L1 elements are a large family of retrotransposons that encode two proteins that can catalyze target-primed, reverse transcription and integration of transcripts. The L1 promoter is found in the 5' untranslated region of the element and initiates transcription within a few base pairs of base 1 2021. The L1 promoter is known to be active in some somatic cell types 2223242526 and therefore new L1 insertions could, in theory, accumulate in somatic cells. The ability of L1 transposition in somatic cells to result in gene mutation was appreciated when a somatic L1 insertion in the MYC proto-oncogene was discovered in a human breast ductal adenocarcinoma 27. However, the contribution of somatic mobilization of endogenous L1 elements to cancer formation may be rare. The only other example of a known somatic insertion of a L1 element in a tumor was found in the tumor suppressor gene APC in a colon cancer 28.

Further research will be necessary to determine how often L1 gene insertions occur in human cancer because many techniques currently used to detect mutations in cancer, for example exon resequencing, would not always detect this kind of mutation. In fact, there is evidence that epigenetic regulation of endogenous L1 elements may influence tumor progression. Many human tumor genomes become globally hypomethylated upon cancer progression 29, and genome hypomethylation can result in upregulated retrotransposon transcription in cancer cells 3031. L1 promoter hypomethylation has been documented in progression of chronic myeloid leukemia to blast crisis. In chronic myeloid leukemia, L1 hypomethylation was associated with an upregulation of L1 transcript levels and poorer long-term survival of patients 31. Certainly, if the levels of transcripts from active L1 elements increase, then it could lead to the accumulation of new insertional mutations in cancer genes. However, activation of L1 transcription could also contribute to tumor initiation or progression by additional mechanisms. Studies have shown that inhibition of L1-encoded reverse transcriptase using RNA interference or small molecules reduces proliferation and promotes differentiation of melanoma, thyroid, and prostate cancer cell lines 3233. It has been speculated that L1 reverse transcriptase can regulate endogenous gene expression by some unknown mechanism, such as chromatin modification or nuclear repositioning 33. In addition, because L1 expression in cell culture can generate double strand breaks 34, it is possible that L1 activity in somatic cells could contribute to genomic instability.

Although the action of transposable elements can be either beneficial or detrimental to the organism, those insertions that harm the organism, by promoting cancer for instance, will be the easiest to identify. However, recent work indicates the potential for somatic mobilization of L1 elements to have a positive impact on the organism by generating diversity within a cell type 26. Specifically, a human L1 element driven from its endogenous promoter was shown to retrotranspose in vitro in rodent neural progenitor cells (NPCs) and in vivo in mouse brain. Furthermore, these insertions were capable of generating mutations that apparently have an impact on cell differentiation.

This study of L1 activity in neuronal cells took advantage of a retrotransposition indicator to detect retrotransposition. This indicator is a transgene that expresses a modified L1 element from its endogenous promoter. The L1 transgene is engineered to contain a reporter that consists of an enhanced green fluorescence protein (EGFP) expression cassette in the opposite orientation to that of the L1 element. The EGFP cassette is interrupted by an intron in the same orientation as L1 transcription, so that EGFP can only be expressed when the element has undergone transcription, splicing, and integration into a new location in the genome. EGFP expression could be detected after nucleofection of the retrotransposition indicator into NPCs in vitro and in neurons of mice harboring the retrotransposition indicator as a transgene. In vitro, cells that had acquired new retrotransposon insertions could be differentiated into the three major neural cell types, namely neurons, astrocytes, and oligodendrocytes. In vivo, cells that expressed EGFP also expressed markers for neurons but not markers for astrocytes or oligodendrocytes. In vitro, the genomic locations of several retrotransposed L1s were cloned using inverse polymerase chain reaction based techniques. Some of the transposed elements had landed in neuronally expressed genes, including Psd-93. The cell clone harboring the Psd-93 insertion did express higher levels of the Psd-93 transcript than the parental cells, and downregulating Psd-93 via small interfering RNA knockdown in this clone resulted in a less differentiated phenotype. This indicates that, at least in vitro, L1 transposition can change somatic genomes in a way that has phenotypic consequences.

Future work will be required to determine whether endogenous L1s (for instance, mouse L1s in mouse cells or human L1s in human cells) transpose in NPCs and whether this truly promotes diversity among these cells. Because retrotransposition of Alu elements in trans by LINE elements has been detected in cell culture 18, it will also be interesting to determine whether somatic activity of L1 promotes somatic retrotransposition of Alu or other non-autonomous elements in vivo.

Although there are no known active DNA transposons in mammals, there are genes that were domesticated from these elements present in their host genomes 7. The recombination-activating gene (RAG)1 and RAG2 proteins are important for generating somatic diversity in the immune system because they play an indispensable role in V(D)J recombination during lymphocyte development. The RAG1 gene is hypothesized to have its origin from the transposase encoded by an ancient Transib superfamily transposon 3536. In fact, recent evidence demonstrates that the RAG proteins still possess the capability to transpose genomic sequences excised during V(D)J recombination to new places in the lymphocyte genome 37.

V(D)J recombination is a complex event, and both RAG proteins as well as other factors are involved in the process. Recombination in developing lymphocytes results in the joining of variable (V), joining (J), and sometimes diversity (D) segments together to form a mature B-cell receptor (BCR) or T-cell receptor (TCR) gene. The recombination events are catalyzed between recombination signal sequences (RSSs). Each RSS is composed of unique heptamer and nonamer sequences separated by 12 or 23 base pairs. Recombination only occurs between RSSs with 12 and 23 base pair sequences separating the heptamer and nonamers. Recombination removes sequences between V and J, or V, D, and J segments, releasing a circularized intervening DNA called the signal joint. V(D)J recombination events share biochemical similarity with the cut-and-paste reaction of DNA transposons, such as the Hermes and other hobo, Activator, Tam3 family elements 38. In fact, the excised fragment with its RSS ends is similar to a DNA transposon, but unlike a DNA transposon it is circularized.

The extrachromosomal array, or signal joint, excised during V(D)J recombination can be reintegrated by the RAG proteins into artificial DNA targets 39404142 and has been detected in vivo at the HPRT locus in T cells isolated from normal human donors 43. However, only recently has an assay been developed that allows measurement of the rate at which RAGs can catalyze insertion of the extrachromosomal array into the host genome 37. This was accomplished by generating a recombination substrate in a pre-B cell line that could detect both rearrangement and transposition. This recombination substrate consists of a puromycin resistance gene that is interrupted by a zeocin-green fluorescent protein (GFP) resistance marker. The zeocin-GFP marker is flanked by RSSs so that it mimics the substrate for V(D)J recombination and is a substrate for RAG activity. When this reporter undergoes RAG-initiated recombination, the zeocin-GFP marker with its RSSs are excised as a signal joint, and the puromycin resistance reading frame is restored, allowing the cells to grow in puromycin selection. The excised signal joint DNA consisting of the zeocin-GFP fragment flanked by RSSs resembles a transposon. One potential fate for this fragment is simply to be lost upon cell division. However, if the fragment undergoes a transposition-like reaction and is reintegrated into the host genome, the cell will also become zeocin resistant. As zeocin resistance can also be acquired if the fragment randomly integrates into DNA, true transposition events can be identified because of the generation of a characteristic integration-associated target DNA repeat.

Using this assay, it was determined that the fragment excised by RAG activity during V(D)J recombination reintegrates in this cell line in one out of every 13,000 recombination events. Some of these events are random integrations, but the estimated rate of bona fide transposition events is one every 50,000 recombination events. If this rate were true for human lymphocytes in vivo, then it would translate into approximately 10,000 transpositions per day.

In addition, the rate detected in the B-cell line could actually be an underestimation because the assay only detects transposition events that insert the zeocin-GFP construct in a genomic location that allows its expression. Nevertheless, the critical action of the RAG proteins in V(D)J rearrangement also results in a potential negative consequence: insertional mutation of the host genome via integration of the excised 'transposon'. The consequences of these events for the genomes of developing lymphocytes remain to be determined but they should be investigated, particularly because they may relate to B-cell or T-cell malignancies.