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• W2928206577 abstract "•High-throughput insertion site profiling of a LINE-1 (L1) element by ATLAS-seq•Insertion is influenced strongly by DNA sequence but only weakly by chromatin state•L1 integration preferences suggest a link with host DNA replication•Post-insertion selection reshapes L1 distribution across functional genomic regions L1 retrotransposons are transposable elements and major contributors of genetic variation in humans. Where L1 integrates into the genome can directly impact human evolution and disease. Here, we experimentally induced L1 retrotransposition in cells and mapped integration sites at nucleotide resolution. At local scales, L1 integration is mostly restricted by genome sequence biases and the specificity of the L1 machinery. At regional scales, L1 shows a broad capacity for integration into all chromatin states, in contrast to other known mobile genetic elements. However, integration is influenced by the replication timing of target regions, suggesting a link to host DNA replication. The distribution of new L1 integrations differs from those of preexisting L1 copies, which are significantly reshaped by natural selection. Our findings reveal that the L1 machinery has evolved to efficiently target all genomic regions and underline a predominant role for post-integrative processes on the distribution of endogenous L1 elements. L1 retrotransposons are transposable elements and major contributors of genetic variation in humans. Where L1 integrates into the genome can directly impact human evolution and disease. Here, we experimentally induced L1 retrotransposition in cells and mapped integration sites at nucleotide resolution. At local scales, L1 integration is mostly restricted by genome sequence biases and the specificity of the L1 machinery. At regional scales, L1 shows a broad capacity for integration into all chromatin states, in contrast to other known mobile genetic elements. However, integration is influenced by the replication timing of target regions, suggesting a link to host DNA replication. The distribution of new L1 integrations differs from those of preexisting L1 copies, which are significantly reshaped by natural selection. Our findings reveal that the L1 machinery has evolved to efficiently target all genomic regions and underline a predominant role for post-integrative processes on the distribution of endogenous L1 elements. Transposable elements are present in almost all species and significantly contribute to shaping host genome structure and function (Chuong et al., 2017Chuong E.B. Elde N.C. Feschotte C. Regulatory activities of transposable elements: from conflicts to benefits.Nat. Rev. Genet. 2017; 18: 71-86Crossref PubMed Scopus (662) Google Scholar). In humans, the only autonomously active family is the long interspersed element-1 (LINE-1 or L1) (Kazazian and Moran, 2017Kazazian Jr., H.H. Moran J.V. Mobile DNA in health and disease.N. Engl. J. Med. 2017; 377: 361-370Crossref PubMed Scopus (195) Google Scholar). Our genome contains ∼500,000 copies of this non-LTR-retrotransposon, occupying 17% of the genome (Kazazian and Moran, 2017Kazazian Jr., H.H. Moran J.V. Mobile DNA in health and disease.N. Engl. J. Med. 2017; 377: 361-370Crossref PubMed Scopus (195) Google Scholar). However, only ∼100 L1 copies are still retrotransposition competent, all of them belonging to the youngest and human-specific L1HS subfamily (Brouha et al., 2003Brouha B. Schustak J. Badge R.M. Lutz-Prigge S. Farley A.H. Moran J.V. Kazazian Jr., H.H. Hot L1s account for the bulk of retrotransposition in the human population.Proc. Natl. Acad. Sci. USA. 2003; 100: 5280-5285Crossref PubMed Scopus (736) Google Scholar). Each individual also has L1 copies not present in the reference genome, which contribute to as much as 20% of all structural variants in humans (Mir et al., 2015Mir A.A. Philippe C. Cristofari G. euL1db: the European database of L1HS retrotransposon insertions in humans.Nucleic Acids Res. 2015; 43: D43-D47Crossref PubMed Scopus (41) Google Scholar, Sudmant et al., 2015Sudmant P.H. Rausch T. Gardner E.J. Handsaker R.E. Abyzov A. Huddleston J. Zhang Y. Ye K. Jun G. Fritz M.H.-Y. et al.1000 Genomes Project ConsortiumAn integrated map of structural variation in 2,504 human genomes.Nature. 2015; 526: 75-81Crossref PubMed Scopus (1250) Google Scholar). Many of these highly polymorphic elements (in terms of presence or absence) are active and can produce new insertions (Beck et al., 2010Beck C.R. Collier P. Macfarlane C. Malig M. Kidd J.M. Eichler E.E. Badge R.M. Moran J.V. LINE-1 retrotransposition activity in human genomes.Cell. 2010; 141: 1159-1170Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, Gardner et al., 2017Gardner E.J. Lam V.K. Harris D.N. Chuang N.T. Scott E.C. Pittard W.S. Mills R.E. Devine S.E. 1000 Genomes Project ConsortiumThe Mobile Element Locator Tool (MELT): population-scale mobile element discovery and biology.Genome Res. 2017; 27: 1916-1929Crossref PubMed Scopus (151) Google Scholar, Philippe et al., 2016Philippe C. Vargas-Landin D.B. Doucet A.J. van Essen D. Vera-Otarola J. Kuciak M. Corbin A. Nigumann P. Cristofari G. Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci.eLife. 2016; 5: 166Crossref Scopus (95) Google Scholar, Scott et al., 2016Scott E.C. Gardner E.J. Masood A. Chuang N.T. Vertino P.M. Devine S.E. A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer.Genome Res. 2016; 26: 745-755Crossref PubMed Scopus (146) Google Scholar, Tubio et al., 2014Tubio J.M.C. Li Y. Ju Y.S. Martincorena I. Cooke S.L. Tojo M. Gundem G. Pipinikas C.P. Zamora J. Raine K. et al.ICGC Breast Cancer GroupICGC Bone Cancer GroupICGC Prostate Cancer GroupMobile DNA in cancer. Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes.Science. 2014; 345 (1251343–1251343)Crossref PubMed Scopus (248) Google Scholar). Retrotransposition is not restricted to the germline (leading to inheritable genetic variations and occasionally novel genetic diseases) and can also drive somatic genome rearrangements during embryogenesis, neural development, and in many cancers (Burns, 2017Burns K.H. Transposable elements in cancer.Nat. Rev. Cancer. 2017; 17: 415-424Crossref PubMed Scopus (246) Google Scholar, Faulkner and Garcia-Perez, 2017Faulkner G.J. Garcia-Perez J.L. L1 mosaicism in mammals: extent, effects, and evolution.Trends Genet. 2017; 33: 802-816Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, Hancks and Kazazian, 2016Hancks D.C. Kazazian Jr., H.H. Roles for retrotransposon insertions in human disease.Mob. DNA. 2016; 7: 9Crossref PubMed Scopus (344) Google Scholar). Intact L1 elements (∼6 kb long) are transcribed from an internal promoter and encode two major proteins, ORF1p and ORF2p, required for L1 retrotransposition (Moran et al., 1996Moran J.V. Holmes S.E. Naas T.P. DeBerardinis R.J. Boeke J.D. Kazazian Jr., H.H. High frequency retrotransposition in cultured mammalian cells.Cell. 1996; 87: 917-927Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar). ORF2p is a combined endonuclease (EN) and reverse transcriptase (RT) (Feng et al., 1996Feng Q. Moran J.V. Kazazian Jr., H.H. Boeke J.D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition.Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (866) Google Scholar, Mathias et al., 1991Mathias S.L. Scott A.F. Kazazian Jr., H.H. Boeke J.D. Gabriel A. Reverse transcriptase encoded by a human transposable element.Science. 1991; 254: 1808-1810Crossref PubMed Scopus (583) Google Scholar). After L1 nuclear import, ORF2p EN activity nicks the genomic DNA at a loosely defined consensus sequence (5′-TTTT/A-3′ or variants of that sequence) (Cost and Boeke, 1998Cost G.J. Boeke J.D. Targeting of human retrotransposon integration is directed by the specificity of the L1 endonuclease for regions of unusual DNA structure.Biochemistry. 1998; 37: 18081-18093Crossref PubMed Scopus (190) Google Scholar, Feng et al., 1996Feng Q. Moran J.V. Kazazian Jr., H.H. Boeke J.D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition.Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (866) Google Scholar, Jurka, 1997Jurka J. Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons.Proc. Natl. Acad. Sci. USA. 1997; 94: 1872-1877Crossref PubMed Scopus (425) Google Scholar). Then, the liberated T-rich 3′ end anneals to the L1 RNA poly(A) and is extended by ORF2p RT activity to synthesize first-strand L1 cDNA (Cost et al., 2002Cost G.J. Feng Q. Jacquier A. Boeke J.D. Human L1 element target-primed reverse transcription in vitro.EMBO J. 2002; 21: 5899-5910Crossref PubMed Scopus (379) Google Scholar, Doucet et al., 2015Doucet A.J. Wilusz J.E. Miyoshi T. Liu Y. Moran J.V. A 3′ poly(A) tract is required for LINE-1 retrotransposition.Mol. Cell. 2015; 60: 728-741Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, Kulpa and Moran, 2006Kulpa D.A. Moran J.V. Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles.Nat. Struct. Mol. Biol. 2006; 13: 655-660Crossref PubMed Scopus (209) Google Scholar, Luan et al., 1993Luan D.D. Korman M.H. Jakubczak J.L. Eickbush T.H. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition.Cell. 1993; 72: 595-605Abstract Full Text PDF PubMed Scopus (913) Google Scholar, Monot et al., 2013Monot C. Kuciak M. Viollet S. Mir A.A. Gabus C. Darlix J.-L. Cristofari G. The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.PLoS Genet. 2013; 9: e1003499Crossref PubMed Scopus (43) Google Scholar). Subsequent steps, which are less well defined, then result in creation of a new genomically inserted dsDNA L1 copy, flanked by short (4- to 16-bp) target-site duplications. Of note, the majority of insertions are 5′ truncated due to DNA repair pathways involved in insertion resolution or host defense (Coufal et al., 2011Coufal N.G. Garcia-Perez J.L. Peng G.E. Marchetto M.C.N. Muotri A.R. Mu Y. Carson C.T. Macia A. Moran J.V. Gage F.H. Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells.Proc. Natl. Acad. Sci. USA. 2011; 108: 20382-20387Crossref PubMed Scopus (178) Google Scholar, Suzuki et al., 2009Suzuki J. Yamaguchi K. Kajikawa M. Ichiyanagi K. Adachi N. Koyama H. Takeda S. Okada N. Genetic evidence that the non-homologous end-joining repair pathway is involved in LINE retrotransposition.PLoS Genet. 2009; 5: e1000461Crossref PubMed Scopus (96) Google Scholar, Zingler et al., 2005Zingler N. Willhoeft U. Brose H.-P. Schoder V. Jahns T. Hanschmann K.-M.O. Morrish T.A. Löwer J. Schumann G.G. Analysis of 5′ junctions of human LINE-1 and Alu retrotransposons suggests an alternative model for 5′-end attachment requiring microhomology-mediated end-joining.Genome Res. 2005; 15: 780-789Crossref PubMed Scopus (83) Google Scholar). Despite a pronounced cis preference for its own RNA, L1 is also responsible for the trans mobilization of non-autonomous retrotransposons, such as Alu or SVA sequences, and cellular mRNAs, following a similar mechanism (Kazazian and Moran, 2017Kazazian Jr., H.H. Moran J.V. Mobile DNA in health and disease.N. Engl. J. Med. 2017; 377: 361-370Crossref PubMed Scopus (195) Google Scholar). L1 elements carry a number of cis-regulatory sequences (sense and antisense promoters, cryptic splice sites, and polyadenylation signals); they can transduce 5′ and 3′ sequences from the donor locus to the site of insertion, potentially leading to exon or cis-regulatory sequence shuffling; and they can attract chromatin or DNA modification complexes, leading to altered epigenetic patterns (Cordaux and Batzer, 2009Cordaux R. Batzer M.A. The impact of retrotransposons on human genome evolution.Nat. Rev. Genet. 2009; 10: 691-703Crossref PubMed Scopus (1094) Google Scholar, Denli et al., 2015Denli A.M. Narvaiza I. Kerman B.E. Pena M. Benner C. Marchetto M.C.N. Diedrich J.K. Aslanian A. Ma J. Moresco J.J. et al.Primate-specific ORF0 contributes to retrotransposon-mediated diversity.Cell. 2015; 163: 583-593Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, Kaer and Speek, 2013Kaer K. Speek M. Retroelements in human disease.Gene. 2013; 518: 231-241Crossref PubMed Scopus (85) Google Scholar, Liu et al., 2018Liu N. Lee C.H. Swigut T. Grow E. Gu B. Bassik M.C. Wysocka J. Selective silencing of euchromatic L1s revealed by genome-wide screens for L1 regulators.Nature. 2018; 553: 228-232Crossref PubMed Scopus (143) Google Scholar, Tubio et al., 2014Tubio J.M.C. Li Y. Ju Y.S. Martincorena I. Cooke S.L. Tojo M. Gundem G. Pipinikas C.P. Zamora J. Raine K. et al.ICGC Breast Cancer GroupICGC Bone Cancer GroupICGC Prostate Cancer GroupMobile DNA in cancer. Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes.Science. 2014; 345 (1251343–1251343)Crossref PubMed Scopus (248) Google Scholar, Walter et al., 2016Walter M. Teissandier A. Pérez-Palacios R. Bourc’his D. An epigenetic switch ensures transposon repression upon dynamic loss of DNA methylation in embryonic stem cells.eLife. 2016; 5: R87Crossref Google Scholar). Thus, L1 insertion can considerably remodel gene structure and networks in a very short evolutionary time frame (Cordaux and Batzer, 2009Cordaux R. Batzer M.A. The impact of retrotransposons on human genome evolution.Nat. Rev. Genet. 2009; 10: 691-703Crossref PubMed Scopus (1094) Google Scholar, Denli et al., 2015Denli A.M. Narvaiza I. Kerman B.E. Pena M. Benner C. Marchetto M.C.N. Diedrich J.K. Aslanian A. Ma J. Moresco J.J. et al.Primate-specific ORF0 contributes to retrotransposon-mediated diversity.Cell. 2015; 163: 583-593Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Additionally, expression and mobilization of inserted L1 copies is restricted to cell-type-dependent permissive loci (Deininger et al., 2017Deininger P. Morales M.E. White T.B. Baddoo M. Hedges D.J. Servant G. Srivastav S. Smither M.E. Concha M. DeHaro D.L. et al.A comprehensive approach to expression of L1 loci.Nucleic Acids Res. 2017; 45: e31Crossref PubMed Scopus (59) Google Scholar, Gardner et al., 2017Gardner E.J. Lam V.K. Harris D.N. Chuang N.T. Scott E.C. Pittard W.S. Mills R.E. Devine S.E. 1000 Genomes Project ConsortiumThe Mobile Element Locator Tool (MELT): population-scale mobile element discovery and biology.Genome Res. 2017; 27: 1916-1929Crossref PubMed Scopus (151) Google Scholar, Philippe et al., 2016Philippe C. Vargas-Landin D.B. Doucet A.J. van Essen D. Vera-Otarola J. Kuciak M. Corbin A. Nigumann P. Cristofari G. Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci.eLife. 2016; 5: 166Crossref Scopus (95) Google Scholar, Scott et al., 2016Scott E.C. Gardner E.J. Masood A. Chuang N.T. Vertino P.M. Devine S.E. A hot L1 retrotransposon evades somatic repression and initiates human colorectal cancer.Genome Res. 2016; 26: 745-755Crossref PubMed Scopus (146) Google Scholar, Tubio et al., 2014Tubio J.M.C. Li Y. Ju Y.S. Martincorena I. Cooke S.L. Tojo M. Gundem G. Pipinikas C.P. Zamora J. Raine K. et al.ICGC Breast Cancer GroupICGC Bone Cancer GroupICGC Prostate Cancer GroupMobile DNA in cancer. Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes.Science. 2014; 345 (1251343–1251343)Crossref PubMed Scopus (248) Google Scholar). Altogether, where L1 integrates into the host genome dictates both its genomic impact and the ability of the novel copy to be subsequently expressed and remobilized. Therefore, elucidating L1 target site selection is critical to understanding genome evolution and somatic genome plasticity in cancer or aging. Although L1 and Alu elements share a common integration machinery and similar target site consensus motifs, they are distributed in differing chromosomal regions within the human genome (Gilbert et al., 2002Gilbert N. Lutz-Prigge S. Moran J.V. Genomic deletions created upon LINE-1 retrotransposition.Cell. 2002; 110: 315-325Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, Korenberg and Rykowski, 1988Korenberg J.R. Rykowski M.C. Human genome organization: Alu, lines, and the molecular structure of metaphase chromosome bands.Cell. 1988; 53: 391-400Abstract Full Text PDF PubMed Scopus (484) Google Scholar, Lander et al., 2001Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. FitzHugh W. et al.International Human Genome Sequencing ConsortiumInitial sequencing and analysis of the human genome.Nature. 2001; 409: 860-921Crossref PubMed Scopus (17764) Google Scholar, Wagstaff et al., 2012Wagstaff B.J. Hedges D.J. Derbes R.S. Campos Sanchez R. Chiaromonte F. Makova K.D. Roy-Engel A.M. Rescuing Alu: recovery of new inserts shows LINE-1 preserves Alu activity through A-tail expansion.PLoS Genet. 2012; 8: e1002842Crossref PubMed Scopus (29) Google Scholar). L1 elements accumulate in AT-rich isochores, while Alu sequences are rather enriched in GC-rich isochores. These observations suggest that the distributions of L1 and Alu may be drastically (and differently) reshaped after integration by recombination, purifying selection, and possibly other processes (Pavlícek et al., 2001Pavlícek A. Jabbari K. Paces J. Paces V. Hejnar J.V. Bernardi G. Similar integration but different stability of Alus and LINEs in the human genome.Gene. 2001; 276: 39-45Crossref PubMed Scopus (89) Google Scholar). Direct analysis of L1 genomic integration and comparison with the landscape of existing copies are essential to understand the interplay between L1 retrotransposon and its observed distribution within the host genome. De novo L1 insertion site maps based on Sanger sequencing have provided mechanistic insight about L1 replication but only limited information on target site preference due to the low numbers of integration sites recovered (Gilbert et al., 2002Gilbert N. Lutz-Prigge S. Moran J.V. Genomic deletions created upon LINE-1 retrotransposition.Cell. 2002; 110: 315-325Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, Gilbert et al., 2005Gilbert N. Lutz S. Morrish T.A. Moran J.V. Multiple fates of L1 retrotransposition intermediates in cultured human cells.Mol. Cell. Biol. 2005; 25: 7780-7795Crossref PubMed Scopus (210) Google Scholar, Symer et al., 2002Symer D.E. Connelly C. Szak S.T. Caputo E.M. Cost G.J. Parmigiani G. Boeke J.D. Human l1 retrotransposition is associated with genetic instability in vivo.Cell. 2002; 110: 327-338Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Here, we assessed whether L1 can integrate homogeneously throughout the genome or whether any genomic features or properties might favor or restrict L1 integration. To this end, we induced de novo L1 retrotransposition in cultured cells by transfecting a plasmid-borne active L1 element, and we mapped novel L1 target sites by a dedicated deep-sequencing approach (Philippe et al., 2016Philippe C. Vargas-Landin D.B. Doucet A.J. van Essen D. Vera-Otarola J. Kuciak M. Corbin A. Nigumann P. Cristofari G. Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci.eLife. 2016; 5: 166Crossref Scopus (95) Google Scholar). We compared the new integration sites with a large collection of publicly available genomic datasets and with the distribution of existing endogenous L1 copies. Our data confirm the primordial role of the local DNA sequence at the target site and reveal how preexisting biases in the genomic distribution of L1 target motifs skew the profile of new integration events. Overall, we find that new L1 insertions broadly target all the regions of the human genome, being insensitive to chromatin organization or transcriptional activity, although with a bias for early-replicating genomic domains. This distribution markedly differs from that of endogenous L1 elements, and we find that this difference predominantly results from evolutionary selection rather than from L1-induced chromatin changes. To facilitate the genomic characterization of pre-integration sites in silico, we screened cell lines well characterized by the ENCODE project for their ability to sustain high levels of retrotransposition (K562, GM12878, HeLa S3, MCF-7, HepG2, and IMR90) using the assay described in Figure 1A. Among them, HeLa S3 cells were the most permissive to L1 retrotransposition (0.1%–1% of transfected cells) and were chosen to obtain a large number of independent L1 insertions. We induced retrotransposition from a plasmid-borne active L1 element expressed from its natural promoter to avoid saturating any cellular factors involved in L1 target site selectivity (Figure 1A). The L1 construct also contains a retrotransposition reporter based on the neomycin-resistance gene (NeoR) in its 3′ UTR, allowing us to discriminate new copies from endogenous ones and the possibility to select cells with new insertions, since it only becomes functional upon transcription, splicing, reverse transcription, and integration (Moran et al., 1996Moran J.V. Holmes S.E. Naas T.P. DeBerardinis R.J. Boeke J.D. Kazazian Jr., H.H. High frequency retrotransposition in cultured mammalian cells.Cell. 1996; 87: 917-927Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar). To locate new engineered L1 insertions in the genome of cultured cells, we modified amplification typing of L1 active subfamilies and sequencing (ATLAS-seq), a technique originally developed to profile endogenous L1 elements (Philippe et al., 2016Philippe C. Vargas-Landin D.B. Doucet A.J. van Essen D. Vera-Otarola J. Kuciak M. Corbin A. Nigumann P. Cristofari G. Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci.eLife. 2016; 5: 166Crossref Scopus (95) Google Scholar). We mapped 1,565 de novo L1 target sites from 28 independent pools of HeLa S3 cells selected with G418 (referred as L1 neo hereafter), as well as a smaller set of 184 insertions obtained from pools of cells without any selection for retrotransposition cassette expression (referred as L1 neo-unsel hereafter), which were used in subsequent downstream analyses (Figure S1; Table S2). New engineered L1 insertions detected by ATLAS-seq display the expected hallmarks of L1 retrotransposition. First, we observe a poly(dA) tract at the junction between L1 and its 3′ flanking sequence (Figure 1B). Since the poly(dA) sequence is not encoded in the plasmid DNA, this feature enables us to discriminate bona fide retrotransposition events from random plasmid integration or chimeric reads (see STAR Methods). Second, sequence analysis of pre-integration sites reveals a consensus motif consistent with a canonical L1 EN-mediated cleavage and with the subsequent annealing of the L1 mRNA poly(A) tail to an extended T-tract to promote reverse transcription priming (Figure 1C) (Feng et al., 1996Feng Q. Moran J.V. Kazazian Jr., H.H. Boeke J.D. Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition.Cell. 1996; 87: 905-916Abstract Full Text Full Text PDF PubMed Scopus (866) Google Scholar, Jurka, 1997Jurka J. Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons.Proc. Natl. Acad. Sci. USA. 1997; 94: 1872-1877Crossref PubMed Scopus (425) Google Scholar, Monot et al., 2013Monot C. Kuciak M. Viollet S. Mir A.A. Gabus C. Darlix J.-L. Cristofari G. The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.PLoS Genet. 2013; 9: e1003499Crossref PubMed Scopus (43) Google Scholar). The L1 target motif was virtually identical whether insertions were recovered with G418 selection or not (Figure S1C). Although the pre-integration site sequence is often a non-perfect match, we nevertheless found that this motif is present at the vast majority of the mapped insertion sites (Figure 1D). To summarize, ATLAS-seq can detect de novo engineered L1 insertions in cultured cells, and the local DNA motif is a primary determinant of L1 integration site selection. In vitro, target DNA structure and assembly into nucleosomes can impact L1 EN activity (Cost et al., 2001Cost G.J. Golding A. Schlissel M.S. Boeke J.D. Target DNA chromatinization modulates nicking by L1 endonuclease.Nucleic Acids Res. 2001; 29: 573-577Crossref PubMed Scopus (53) Google Scholar). To examine whether nucleosome occupancy influences L1 local integration site selection in vivo, we examined publicly available micrococcal-nuclease digestion and sequencing (MNase-seq) data from HeLa S3 at and around pre-integration sites (Lacoste et al., 2014Lacoste N. Woolfe A. Tachiwana H. Garea A.V. Barth T. Cantaloube S. Kurumizaka H. Imhof A. Almouzni G. Mislocalization of the centromeric histone variant CenH3/CENP-A in human cells depends on the chaperone DAXX.Mol. Cell. 2014; 53: 631-644Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Because the L1 machinery preferentially targets a specific AT-rich motif (Figures 1C and 1D), we computationally constructed three distinct types of control datasets, each comprising the same number of sites as the experimental data (1,565): (1) 1,000 purely random control datasets (termed random); (2) 1,000 base-composition-matched control datasets (termed BMC), also generated randomly but with a base-composition around the target site (±5 bp) identical to the experimental dataset; and (3) 1,000 motif-matched control datasets (termed MMC) consisting of a random collection of sites with an L1 target motif. L1 target sites are significantly depleted in nucleosomes (Figure 2, left). This depletion can be largely explained by low nucleosome occupancy at AT-rich sequences, since it is also observed for the BMC and the MMC (Figure 2, middle), but not for the random control (Figure 2, right), consistent with previous observations indicating that these sequences disfavor nucleosome positioning (Valouev et al., 2011Valouev A. Johnson S.M. Boyd S.D. Smith C.L. Fire A.Z. Sidow A. Determinants of nucleosome organization in primary human cells.Nature. 2011; 474: 516-520Crossref PubMed Scopus (432) Google Scholar). We confirmed the trend of nucleosome depletion at AT-rich regions characteristic of L1 target sites using data obtained with other cell lines (Descostes et al., 2014Descostes N. Heidemann M. Spinelli L. Schüller R. Maqbool M.A. Fenouil R. Koch F. Innocenti C. Gut M. Gut I. et al.Tyrosine phosphorylation of RNA polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells.eLife. 2014; 3: e02105Crossref PubMed Scopus (56) Google Scholar, Schwartz et al., 2018Schwartz U. Németh A. Diermeier S. Exler J.H. Hansch S. Maldonado R. Heizinger L. Merkl R. Längst G. Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin.Nucleic Acids Res. 2018; 154: 515Google Scholar) (Figure S2). Insertions can still occur at regions in which nucleosomes are more dense (lower part of the heatmaps), implying that nucleosomal DNA is not refractory to L1 integration per se. Thus, L1 preferentially inserts into nucleosome-depleted DNA primarily due to sequence context. HeLa cells possess an abnormal karyotype and are hypertriploid (Adey et al., 2013Adey A. Burton J.N. Kitzman J.O. Hiatt J.B. Lewis A.P. Martin B.K. Qiu R. Lee C. Shendure J. The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line.Nature. 2013; 500: 207-211Crossref PubMed Scopus (235) Google Scholar). To analyze the genomic distribution of L1 insertions, we corrected for aneuploidy and local copy-number variations (CNV), using low-coverage whole-genome sequencing (WGS) of the HeLa S3 stock used in our retrotransposition assays (∼1.6×; Figure 3C, CNV track). We find that the chromosomal distribution of new L1 insertions sites largely reflects chromosome size and copy number (Figure 3A). Endogenous L1 copies are enriched in the X chromosome, where they have been proposed to contribute to X chromosome inactivation (Bailey et al., 2000Bailey J.A. Carrel L. Chakravarti A. Eichler E.E. Molecular evidence for a relationship between LINE-1 elements and X chromosome inactivation: the Lyon repeat hypothesis.Proc. Natl. Acad. Sci. USA. 2000; 97: 6634-6639Crossref PubMed Scopus (320) Google Scholar, Lyon, 1998Lyon M.F. X-chromosome inactivation: a repeat hypothesis.Cytogenet. Cell Genet. 1998; 80: 133-137Crossref PubMed Scopus (352) Google Scholar) (see also Figure 3C, endogenous L1 tracks). However, new L1 insertions are not significantly enriched in the X chromosome under our experimental conditions, suggesting that the evolutionary accumulation of L1 in the X chromosome results either from post-integration selection or requires a stably inactivated X (Xi) chromosome (like many cancer cell lines, HeLa cells do not express the Xist RNA and only contain activated X chromosomes; Kawakami et al., 2004Kawakami T. Zhang C. Taniguchi T. Kim C.J. Okada Y. Sugihara H. Hattori T. Reeve A.E. Ogawa O. Okamoto K. Characterization of loss-of-inactive X in Klinefelter syndrome and female-derived cancer cells.Oncogene. 2004; 23: 6163-6169Crossref PubMed Scopus (84) Google Scholar). New insertions are overrepresented in chromosome 1, indicating that L1 integration is not perfectly random (Figure 3A). We next examined the overall spacing of de novo L1 insertions (Figure 3B) and compared it to each simulated control dataset or to 1,000 random samplings of 1,565 reads from HeLa S3 WGS (random, BMC, and MMC controls [see above] and WGS). New L1 insertion" @default.
• W2928206577 created "2019-04-11" @default.
• W2928206577 creator A5016607425 @default.
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• W2928206577 creator A5081420809 @default.
• W2928206577 date "2019-05-01" @default.
• W2928206577 modified "2023-10-12" @default.
• W2928206577 title "The Landscape of L1 Retrotransposons in the Human Genome Is Shaped by Pre-insertion Sequence Biases and Post-insertion Selection" @default.
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