Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres

Long interspersed element-1 (LINE-1 or L1) elements are abundant, non-long-terminal-repeat (non-LTR) retrotransposons that comprise 17% of human DNA(1). The average human genome contains similar to 80-100 retrotransposition- competent L1s (ref. 2), and they mobilize by a process that uses both the L1 endonuclease and reverse transcriptase, termed target-site primed reverse transcription(3-5). We have previously reported an efficient, endonuclease-independent L1 retrotransposition pathway (ENi) in certain Chinese hamster ovary (CHO) cell lines that are defective in the non-homologous end-joining (NHEJ) pathway of DNA double-strand-break repair(6). Here we have characterized ENi retrotransposition events generated in V3 CHO cells, which are deficient in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and have both dysfunctional telomeres and an NHEJ defect. Notably, similar to 30% of ENi retrotransposition events insert in an orientation-specific manner adjacent to a perfect telomere repeat (5'-TTAGGG-3'). Similar insertions were not detected among ENi retrotransposition events generated in controls or in XR-1 CHO cells deficient for XRCC4, an NHEJ factor that is required for DNA ligation but has no known function in telomere maintenance. Furthermore, transient expression of a dominant-negative allele of human TRF2 ( also called TERF2) in XRCC4-deficient XR-1 cells, which disrupts telomere capping, enables telomere-associated ENi retrotransposition events. These data indicate that L1s containing a disabled endonuclease can use dysfunctional telomeres as an integration substrate. The findings highlight similarities between the mechanism of ENi retrotransposition and the action of telomerase, because both processes can use a 3' OH for priming reverse transcription at either internal DNA lesions or chromosome ends(7,8). Thus, we propose that ENi retrotransposition is an ancestral mechanism of RNA-mediated DNA repair associated with non-LTR retrotransposons that may have been used before the acquisition of an endonuclease domain.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62964/1/nature05560.pd

Short Telomeres Initiate Telomere Recombination in Primary and Tumor Cells

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase—some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR−/− and Eμmyc+mTR−/−) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR−/− tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR−/− cells that had short telomeres. Using mouse mTR+/− and human hTERT+/− primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length

Endonuclease-independent LINE-1 retrotransposition.

Transposable element derived sequences comprise -40% of the human genome. Long Interspersed Elements (L1s or LINEs) comprise ∼17% of the genome and mobilize via an RNA intermediate by a process termed retrotransposition. L1 encodes two proteins (ORF1p and ORF2p), which are essential for mobility. ORF1p is an RNA binding protein, whereas ORF2p exhibits reverse transcriptase and endonuclease activities. L1 integration is thought to occur by target-site primed reverse transcription (TPRT). During TPRT, the L1 endonuclease cleaves the genomic DNA at the consensus sequence 5'TTTT/A (where the / indicates the cleavage site), exposing a 3' hydroxyl residue that serves as a primer for reverse transcription of the L1 RNA by the L1 reverse transcriptase. The nascent L1 cDNA then joins to genomic DNA generating LINE-1 structural hallmarks such as frequent 5' truncations, a 3'poly(A) tail, and variable-length target site duplications (TSDs). An additional pathway for L1 insertion appears to act independently of endonuclease cleavage, but is dependent upon reverse transcriptase. Endonuclease-independent (ENi) retrotransposition occurs at near wild type levels in DNA-PKcs and XRCC4 deficient cell lines, two components utilized for the DNA repair pathway of non-homologous end joining (NHEJ). Analysis of the pre- and post-integration sites revealed that ENi retrotransposition results in unusual structures because the L1s integrate at atypical target sequences, are frequently truncated at both their 5' and 3' ends, and lack TSDs. We also detect ∼30% of ENi retrotransposition events insert in an orientation-specific manner adjacent to telomere repeats, in DNA-PKcs deficient cells which also display a telomere defect, whereas none were detected from XRCC4-deficient cells. Finally, we find L1 retrotransposition in NHEJ deficient cells only requires ORF1p and the RT domains, since mutations in the C-domain are also retrotransposition competent in NHEJ deficient cells and appear to integrate by a similar mechanism as ENi retrotransposition. In sum, L1 may integrate into DNA lesions, resulting in retrotransposon-mediated DNA repair in mammalian cells.Ph.D.Biological SciencesGeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124894/2/3163893.pd

Mobile Genetic Elements in Cellular Differentiation, Genome Stability, and Cancer

The human genome, as with the genome of most organisms, is comprised of various types of mobile genetic element derived repeats. Mobile genetic elements that mobilize by an RNA intermediate, include both autonomous and non-autonomous retrotransposons, and mobilize by a “copy and paste” mechanism that relies of the presence of a functional reverse transcriptase activity. The extent to which these different types of elements are actively mobilizing varies among organisms, as revealed with the advent of Next Generation DNA sequencing (NGS).To understand the normal and aberrant mechanisms that impact the mobility of these elements requires a more extensive understanding of how these elements interact with molecular pathways of the cell, including DNA repair, recombination and chromatin. In addition, epigenetic based-mechanisms can also influence the mobility of these elements, likely by transcriptional activation or repression in certain cell types. Studies regarding how mobile genetic elements interface and evolve with these pathways will rely on genomic studies from various model organisms. In addition, the mechanistic details of how these elements are regulated will continue to be elucidated with the use of genetic, biochemical, molecular, cellular, and bioinformatic approaches. Remarkably, the current understanding regarding the biology of these elements in the human genome, suggests these elements may impact developmental biology, including cellular differentiation, neuronal development, and immune function. Thus, aberrant changes in these molecular pathways may also impact disease, including neuronal degeneration, autoimmunity, and cancer

Multiple Fates of L1 Retrotransposition Intermediates in Cultured Human Cells

LINE-1 (L1) retrotransposons comprise ∼17% of human DNA, yet little is known about L1 integration. Here, we characterized 100 retrotransposition events in HeLa cells and show that distinct DNA repair pathways can resolve L1 cDNA retrotransposition intermediates. L1 cDNA resolution can lead to various forms of genetic instability including the generation of chimeric L1s, intrachromosomal deletions, intrachromosomal duplications, and intra-L1 rearrangements as well as a possible interchromosomal translocation. The L1 retrotransposition machinery also can mobilize U6 snRNA to new genomic locations, increasing the repertoire of noncoding RNAs that are mobilized by L1s. Finally, we have determined that the L1 reverse transcriptase can faithfully replicate its own transcript and has a base misincorporation error rate of ∼1/7,000 bases. These data indicate that L1 retrotransposition in transformed human cells can lead to a variety of genomic rearrangements and suggest that host processes act to restrict L1 integration in cultured human cells. Indeed, the initial steps in L1 retrotransposition may define a host/parasite battleground that serves to limit the number of active L1s in the genome

Analysis of 5′ junctions of human LINE-1 and Alu retrotransposons suggests an alternative model for 5′-end attachment requiring microhomology-mediated end-joining

Insertion of the human non-LTR retrotransposon LINE-1 (L1) into chromosomal DNA is thought to be initiated by a mechanism called target-primed reverse transcription (TPRT). This mechanism readily accounts for the attachment of the 3′-end of an L1 copy to the genomic target, but the subsequent integration steps leading to the attachment of the 5′-end to the chromosomal DNA are still cause for speculation. By applying bioinformatics to analyze the 5′ junctions of recent L1 insertions in the human genome, we provide evidence that L1 uses at least two distinct mechanisms to link the 5′-end of the nascent L1 copy to its genomic target. While 5′-truncated L1 elements show a statistically significant preference for short patches of overlapping nucleotides between their target site and the point of truncation, full-length insertions display no distinct bias for such microhomologies at their 5′-ends. In a second genome-wide approach, we analyzed Alu elements to examine whether these nonautonomous retrotransposons, which are thought to be mobilized through L1 proteins, show similar characteristics. We found that Alu elements appear to be predominantly integrated via a pathway not involving overlapping nucleotides. The results indicate that a cellular nonhomologous DNA end-joining pathway may resolve intermediates from incomplete L1 retrotransposition events and thus lead to 5′ truncations

A Comprehensive Analysis of Recently Integrated Human Ta L1 Elements

The Ta (transcribed, subset a) subfamily of L1 LINEs (long interspersed elements) is characterized by a 3-bp ACA sequence in the 3′ untranslated region and contains ∼520 members in the human genome. Here, we have extracted 468 Ta L1Hs (L1 human specific) elements from the draft human genomic sequence and screened individual elements using polymerase-chain-reaction (PCR) assays to determine their phylogenetic origin and levels of human genomic diversity. One hundred twenty-four of the elements amenable to complete sequence analysis were full length (∼6 kb) and have apparently escaped any 5′ truncation. Forty-four of these full-length elements have two intact open reading frames and may be capable of retrotransposition. Sequence analysis of the Ta L1 elements showed a low level of nucleotide divergence with an estimated age of 1.99 million years, suggesting that expansion of the L1 Ta subfamily occurred after the divergence of humans and African apes. A total of 262 Ta L1 elements were screened with PCR-based assays to determine their phylogenetic origin and the level of human genomic variation associated with each element. All of the Ta L1 elements analyzed by PCR were absent from the orthologous positions in nonhuman primate genomes, except for a single element (L1HS72) that was also present in the common (Pan troglodytes) and pygmy (P. paniscus) chimpanzee genomes. Sequence analysis revealed that this single exception is the product of a gene conversion event involving an older preexisting L1 element. One hundred fifteen (45%) of the Ta L1 elements were polymorphic with respect to insertion presence or absence and will serve as identical-by-descent markers for the study of human evolution