Characterization of pre-insertion loci of de novo L1 insertions

The human Long Interspersed Element-1 (LINE-1) and the Short Interspersed Element (SINE) Alu comprise 28% of the human genome. They share the same L1-encoded endonuclease for insertion, which recognizes an A+T-rich sequence. Under a simple model of insertion distribution, this nucleotide preference would lead to the prediction that the populations of both elements would be biased towards A+T-rich regions. Genomic L1 elements do show an A+T-rich bias. In contrast, Alu is biased towards G+C-rich regions when compared to the genome average. Several analyses have demonstrated that relatively recent insertions of both elements show less G+C content bias relative to older elements. We have analyzed the repetitive element and G+C composition of more than 100 pre-insertion loci derived from de novo L1 insertions in cultured human cancer cells, which should represent an evolutionarily unbiased set of insertions. An A+T-rich bias is observed in the 50 bp flanking the endonuclease target site, consistent with the known target site for the L1 endonuclease. The L1, Alu, and G+C content of 20 kb of the de novo pre-insertion loci shows a different set of biases than that observed for fixed L1s in the human genome. In contrast to the insertion sites of genomic L1s, the de novo L1 pre-insertion loci are relatively L1-poor, Alu-rich and G+C neutral. Finally, a statistically significant cluster of de novo L1 insertions was localized in the vicinity of the c-myc gene. These results suggest that the initial insertion preference of L1, while A+T-rich in the initial vicinity of the break site, can be influenced by the broader content of the flanking genomic region and have implications for understanding the dynamics of L1 and Alu distributions in the human genome.     

Characterization of full-length LINE-1 insertions in 154 genomes

Long interspersed nuclear elements (LINEs) are retrotransposons that contribute to genetic variation in the human genome. LINE-1 elements in larger-scale studies are challenging to identify using sequencing technologies due to cost and scalability. We developed an approach using optical mapping for detection of full-length LINE-1 insertions and 10× sequencing for confirmation. We found 51 true positive full-length LINE-1 insertions, of which 4 are novel insertions, in NA12878. Repeating our analysis on a larger sample set representing 26 populations, we identified 329 full-length LINE-1 elements, of which 123 are novel. 24.8% of these 329 LINE-1 insertions were shared amongst all 5 superpopulations (AFR, AMR, EUR, EAS, SAS). The African superpopulation has a higher percentage of population-specific LINE-1 insertions than any other superpopulation. These data indicate that our approach can provide high-speed, cost-effective, and increased accuracy for LINE-1 detection. These data also provide an insight into variations of LINE-1 elements between different populations.     

A systematic analysis of LINE-1 endonuclease-dependent retrotranspositional events causing human genetic disease

Diverse long interspersed element-1 (LINE-1 or L1)-dependent mutational mechanisms have been extensively studied with respect to L1 and Alu elements engineered for retrotransposition in cultured cells and/or in genome-wide analyses. To what extent the in vitro studies can be held to accurately reflect in vivo events in the human genome, however, remains to be clarified. We have attempted to address this question by means of a systematic analysis of recent L1-mediated retrotranspositional events that have caused human genetic disease, with a view to providing a more complete picture of how L1-mediated retrotransposition impacts upon the architecture of the human genome. A total of 48 such mutations were identified, including those described as L1-mediated retrotransposons, as well as insertions reported to contain a poly(A) tail: 26 were L1 trans-driven Alu insertions, 15 were direct L1 insertions, four were L1 trans-driven SVA insertions, and three were associated with simple poly(A) insertions. The systematic study of these lesions, when combined with previous in vitro and genome-wide analyses, has strengthened several important conclusions regarding L1-mediated retrotransposition in humans: (a) approximately 25% of L1 insertions are associated with the 3' transduction of adjacent genomic sequences, (b) approximately 25% of the new L1 inserts are full-length, (c) poly(A) tail length correlates inversely with the age of the element, and (d) the length of target site duplication in vivo is rarely longer than 20 bp. Our analysis also suggests that some 10% of L1-mediated retrotranspositional events are associated with significant genomic deletions in humans. Finally, the identification of independent retrotranspositional events that have integrated at the same genomic locations provides new insight into the L1-mediated insertional process in humans.     

Characterization of pre-insertion loci of de novo L1 insertions

The human Long Interspersed Element-1 (LINE-1) and the Short Interspersed Element (SINE) Alu comprise 28% of the human genome. They share the same L1-encoded endonuclease for insertion, which recognizes an A+T-rich sequence. Under a simple model of insertion distribution, this nucleotide preference would lead to the prediction that the populations of both elements would be biased towards A+T-rich regions. Genomic L1 elements do show an A+T-rich bias. In contrast, Alu is biased towards G+C-rich regions when compared to the genome average. Several analyses have demonstrated that relatively recent insertions of both elements show less G+C content bias relative to older elements. We have analyzed the repetitive element and G+C composition of more than 100 pre-insertion loci derived from de novo L1 insertions in cultured human cancer cells, which should represent an evolutionarily unbiased set of insertions. An A+T-rich bias is observed in the 50 bp flanking the endonuclease target site, consistent with the known target site for the L1 endonuclease. The L1, Alu, and G+C content of 20 kb of the de novo pre-insertion loci shows a different set of biases than that observed for fixed L1s in the human genome. In contrast to the insertion sites of genomic L1s, the de novo L1 pre-insertion loci are relatively L1-poor, Alu-rich and G+C neutral. Finally, a statistically significant cluster of de novo L1 insertions was localized in the vicinity of the c-myc gene. These results suggest that the initial insertion preference of L1, while A+T-rich in the initial vicinity of the break site, can be influenced by the broader content of the flanking genomic region and have implications for understanding the dynamics of L1 and Alu distributions in the human genome.     

The Zinc-Finger Antiviral Protein ZAP Inhibits LINE and Alu Retrotransposition

Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements.     

Higher Alu Methylation Levels in Catch-Up Growth in Twenty-Year-Old Offsprings

Alu elements and long interspersed element-1 (LINE-1 or L1) are two major human intersperse repetitive sequences. Lower Alu methylation, but not LINE-1, has been observed in blood cells of people in old age, and in menopausal women having lower bone mass and osteoporosis. Nevertheless, Alu methylation levels also vary among young individuals. Here, we explored phenotypes at birth that are associated with Alu methylation levels in young people. In 2010, 249 twenty-years-old volunteers whose mothers had participated in a study association between birth weight (BW) and nutrition during pregnancy in 1990, were invited to take part in our present study. In this study, the LINE-1 and Alu methylation levels and patterns were measured in peripheral mononuclear cells and correlated with various nutritional parameters during intrauterine and postnatal period of offspring. This included the amount of maternal intake during pregnancy, the mother’s weight gain during pregnancy, birth weight, birth length, and the rate of weight gain in the first year of life. Catch-up growth (CUG) was defined when weight during the first year was >0.67 of the standard score, according to WHO data. No association with LINE-1 methylation was identified. The mean level of Alu methylation in the CUG group was significantly higher than those non-CUG (39.61% and 33.66 % respectively, P < 0.0001). The positive correlation between the history of CUG in the first year and higher Alu methylation indicates the role of Alu methylation, not only in aging cells, but also in the human growth process. Moreover, here is the first study that demonstrated the association between a phenotype during the newborn period and intersperse repetitive sequences methylation during young adulthood.     

Exon skipping caused by an intronic insertion of a young Alu Yb9 element leads to severe hemophilia A

Short interspersed elements, such as Alu elements, have propagated to more than one million copies in the human genome. They affect the genome in several ways, caused by retrotransposition, recombination between elements, gene conversion, and alterations in gene expression. These events, including novel insertions into active genes, have been associated with a number of human disorders. Hemophilia A is an X-linked severe bleeding disorder and is caused by mutations in the Factor VIII gene. The spectrum of mutations includes point mutations, rearrangements, insertions, and deletions. Recently, an Alu retrotransposition event in a coding exon has been reported in a family with a severe form of hemophilia A. This was the first report of an Alu insertion in the Factor VIII gene. Here, we report a second Alu insertion event that lies in an intron of the same gene that causes exon skipping and the complete disruption of gene expression.     

Long L1 insertions in human gene introns specifically reduce the content of corresponding primary transcripts

LINE-1 (L1) retrotransposons comprise about 17% of the human genome and include a recently transposed set of Ta-L1 elements that are polymorphic in humans. Although it is widely believed that L1s play an essential role in shaping and functioning of mammalian genomes, the understanding of the impact of L1 insertions on gene expression is far from being comprehensive. Here we compared hnRNA contents for allele pairs of genes heterozygous for Ta-L1 insertions in their introns in human cell lines of various origin. We demonstrated that some Ta-L1 insertions correlated with decreased content of the corresponding hnRNAs. This effect was characteristic of only nearly full-sized L1s and seemed to be tissue specific.     

Subfamily structure and evolution of the human L1 family of repetive sequences

Summary Comparative analysis of the available 3′-portions of the human L1 (LINE-1) family of repeated sequences indicates that all the sequences can be classified in two major subfamilies. The division is based on patterns of diagnostic bases shared within L1 subfamilies of sequences but differing between them. The overall ratio of replacement to synonymous positions, occupied by the diagnostic bases in the large open reading frame of the L1 sequence, is 1.15. This indicates that both subfamilies were obtained from genes coding for functional proteins. The L1 subfamilies appear to be of different ages and may represent a “fossil record” of the same active gene at different times in the history of primates. The younger subfamily can be split further into at least two closely related branches of sequences. The above facts combined with the recent data for the Alu subfamily structure show that LINE and SINE families of interspersed repeats share discontinuous patterns in their evolution. These data are consistent with the model that both Alu and L1 families, as well as other pseudogene families, contain active genes producing discrete layers of pseudogenes throughout the history of primates. Models of evolutionary processes that could generate these discontinuities are discussed together with the possible biological role of Alu and L1 genes.     

Selective inhibition of Alu retrotransposition by APOBEC3G

The non-LTR retrotransposon LINE-1 (L1) comprises  17% of the human genome, and the L1-encoded proteins can function in trans to mediate the retrotransposition of non-autonomous retrotransposons (i.e., Alu and probably SVA elements) and cellular mRNAs to generate processed pseudogenes. Here, we have examined the effect of APOBEC3G and APOBEC3F, cytidine deaminases that inhibit Vif-deficient HIV-1 replication, on Alu retrotransposition and other L1-mediated retrotransposition processes. We demonstrate that APOBEC3G selectively inhibits Alu retrotransposition in an ORF1p-independent manner. An active cytidine deaminase site is not required for the inhibition of Alu retrotransposition and the resultant integration events lack G to A or C to T hypermutation. These data demonstrate a differential restriction of L1 and Alu retrotransposition by APOBEC3G, and suggest that the Alu ribonucleoprotein complex may be targeted by APOBEC3G.     

LINE-1 retrotransposition events affect endothelial proliferation and migration

Long interspersed nuclear element-1 (LINE-1, L1) is a retrotransposon which affects the human genome by a variety of mechanisms. While LINE-1 expression is suppressed in the most somatic human cells, LINE-1 elements are activated in human cancer. Recently, high accumulation of LINE-1-encoded ORF1p and ORF2p in endothelial cells of mature human blood vessels was described. Here, we demonstrate that LINE-1 de novo retrotransposition events lead to a reduction of endothelial cell proliferation and migration in a porcine aortic endothelial (PAE) cell model. Cell cycle studies show a G0/G1 arrest in PAE cells harboring LINE-1 de novo retrotransposition events. Remarkably, in in situ analysis LINE-1-encoded ORF2p was not detectable in tumor blood vessels of different human organs while vascular endothelial cells of corresponding normal organs strongly expressed LINE-1 ORF2p. Quantitative RT-PCR analysis revealed that LINE-1 de novo retrotransposition influences selectively the expression of some angiogenic factors such as VEGF and Tie-2. Thus, our data suggest that LINE-1 de novo retrotransposition events might suppress angiogenesis and tumor vascularisation by reducing the angiogenic capacity of vascular endothelial cells.     

Retrofitting the genome: L1 extinction follows endogenous retroviral expansion in a group of muroid rodents

Long interspersed nuclear element 1 (LINE-1; L1) retrotransposons are the most common retroelements in mammalian genomes. Unlike individual families of endogenous retroviruses (ERVs), they have remained active throughout the mammalian radiation and are responsible for most of the retroelement movement and much genome rearrangement within mammals. They can be viewed as occupying a substantial niche within mammalian genomes. Our previous demonstration that L1s and B1 short interspersed nuclear elements (SINEs) are inactive in a group of South American rodents led us to ask if other elements have amplified to fill the empty niche. We identified a novel and highly active family of ERVs (mysTR). To determine whether loss of L1 activity was correlated with expansion of mysTR, we examined mysTR activity in four South American rodent species that have lost L1 and B1 activity and four sister species with active L1s. The copy number of recent mysTR insertions was extremely high, with an average of 4,200 copies per genome. High copy numbers exist in both L1-active and L1-extinct species, so the mysTR expansion appears to have preceded the loss of both SINE and L1 activity rather than to have filled an empty niche created by their loss. It may be coincidental that two unusual genomic events--loss of L1 activity and massive expansion of an ERV family--occur in the same group of mammals. Alternatively, it is possible that this large ERV expansion set the stage for L1 extinction.     

Proteome Profile of Endogenous Retrotransposon‐Associated Complexes in Human Embryonic Stem Cells

Long Interspersed Element‐1 (LINE‐1 or L1) are transposable elements similar to retroviruses that have existed in the genome of primates for millions of years. They encode two Open Reading Frame (ORF) proteins (ORF1p and ORF2p) that bind L1 RNA to form a ribonucleoprotein (RNP) complex and are required for L1 integration into the host genome. Humans have evolved with L1 and found ways to limit L1 activity. To identify partners of the L1 RNP, previous studies used ectopic expression of L1 ORF1/2p or RNA in various cancer cells, which express low levels of the ORF proteins. Whether naturally occurring L1 RNP interacts with the same proteins in non‐cancer cells is unknown. Here, the aim is to examine the natural assembly of endogenous L1 RNPs in normal human cells. L1 elements are expressed in human embryonic stem cells (hESCs), derived from pre‐implantation embryos. Therefore, these cells are used to immunoprecipitate ORF1p followed by MS to identify proteins that associate with the naturally‐occurring L1 ORF1p. Some of the same proteins as well as unique proteins are found interacting with the endogenous L1 ORF1p complexes. The analysis of ORF1p‐associated proteins in hESCs can help address important questions in both retrotransposon biology and the biology of hESCs.