs may be categorized by their mobilization

echanism as either DNA transposons or retrotransposons. DNA ransposons propagate via a cut-and-paste mechanism. While ctive in very early primate evolution, these elements essentially eased activity in the primate lineage approximately 37 million ears ago [19]. In contrast, retrotransposons use an RNA intermedi- te; are reverse transcribed; and move within the genome through copy-and-paste mechanism [15,20]. Retrotransposons are further ubdivided into two groups on the basis of presence or absence f long terminal repeats (LTRs). The most prominent members of TR-retrotransposons are endogenous retroviruses (ERVs), which omprise about 8% of the human genome [1]. There is very lit- le (if any) evidence of ongoing ERV retrotransposition in humans 1,7,21]. Their potential role in tumorgenesis is subject to an ongo- ng debate and covered elsewhere (e.g., [22]). The other group ncompasses non-LTR retrotransposons, and is discussed in more etail in this review.

.2. Non-LTR retrotransposon biology

Three different families of non-LTR retrotransposons are ctively mobilized in the human genome. These are long nterspersed elements 1 (LINE1s, L1s); Alu elements (a short inter- persed element, or SINE); and SVAs (named after their composite arts: SINE-R, VNTR (variable number of tandem repeats), and an lu-like sequence) [1,7,12,15,23]. Their success is evident through he fact that non-LTR retrotransposons occupy about one-third of he human genome, making them the most populous TE group in he human genome [1].

L1s are the only currently known autonomous (providing ts own enzymatic machinery for retrotransposition) retrotrans- osons that are currently mobilizing within the human genome. hey comprise about 17% (∼500,000 copies) of the human genome ith evidence of ongoing activity dating back roughly 160 million

ears [1]. A full-length L1 is about 6 kb in length and contains an nternal Polymerase II promoter, two open reading frames (ORFs), nd ends in a polyadenylation signal followed by a homopolymeric ract of Adenosines (also known as a polyA-tail; see Fig. 1) [24,25]. RF1 protein is an RNA-binding protein [26] while ORF2 encodes a rotein with both endonuclease and reverse transcriptase activity 27–29]. The majority of L1 insertions are retrotransposition-

lly incompetent due to variable truncation upon insertion and ebilitating mutations [1]. Consequently, only about 80–100 retro- ransposition competent L1s have been identified in the human enome [30]. Of those, a few (6–8) “hot” L1s appear to be respon- ible for the bulk of new insertions [30].

two antisense Alu fragments including other sequence of unknown origin, a variable number of tandem repeat (VNTR) region, and ends in a SINE region from parts of HERV-K10, an human endogenous retrovirus.

The human genome contains two actively mobilizing non- autonomous non-LTR retrotransposons: Alu elements (member of the SINE family) and SVAs. Non-autonomous elements are believed to rely on the enzymatic machinery of L1s for retrotransposition; e.g., as shown for Alu elements [31,32]. With more than 1,000,000 insertions, Alu elements are the most successful TE in the human genome by number [1]. This accomplishment is even more remark- able given that Alu elements are primate-specific and originated only about 65 million years ago [15]. Alu elements are heterodimers made of two non-identical monomers connected by an Adenosine- rich linker [15,33,34]. As shown in Fig. 1, an approximately 300 bp long Alu element contains an internal Polymerase III promoter at its 5′ end, and ends in a polyA-tail.

SVA elements, which are altogether less well characterized than other non-LTR retrotransposons, represent the second group of currently mobilizing non-autonomous elements in the human genome. Similar to L1s, SVA insertions are often truncated and ter- minate in a polyadenylation signal followed by a polyA-tail (Fig. 1) [35,36]. It is now generally believed that SVA elements are tran- scribed by Polymerase II. However, an internal promoter has not been detected, and SVA transcription might – at least occasionally – take place through promoter activity in the vicinity of the SVA [35–37]. Due to their relatively recent origin (originating less than 25 million years ago), with ∼3000 copies, SVA elements show the lowest retrotransposon density in the human genome [7,36].

Non-LTR retrotransposons are thought to typically insert into the human genome through a mechanism referred to as Target Primed Reverse Transcription (TPRT) [7,12,38,39]. During TPRT, the L1-derived endonuclease cuts the minus strand of the host DNA at a loosely recognized target site (5′-TTTT/AA-3′) [28,40]. The polyA- tail of the non-LTR retrotransposon mRNA is proposed to bind to the free 3′ end of the host DNA, and the mRNA is reverse tran- scribed by the reverse transcriptase encoded by L1 [41]. The next steps of second strand cleavage, second strand synthesis, and lig- ation are the subjects of ongoing research. However, host repair systems have been implicated in the later stages of L1 retrotranspo- sition [42–46]. A recent tissue culture-based study further supports this, as proteins of the non-homologous end joining (NHEJ) path- way were shown to be involved in L1 retrotransposition [47]. Due

to a staggered break of the host DNA at the insertion site, the non- LTR retrotransposon insertion is flanked by short stretches (usually between 6 and 20 bp) of identical host DNA, referred to as target site duplications (TSD) [27,48].

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. Insertional mutagenesis

.1. Potential impact upon the human genome

Inherited de novo TE insertions occur in the germline and/or uring early embryogenesis [49–52]. De novo retrotransposon

nsertions account for about 0.3% of all human mutations [7]. The etrotransposition rate compatible with live birth varies greatly etween the three retrotransposon families. Alu elements have the ighest estimated retrotransposition rate, with ∼1 in 20 live births,

ollowed by L1 with about 1 in 200, and SVA with ∼1 in 900 [7,11]. onsequently, non-LTR retrotransposons have a remarkable impact n genome plasticity and stability.

With few exceptions, retrotransposon insertions are neutral or n some cases even deleterious to the host [53]. Due to the unique roperties of non-LTR retrotransposon insertions, an independent e novo insertion can be easily discriminated from a shared TE inser- ion that has been passed down over generations. These include andom insertion site (apart from the loose recognition motif of he endonuclease cleavage site), unique TSDs, and identity of sub- amily affiliation [15,54]. In the case of L1, the length of the insertion s yet another discriminating factor to discern shared from separate nsertions [55]. Because the precise excision of non-LTR retrotrans- osons is exceedingly rare, the ancestral state is known to be the bsence of the element; thus, TEs are generally homoplasy free [56], reviewed in [57]). Consequently, an insertion shared between wo humans at exactly the same genomic location with identical SDs is testimony to an inherited insertion and a common ancestor.

Deleterious insertions include the disruption of coding or regu- atory sequences (reviewed in [12–14,16]). The coding sequence of enes can be disrupted when a non-LTR retrotransposon inserts nto an exon, but also can be affected if the insertion occurs

ithin an intron. While the impact of the latter event is often ore difficult to demonstrate and may be overlooked, these inser-

ions can potentially disrupt splice sites and cause exon skipping. ntronic TE insertions have been implicated to potentially alter he expression of a gene through introduction of alternative splice ites or polyadenylation signals [58–61]. Intronic insertions have lso been associated with destabilization of the mRNA result- ng in reduced expression [62]. In addition, insertions into the 5′

nd 3′ prime region of genes can possibly alter their expression 63–65], reviewed in [66]. Alterations in gene expression increase he potential for altering equilibrium of regulatory networks, and hus augment susceptibility to certain diseases – including cancer.

.2. Insertional mutagenesis and disease

All three currently actively mobilizing non-LTR retrotransposon amilies – L1, SVA, and Alu – have been identified as the causative gent of several genetic disorders. These include hemophilia, lpert syndrome, familial hypercholesterolemia, and colon and reast cancer (reviewed in [7,12,13,16]). Several cancer predispos-

ng mutations caused by retrotransposon insertions are shown in able 1. Of all TE-causing genetic disorders identified to date, X- inked diseases are disproportionally over-represented compared o the autosomal diseases [7,12,62]. The X-chromosome is partic- larly enriched in inherited diseases caused by L1. The underlying easons are the subject of ongoing debate (e.g. [7,12,62,67]). An scertainment bias likely contributes (at least in part) to this find- ng, as the insertion on autosomes is commonly masked by the

ild-type allele when standard PCR procedures are used. In par- icular, this is the case for longer TE insertions (L1 and SVA) where he wild-type allele has a much shorter PCR amplicon. However, n insertional bias of L1 insertions toward the X-chromosome has lso been reported [67].

ancer Biology 20 (2010) 211–221 213

The coding sequence of some genes has been disrupted more than once by independent non-LTR retrotransposon insertions [14]. An example is the BRCA2 gene, which is associated with breast/ovarian cancer susceptibility [68,69]. Moreover, some genes have been targeted twice at exactly the same location. The APC gene (associated with colon cancer predisposition) is an intriguing example, as one insertion was caused by an L1 and the other by an Alu element (Table 1) [14]. The fact that different genes have been identified with recurrent disease-causing retrotransposon insertions indicates that insertions do not solely occur by chance. Instead, this implies varying susceptibility to non-LTR retrotrans- poson insertions. The exact reasons for this finding are elusive. However, certain characteristics of these genes likely contribute to their predisposition for multiple retrotransposon insertions. For example, the presence of TEs and the nucleotide composition of introns prone to recurrent de novo TE insertions might play a piv- otal role. The TE content of genes might be a contributing factor, as genes enriched in TE sequences harbor, on average, more sequences that resemble endonuclease cleavage sites, possibly increasing the proliferation of TEs.

For example, Alu elements have been shown to insert upstream of another element or within the polyA-tail of an existing ele- ment containing a less than perfect endonuclease cleavage site [70]. The TSD itself, created by classical TPRT, is a source for an addi- tional endonuclease cleavage site. The spacer region (see Fig. 1) of Alu elements also closely resembles an endonuclease recogni- tion site. Thus, one byproduct of Alu insertions is the creation of additional L1 endonuclease target sites suitable for the insertion of non-LTR retrotransposons. Also, recently inserted full-length L1s in particular contain several nucleotide sequences closely resembling endonuclease cleavage sites. However, TE density alone is likely not a sufficient explanation, as some genes with high TE density do not encounter recurrent de novo insertions. The methylation status and expression level of a gene may be other contributing factors as the DNA of genes that are actively transcribed may exist in more open chromatin structures that are more accessible to retrotransposition machinery and, therefore, may be more prone to de novo non-LTR insertions.

The origin of a TE insertion can be reconstructed based on the geographical distribution of a TE insertion and its frequency within a population. In the case of cancer, several founder muta- tions involving Alu elements have been identified. For example, an Alu insertion into the BRCA2 gene has been identified in the Por- tuguese population [69]. The Alu insertion disrupts exon 3 which results in exon skipping. This skipping of exon 3 has also been found in individuals without the Alu insertion, and thus an asso- ciation of the Alu insertion and cancer susceptibility has been questioned. However, a recent study convincingly linked the Alu founder mutation in the Portuguese population with cancer sus- ceptibility [71].

3.3. Insertional mutagenesis of TEs in somatic cells

The investigation of TE retrotransposition activity in somatic cells at a comprehensive level was until recently out of reach. Advancements in detection technologies, in particular high- throughput sequencing approaches, are on the verge of changing this. While knowledge about somatic retrotransposition is still sparse, there is increasing evidence of ongoing L1 mobilization in healthy somatic tissues. For example, L1 protein has been identified in adult cells [72], and ongoing retrotransposition causing somatic

mosaicism has been demonstrated in embryogenesis and within developing neuronal precursor cells [49–51]. In vitro assays using an L1 cassette with an adenovirus vector further indicate that L1 retrotransposition can occur in differentiated human primary cells in G1/S-arrested cells but not in G0-arrested cells [73]. Another

214 M.K. Konkel, M.A. Batzer / Seminars in Cancer Biology 20 (2010) 211–221

Table 1 Selection of TE insertions associated with cancer predisposition.

Cancer-associated insertional mutagenesis of transposable elements (TEs)

Locus Meaning Cancer association TE Distribution References

Classical TPRT insertions APC Adenomotous polyposis coli gene Colon cancer Alu Germline [135] APC L1Ta Somatic [132] BRCA1 Breast cancer 1 gene Breast cancer/ovarian cancer Alu Germline [69] BRCA2 Breast cancer 2 gene Breast cancer/ovarian cancer Alu Germline [68] BRCA2 Alu Germline [69] MLVI2 Moloney leukemia virus integration 2 homolog Leukemia Alu Germline [136] NF1 Neurofibromatosis 1 gene Neurofibromatosis type 1a Alu Germline [137]

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While the bulk of retrotransposon insertions are endonuclease- dependent and thus show the typical hallmarks of TPRT, a small fraction of endonuclease-independent L1 insertions has been observed in tissue culture cells deficient in NHEJ [78]. Although these insertions are also often associated with deletions of host

Fig. 2. Illustration of non-LTR retrotransposon insertion mechanisms. TSDs are shown in green; AAAAA stands for polyA-tail; A) Illustrates a typical non- LTR retrotransposon insertion. These insertions are thought to occur via TPRT. The insertion is 3′ intact (contains a polyA-tail) and is flanked by TSDs. No host sequence

APC Adenomotous polyposis coli gene Canc

a ∼10% of patients diagnosed with NF1 develop cancer.

tudy found that L1 retrotransposition using its endogenous L1 romoter requires cell divisions [74].

Furthermore, this research indicates that L1 retrotransposition ccurs at (very) low levels in primary human fibroblast cell lines 74]. On the basis of these initial results, it appears likely that the etrotransposition frequency varies individually and also between ifferent tissues. The TE mobilization rate in cancer cells is likely ore pronounced in comparison to “normal” tissue due to the

ikely activation of L1s through demethylation of their promoters see Section 7). In addition, deleterious retrotransposon insertions

ight not underlie the same selection criteria in cancer cells com- ared to healthy tissue. For example, retrotransposon insertions hat might typically result in apoptosis in normal cells may not ause cell death in cancer, given that the apoptosis pathway is often mpaired in cancerous cells [75]. However, in vitro studies of human arcinoma cells suggest that apoptosis is positively correlated with he presence of retrotransposition-competent L1 [76]. Conceivably, he cell reaction to L1 reactivation is dosage-dependent.

.4. TE insertions associated with deletion of host DNA

The deletion of host DNA associated with the de novo inser- ion of an L1 or Alu element was first demonstrated for L1 in issue culture and confirmed by comparative genomics studies or both L1 and Alu insertions [77–82]. SVA elements have not een sufficiently studied in this context, but probably are equally

nvolved in this mechanism. Two vastly different mechanisms, each ith characteristic properties, have been identified causing dele-

ions upon the insertion of non-LTR retrotransposons. These are PRT-dependent insertion-mediated deletions and endonuclease- ndependent insertions [41,77,79–82]. The primary difference etween the two mechanisms is the dependence on the L1 endonu- lease.

.4.1. Insertion-mediated deletions Insertion-mediated deletions are endonuclease-dependent and

re thought to make use of TPRT [41,77,79,80]. In these instances, n endonuclease cleavage site can be commonly identified at the nsertion site. In addition, the 3′ end of non-LTR retrotransposon nsertions involved in insertion-mediated deletions (the insertion nds in a polyA-tail) is generally intact [77,79,80]. However, due o the deletion of the host sequence, TSDs are absent in these nsertion-mediated events (see Fig. 2). While the precise insertion

echanism(s) of endonuclease-dependent TE insertion-mediated

eletions remains elusive, two different mechanisms have been roposed depending on the size of the deleted host DNA [77]. Small eletions of only a few nucleotides could be caused through a nick f the top strand of the DNA to the right of the initial cleavage site esulting in a 5′ overhang [77]. Larger deletions have been proposed

he colon Alu Germline [14,84]

to occur when a TE inserts downstream of a double strand break (DSB) [77].

A new line of evidence using in vitro assays indicates that dis- ruption of the NHEJ pathway commonly results in deletion of host DNA upon endonuclease-dependent L1insertion [47]. This part of the study was performed with zebrafish L1s in DT40 cells deficient in Ku70. Intriguingly, Ku70 is associated with the protection of DNA from exonucleolytic degradation [83]. This indi- cates that the host DNA of insertion-mediated deletions is not sufficiently protected from degradation at the endonuclease cleav- age site. It is the subject of future studies to determine if this finding represents a typical in vivo mechanism of human L1 retro- transposition. However, it strongly supports the hypothesis of competition between the retrotransposon insertion event itself and an attempt by the host to repair the nascent insertion site [10,41].

A number of genetic disorders caused by TE insertion-mediated deletions have been identified (reviewed in [13,14]). Alu insertion- mediated deletion has been suggested as a mechanism for deletion in the APC gene, which is associated with colon cancer predisposi- tion [14,84]. Somatic mutations involving this mechanism have not yet been identified. Altogether, the combination of host sequence deletion and a de novo retrotransposon insertion represents a large threat to the integrity of the human genome and has a higher potential to be deleterious to the host than insertional mutagenesis alone.

3.4.2. Endonuclease-independent TE insertions

is deleted. B) Shown are the typical hallmarks of an insertion-mediated deletion. The non-LTR retrotransposon is 3′ intact, indicated by the polyA-tail. TSDs are absent; upstream (left) of the element, host DNA is deleted. C) Illustrated is an endonuclease- independent insertion with deletion of host DNA 3′ and 5′ of the insertion. However, deletions can be limited to 3′ or 5′ host DNA sequence. The non-LTR retrotransposon usually does not contain a polyA-tail and is not flanked by TSDs.

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NA, these insertion events are not thought to be the products of he retrotransposition pathway; rather they occur by other mech- nisms such as DSBs. Recently, comparative genomic studies have hown that endonuclease-independent L1 insertions are not tis- ue culture artifacts and also occur in vivo [81,82]. Up to 0.5% and .7% of L1 and Alu insertions respectively could be attributed to ndonuclease-independent insertion mechanisms [7,81,82]. Apart rom the deletion of host sequence and absence of a typical endonu- lease cleavage site, these non-LTR insertions are commonly 3′ and ′ truncated and do not contain TSDs (Fig. 2) [78,81,82]. The aver- ge size of endonuclease-independent insertions is also in general maller (e.g. 572 bp for L1 [81]) than that of recent classical non- TR retrotransposon insertion events (e.g. 900 bp for L1 [1]) that re thought to have inserted using TPRT.

The deletion of host DNA in conjunction with the structure and echanism of the TE insertion and the commonly found microho- ology between the L1 insertion and the host DNA indicates that

ndonuclease-independent TE insertions are involved in DSB repair 78,81,82]. Hence, a very small fraction of TEs potentially contribute o cell integrity. A few endonuclease-independent L1 insertions ave been identified in genetic disorders (reviewed in [62]). It emains an unanswered question if endonuclease-independent nsertions occur only in the germline or if they are also common

n somatic cells. While these insertions are probably involved in NA repair and thus stabilizing, the deletion of host sequence can

till be deleterious to the host and (for example) be associated with ancer predisposition. In cancer cells their contribution could be imilar to other DSB repair mechanisms that can cause new onco-

ig. 3. Typical TE-mediated NAHR models. he colored arrows represent non-LTR retrotransposons of a given family; e.g. Alu eleme chimeric TE element (indicated by two different colors within element). The breakage

or two adjacent elements, these events can occur between far removed TE elements in t ) TEs involved in NAHR are inverted. Del stands for deletion, dup for duplication, and inv nd duplication. (If two non-homologous chromosomes are involved a translocation can reates reciprocal deletion and duplication. C) Intrachromosomal, intrachromatid TE-me n inversion of DNA between involved TEs.

ancer Biology 20 (2010) 211–221 215

genes through fusion of two genes or disrupt tumor suppressor genes.

4. Inverted Alu elements cause genomic instability

On average, the human genome contains approximately one Alu insertion per every 3 kb [1]. However, Alu elements are not evenly distributed throughout the human genome [1]. Areas of higher than average Alu density have been particularly associ- ated with genomic instability. The abundance of Alu insertions and high sequence homology between Alu elements (average 71%) [85] makes them targets for genome rearrangements (see section 5). Moreover, inverted Alu elements in close proximity to each other are less frequently identified in the human genome than Alu inser- tions in the same orientation [86,87]. This can only partially be attributed to insertional bias of Alu insertions in the same orien- tation [65,83]. Instead, inverted repeats likely represent hotspots of genomic instability, as seen in studies with yeast [86].

Inverted Alu elements that are closely spaced appear to build hairpin structures, which can cause DSBs of the DNA and excision of inverted Alu elements from the human genome [86,88]. More- over, hairpin structures involving Alu elements appear to cause replication stalling and collapse of the replication fork, which can

lead to DSBs and/or intra- or intermolecular template switch [88]. Apart from the distance of two inverted Alu insertions, the size of sequence identity between two Alu insertions seems to be an important contributing factor [87]. Despite their underrepresen- tation in the human genome, inverted repeats continue to cause

nts. The tip of the arrow indicates the 3′ end of the TE. TE-mediated NAHRs create point can be anywhere within the TE. While TE-mediated NAHRs are here shown he geography of the chromosome. For A) to C) TEs are in the same orientation; for

for inversion. A) Interchromosal TE-mediated NAHR results in reciprocal deletion occur.) B) Intrachromosomal TE-mediated NAHR between two sister chromatids

diated NAHR produces only a deletion. D) NAHR between two inverted TEs results

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enomic instability. Moreover, de novo Alu insertions can generate ew inverted loci, which pose a potential threat for rearrangements

n future human generations. Conceivably, these rearrangements an also occur in somatic cells and potentially contribute to cancer evelopment.

. TE-mediated recombination and genomic instability

.1. Role of TE-mediated recombination in the human genome

Chromosomal structural variation is caused by two different eneral mechanisms: homologous recombination (HR) and NHEJ 89]. HR is highly conserved in a wide array of species, including rokaryotes and eukaryotes, suggesting that HR is a fundamen- al biological mechanism. Deficiencies involving HR have been ssociated with cancer development [90,91]. While programmed R occurs only once during chromosomal crossover in meiosis, R is commonly involved in the repair of DSBs, preventing an

ndividual from DNA damage (reviewed in [92]). For further infor- ation regarding DSB repair mechanisms, we refer to other recent

eviews (e.g. [10,92]). If performed accurately, HR will repair the NA without a trace. However, the process is often undermined y abundant and/or highly homologous sequences including TEs 10,93]. In these instances, two homologous sequences from dif- erent genomic locations recombine in a process called non-allelic omologous recombination (NAHR) and consequently cause dele- ions, duplications, or inversions, as illustrated in Fig. 3 [93–95].

In addition, TE-mediated NAHR can also result in translocations. t would seem reasonable to assume that given the abundance f TEs in the human genome, TE-mediated NAHR translocations ould commonly occur by chance. TEs, in particular Alu elements,

re often found in the vicinity or even within the breakage points f translocations [96]. However, few disease-associated translo- ations have been identified that clearly suggest the involvement f TE-mediated NAHR (e.g. [97]). This may be due to the require- ents of the DSB repair pathways that cause translocations: NHEJ

nd single strand annealing (SSA) [98]. SSA – which can create lu-mediated NAHR translocations – is likely rarely utilized due o higher sequence homology requirements than usually found etween two TEs [98,99]. Instead, NHEJ appears to be the main echanism in the creation of translocations. While insertional mutagenesis caused by TEs is occasionally

eleterious to the host resulting in genetic disorders including ancer, post-insertional rearrangements of TEs pose altogether a ar greater threat to the integrity of the genome [10]. While a E insertion may disrupt the function of one gene, recombina- ion between two TEs might result in the deletion of a functional egion – including several genes, especially if two distant (not ecessarily consecutive) TEs are involved. So far, most of our knowl- dge regarding TE-mediated recombination events is based on ermline mutations, some of which cause genetic diseases (also eferred to as genomic disorders). Rearrangements involving the ermline can be de novo or inherited and passed on to future gen- rations [100]. However, somatic structural variation also does ccur [101]. TE-mediated NAHRs – both somatic and inherited – ave been associated with cancer predisposition and development e.g. [102–105]). Still, TE-mediated NAHR events are most likely nderrepresented in studies involving cancer susceptibility, devel- pment, and progression as a consequence of detection challenges.

Comprehensive comparative genomics studies of the human

nd chimpanzee genomes have shown that L1 and Alu elements are ommonly involved in TE-mediated recombination events caus- ng deletions [93,94,106]. Alu elements are also often involved in AHR-mediated duplications, as they have been found at chro- osomal breakpoints of segmental duplications with a higher

ancer Biology 20 (2010) 211–221

frequency (∼27%) compared to the average Alu density (∼10%) in the human genome [107]. Beyond genomic instability caused by Alu-mediated NAHRs that cause duplications, segmental duplica- tions themselves represent hotspots for structural variation and genomic instability as they share a very high homology. As a result, they can lead to genetic disease and altered gene expression of genes located within these regions [58,92].

Analysis of the human genome using the chimpanzee genome as a reference indicates that deletions caused by Alu-mediated NAHR occur about nine times more often (492 versus 55) than L1-mediated NAHRs in the human genome [93,94]. The anal- ysis of L1-mediated recombination deletions also revealed 18 chimeric L1s showing hallmarks of NHEJ, raising the number of recombination events to 73. In terms of frequency, L1 recom- bination plays a relatively minor role in the human genome. However, L1-mediated recombination events are significantly larger in size than Alu-mediated NAHRs (6132 bp versus 806 bp) [93,94]. L1 recombination-mediated deletions have deleted more DNA sequence from the human genome than the sum of all other TE deletion related events [94]. This includes DNA loss of Alu insertion-mediated deletions, L1 insertion-mediated deletions, and Alu-mediated NAHR deletions from the human genome over the last 5–6 million years [94]. Thus, L1 recombination-mediated dele- tions – even though less frequent – are a major contributor to genome instability. In addition, larger recombination events are more likely to involve greater disruption of functional genomic regions, making them more deleterious to the host. Consequently, these events are likely under significant negative selection, which commonly results in the loss of these events from the human popu- lation. These deletion events may commonly be so deleterious that affected individuals do not survive to birth. Indeed, to date only a few L1-mediated recombinations have been detected (reviewed in [14]).

5.2. TE-mediated NAHRs in human genetic disorders

TE-mediated NAHR events, in particular those involving Alu ele- ments, have been identified in a variety of genetic disorders and play an important role in their genesis (reviewed in [13,16]). Also, several Alu-mediated NAHRs with implications for cancer suscep- tibility have been identified; a subset is shown in Table 2. With few exceptions, NAHR-mediated deletions are more commonly detected than duplications. While in part this might be caused by a detection bias, as deletions are more easily identified compared to duplications, there is also evidence that overall deletions occur more often than duplications (reviewed in [92]). This may in part be explained by the intrachromatid NAHR mechanism which causes only deletions (see Fig. 3).

Through advancements in detection methods for SVs, genomic rearrangements are now recognized in a number of genetic disor- ders. However, SVs are likely still severely underrepresented due to the required use of more complicated, lengthier, and resource con- sumptive methods to detect genomic rearrangements. The analysis of the CDH1 gene – associated with hereditary diffuse gastric can- cer (HDGC) – is an intriguing example. Up until 2009, only germline single nucleotide or small frameshift mutations were associated with HDGC [108,109]. Yet in individuals with HDGC, mutations were identified in only 30–50% of the cases. Oliveira et al. [108] have convincingly shown that some individuals with HDGC har- bor structural variations including two Alu-mediated NAHRs that disrupt the CDH1 gene. The identification of all mutations includ-

ing SV in genes associated with cancer is of great importance for the identification of carriers, counseling, appropriate individ- ualized screening and preventive measures. Counseling of breast cancer patients is another intriguing example, as a recent retro- spective study found that close to 50% of breast cancer patients with

M.K. Konkel, M.A. Batzer / Seminars in Cancer Biology 20 (2010) 211–221 217

Table 2 Overview of cancer-associated Alu-mediated non-allelic homologous recombination (NAHR) events.

Alu-Alu non-allelic homologous recombination (NAHR) and tumors

Locus Meaning Tumor association Occurrence # of events References

Deletions VHL von Hippel Lindau gene von Hippel Lindau disease Germline Multiplea [102,138] BRCA1 Breast cancer 1 gene Breast/ovarian cancer Germline Multiplea [105,113], reviewed in [112] BRCA2 Breast cancer 2 gene Breast/ovarian cancer Germline Fewb [105,139] CHEK Checkpoint kinase 2 gene Breast cancer Germlne 1 [105] CHEK Checkpoint kinase 2 gene Prostate cancer Germline 1 [140] MLH 1 MutL E. coli homolog 1

gene Hereditary non-polyposis colorectal cancer

Germline Fewc [111,141–144]

MSH2 MutS E. coli homolog 2 gene

Hereditary non-polyposis colorectal cancer

Germline Multiplea [111,142,145–147]

MEN1 Multiple endocrine neoplasia type 1 gene

Multiple endocrine neoplasia type 1

Germline 1 [148]

CDH1 Cadherin 1 gene Hereditary diffuse gastric cancer

Germline Fewc [108]

RB Retinobalstoma gene Association with glioma Germline 1 [149] CAD Caspase activated dnase

gene Hepatoma Somatic Recurrenta ? [150]

NF1 Neurofibromatosis 1 gene Neurofibromatosis type 1 Germline 1 [151]

Duplications MLL1 Myeloid/lymphoid mixed

lineage leukemia gene Acute myeloid leukemia (AML) Somatic Recurrenta,d [103,104,119]

MYB Transcription factor T-acute lymphoblasctic leukemia

Somatic Recurrenta ? [118]

BRCA1 Breast cancer 1 gene Breast cancer/ovarian cancer Germline Fewc [105,152], reviewed in [112] BRCA2 Breast cancer 2 gene Germline 1 [105]

Translocations EWSR1-ETV Ewing sarcoma breakpoint

region 1 – ETS variant gene Ewing sarcoma Somatic Recurrenta ? [97]

#Number. a More than 10. b More than 1 and less 10.

B [

5

N b e M N c a a t a m e a

t a a t B i g a a m

c Commonly detected. d Also detected in healthy blood donors.

RCA1 mutations eventually develop contra-lateral breast cancer 110].

.3. Predisposition to TE-mediated NAHRs

It appears that some genes are more prone to TE-mediated AHRs than others, as multiple recurrent independent recom- ination events have been identified in these genes [16]. For xample, the BRCA1 (and to a lesser extent BRCA2), VHL, MLL1, LH1, and MSH2 genes encountered recurrent Alu-mediated AHRs (e.g. [102,111,112]). In some genes, 25% or more of can- er predisposing mutations in certain demographic populations re caused by Alu-mediated NAHRs (e.g [102,113]). The over- ll density of Alu elements seems to be a contributing factor o recurrent Alu-mediated NAHRs as genes with higher than verage Alu density in general show evidence of a higher Alu- ediated NAHR rate. Another factor could be the absence of

pigenetic constraints such as CpG methylation and/or histone cetylation.

MLH1 and MSH2, both involved in the mismatch repair sys- em, are two intriguing examples. Point mutations, small insertions nd deletions, and genomic rearrangements within these genes re associated with hereditary non-polyposis colorectal cancer, he most common form of inherited colon cancer (e.g. [111,114]). oth genes have a higher than average Alu density within their

ntronic sequence (20% MLH1 and 40% MSH2 compared to 10% enome average) [111]. In the MSH2 gene ∼3/4 of rearrangements re caused by Alu-mediated NAHRs [111]. In contrast, only about fourth of the rearrangements of the MLH1 gene contained hall- arks of unequal Alu recombination [111]. The majority of the

other events show characteristics of NHEJ often with involve- ment of TEs (in particular Alu elements). Another contributing factor to multiple independent Alu-mediated NAHRs is the age of the involved Alu insertions, as members of the youngest Alu subfamily, AluY, have been disproportionately detected in NAHRs relative to their density in the genome [93]. Occasionally, the same (often young) Alu element is involved in several indepen- dent NAHRs [102]. This is likely due to a higher homology of the Alu element, as younger insertions have on average accumu- lated fewer point mutations. This is further supported by a recent study that showed that AluY elements that are fixed in the human genome are associated with an increased local recombination rate [115].

The majority of TE-mediated rearrangements are sporadic and specific to the individual in whom the event was detected, and possibly also in immediate family members. However, some Alu- mediated NAHRs founder mutations have been identified in certain populations. For example, out of more than 60 reported BRCA1 rearrangements, the majority of which were caused by unequal recombination of Alu elements, six founder mutations have been identified [113,116]. One Alu-mediated NAHR event – identified in the Dutch population – accounts for about a quarter of all varia- tions identified in the BRCA1 gene in this demographic population [113]. Due to this founder mutation, the Dutch population has to date the largest fraction of mutations caused by structural vari-

ation in the BRCA1 gene. Analysis of the breakage point at the DNA sequence level can discriminate between identical and inde- pendent rearrangements. Inherited genomic rearrangements share identical DNA breakage points. The occurrence of two separate identical NAHRs is extraordinarily unlikely. In addition, SNP and/or

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18 M.K. Konkel, M.A. Batzer / Semina

icrosatellite information in the vicinity of the rearrangement can e used as further support for a shared or separate event.

Rearrangements in the MLL gene are in several ways intrigu- ng. SVs involving this gene are commonly identified in acute

yeloid leukemia (AML) [103,104,117]. Beside the recurrence of ranslocations in certain AML cancers, partial Alu-mediated NAHR uplications commonly occur within the coding sequence of this ene [103,117]. The most common in-frame duplication events cre- te in-frame fusions of exons 11 or 12 upstream of exon 5 [103,117]. t the same time, partial deletions involving this region of the enome have not been identified [104]. Thus, it seems likely that nother recombination mechanism is involved in the genesis of hese partial duplications. Synthesis-dependent strand annealing see [10]), a repair mechanism of DSBs in mammalian cells, could enerate a partial duplication in the MLL1 gene. This mechanism as suggested for the creation of somatic tandem duplications in YB, a transcription factor that can be associated with T-cell acute

ymphoblastic leukemia (T-ALL) [118]. In addition, the MLL gene epresents one of a few genes in which several independent somatic lu-mediated NAHRs have been detected. While partial duplica- ions show a high prevalence in patients with AML [103,117], these vents have also been identified in hematopoietic cells of healthy lood donors [119]. More recently, it has been shown that sup- ression of the wild-type allele with expression of the MLL partial uplication allele contributes to the leukemic phenotype [120].

. L1 induces double strand breaks

Another mechanism by which L1s potentially contribute to enome instability in their human host, is the observation that he L1 endonuclease creates far more DSBs than required for retro- ransposition in mammalian cells [43]. To what extent these tissue ulture observations will translate to human genomic instabil- ty remains elusive, as L1 expression under these experimental onditions was also far greater than under normal physiological onditions. It may not be possible to confirm or refute this mecha- ism in vivo because DSBs caused by L1 are indistinguishable from SBs caused by other mechanisms [10,43]. Regardless, DSBs com- romise DNA integrity, are highly recombinogenic, and exacerbate enomic instability.

. Methylation status and retrotransposition

In various tumors studied, global demethylation with site- pecific hypermethylation has been associated with cancer evelopment and progression (reviewed in [121]). Hypomethyla- ion varies considerably between different cancers, and in some umor cells hypomethylation is associated with cancer develop-

ent, while in others with progression [121]. The current model orrelates global demethylation with an elevated mutation rate nd chromosomal instability [122–124]. TEs including the pro- oters of L1 elements are often demethylated in cancer cells

[125,126], reviewed in [121]). The methylation of retrotrans- osons is believed to be a host defense mechanism in somatic cells gainst ongoing retrotransposition [127]. Indeed, for several can- er cell lines an increased L1 transcription rate has been detected n hypomethylated cancer cells [125,128]. One possible exception

ay be hepatocellular carcinoma cells, as these cells did not show vidence of this correlation [129].

Several points of evidence further support a correlation between

ypomethylation and TE activation and increased recombination ates. The lack of regular de novo methylation of LTR and non-LTR etrotransposons through Dnmt3L (DNA methltransferase 3-like) n non-dividing precursors of spermatogonial mouse stem cells esulted in high transcription levels of these TEs and meiotic fail-

ancer Biology 20 (2010) 211–221

ure in spermatocytes [130]. Hypomethylated thymic lymphomas in transgenic mice carrying a hypomorphic DNA methyltransferase Dnmt1 allele showed evidence of chromosomal instability [123]. Moreover, somatic retrotransposition of an Intracisternal A Parti- cle, an endogenous retrovirus, into the Notch1 gene was detected in several lymphomas in mice, indicating an activation of endogenous retroviruses through hypomethylation [131]. Finally, a somatic de novo L1 insertion into the APC gene has been identified in a human colon tumor [132].

The demethylation of TE promoters (e.g. L1 and ERVs) has pos- sible implications beyond the activation of TE retrotransposition. Through activation of potent TE promoters, transcription factor levels might be globally modified and/or the expression of genes might be altered in the vicinity of demethylated promoters [121]. Moreover, demethylation might result in the activation of the L1 antisense promoter that is also located within the 5′ UTR of a full- length element and which in turn can create chimeric transcripts [133]. Cancer-specific chimeric transcripts derived from L1 anti- sense promoters have been recently detected [134]. Intriguingly, these transcripts were derived from fixed, older L1s that are likely no longer capable of retrotransposition [134].

8. Conclusions

We have discussed the known manifold roles of TEs with respect to genome instability. Among other things, we have shown that TEs are major contributors to genomic rearrangements. We are just beginning to understand the full impact of TEs upon the genome architecture. It is now recognized that TEs play a role in cancer predisposition, development, and progression. The comprehensive use of recently available technologies such as second-generation sequencing and advanced computational algorithms will allow us to understand the role of TEs in the human genome more deeply. Moreover, it seems highly likely we will soon enter an era of personal genomics. Future projects will greatly increase our under- standing of human inter-variability, hotspots of alteration in the human genome, and disease associations. Soon, medical diagnos- tics and treatment decisions will greatly rely on the analysis of the individual’s genome. We are already witnessing the emergence of personalized cancer treatments. In addition, detailed analyses of single cells and/or different tissues are about to become possible for the first time. Using these advancements, we will likely dis- cover a great deal of variation within individual humans. Cancer in particular seems a prime candidate for detailed structural variation studies, as genomic instability and dynamics in genome structure are typical characteristics of cancer cells. A fascinating era employ- ing use of these rapidly evolving technologies lies ahead of us, along with the opportunity to deepen our understanding of TE biology and genome evolution.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

We thank Jerilyn A. Walker, Thomas J. Meyer, George Cook, and Brygg Ullmer for their thoughtful advice during the preparation the manuscript. This work was supported by National Institutes of Health grants ROI GM59290 and PO1 AG0022064 (M.A.B).

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