The third international conference on the genomic impact of eukaryotic transposable elements (TEs... more The third international conference on the genomic impact of eukaryotic transposable elements (TEs) was held 24 to 28 February 2012 at the Asilomar Conference Center, Pacific Grove, CA, USA. Sponsored in part by the National Institutes of Health grant 5 P41 LM006252, the goal of the conference was to bring together researchers from around the world who study the impact and mechanisms of TEs using multiple computational and experimental approaches. The meeting drew close to 170 attendees and included invited floor presentations on the biology of TEs and their genomic impact, as well as numerous talks contributed by young scientists. The workshop talks were devoted to computational analysis of TEs with additional time for discussion of unresolved issues. Also, there was ample opportunity for poster presentations and informal evening discussions. The success of the meeting reflects the important role of Repbase in comparative genomic studies, and emphasizes the need for close interactions between experimental and computational biologists in the years to come.
Malformations of cortical development (MCD) are neurological conditions displaying focal disrupti... more Malformations of cortical development (MCD) are neurological conditions displaying focal disruption of cortical architecture and cellular organization arising during embryogenesis, largely from somatic mosaic mutations. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat epilepsy. Here, we report a genetic atlas from 317 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation and single-cell sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associating distinct pathophysiological and clinical phenotypes. The unique spatiotemporal expression patterns identified by comparing single-nucleus transcriptional sequences of mutated genes in control and patient brains implicate critical roles in excitatory neurogenic pools during brain development, a...
In this article the affiliation 'Laboratory for Retrotransposon Dynamics, RIKEN Center for Integr... more In this article the affiliation 'Laboratory for Retrotransposon Dynamics, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan' for Tomoichiro Miyoshi was missing. The original article has been corrected.
Enabling the genomic revolution in Africa: H3Africa is developing capacity for health-related gen... more Enabling the genomic revolution in Africa: H3Africa is developing capacity for health-related genomics research in Africa The H3Africa Consortium *, † * H3Africa Consortium research investigators, funding agencies, and institutions appear in the supplementary materials Our understanding of genome biology, genomics, and disease, and even human history, has advanced tremendously with the completion of the Human Genome Project. Technological advances coupled with significant cost reductions in genomic research have yielded novel insights into disease etiology, diagnosis, and therapy for some of the world's most intractable and devastating diseases-including malaria, HIV/AIDS, tuberculosis, cancer, and diabetes. Yet, despite the burden of infectious diseases and, more recently, noncommunicable diseases (NCDs) in Africa, Africans have only participated minimally in genomics research. Of the thousands of genome-wide association studies (GWASs) that have been conducted globally, only seven (for HIV susceptibility, malaria, tuberculosis, and podoconiosis) have been conducted exclusively on African participants; four others (for prostate cancer, obsessive compulsive disorder, and anthropometry) included some African participants (www.genome.gov/gwastudies/). As discussed in 2011 (www.h3africa.org), if the dearth of genomics research involving Africans persists, the potential health and economic benefits emanating from genomic science may elude an entire continent.
Retrotransposition of LINEs and other retroelements increases repetition in mammalian genomes and... more Retrotransposition of LINEs and other retroelements increases repetition in mammalian genomes and can cause deleterious mutations. Recent insertions of two full-length L1s, L1 spa and L1 Orl , caused the disease phenotypes of the spastic and Orleans reeler mice respectively. Here we show that these two recently retrotransposed L1s are nearly identical in sequence, have two open reading frames and belong to a novel subfamily related to the ancient F subfamily. We have named this new subfamily T F (for transposable) and show that many full-length members of this family are present in the mouse genome. The T F 5Ј untranslated region has promoter activity, and T F-type RNA is abundant in cytoplasmic ribonucleoprotein particles, which are likely intermediates in retrotransposition. Both L1 spa and L1 Orl have reverse transcriptase activity in a yeast-based assay and retrotranspose at high frequency in cultured cells. Together, our data indicate that the T F subfamily of L1s contains a major class of mobile elements that is expanding in the mouse genome.
Some interferon stimulated genes (ISGs) encode proteins that inhibit LINE-1 (L1) retrotranspositi... more Some interferon stimulated genes (ISGs) encode proteins that inhibit LINE-1 (L1) retrotransposition. Here, we used immunoprecipitation followed by liquid chromatography-tandem mass spectrometry to identify proteins that associate with the L1 ORF1-encoded protein (ORF1p) in ribonucleoprotein particles. Three ISG proteins that interact with ORF1p inhibit retrotransposition: HECT and RLD domain containing E3 ubiquitin-protein ligase 5 (HERC5); 2’-5’-oligoadenylate synthetase-like (OASL); and helicase with zinc finger 2 (HELZ2). HERC5 destabilizes ORF1p, but does not affect its cellular localization. OASL impairs ORF1p cytoplasmic foci formation. HELZ2 recognizes sequences and/or structures within the L1 5’UTR to reduce L1 RNA, ORF1p, and ORF1p cytoplasmic foci levels. Overexpression of WT or reverse transcriptase-deficient L1s led to a modest induction of IFN-α expression, which was abrogated upon HELZ2 overexpression. Notably, IFN-α expression was enhanced upon overexpression of an OR...
transfection, cells were collected and processed for CAT or luciferase activity using standard te... more transfection, cells were collected and processed for CAT or luciferase activity using standard techniques 14. GST pull downs and immunoprecipitations GST±Rb (wild type and mutants 15) and other GST fusion proteins were expressed and puri®ed from Escherichia coli XA90 (ref. 16). GST fusion proteins that were immobilized on glutathione-sepharose (Pharmacia), or H3-derived peptides 3 bound to Sulfolink beads (Pierce), were incubated with extract in IPH buffer 16. Complexes were washed four times in IPH buffer before processing for methylase assays or western blotting. Antibodies against HA (12CA5, Roche), Gal4±DBD (DNA-binding domain; sc-510, Santa Cruz), SUV39H1 (M. Cleary), Rb (G3-245; XZ55, PharMingen) or HP1 (ref. 3) were used. For immunoprecipitations antibodies were incubated with HeLa nuclear extract (Cell Culture Center) or U2OS nuclear extract in IPH buffer at 4 8C (ref. 17). After 2 h a 50:50 mixture of protein A/G-sepharose beads (Pharmacia) was added. To avoid the possibility that DNA mediates the interaction between SUV39H1 and Rb, the immunoprecipitations were probed for the presence of histones with negative results. Histone methylase assays and protein sequencing Precipitations from pull downs or immunoprecipitations were incubated with 20 mg histones (Sigma) and 1 ml [3H-Me]-S-adenosyl methionine (NEN, 80 Ci mmol-1) in PBS at 30 8C for 1 h. Assays were analysed by SDS±PAGE followed by western blotting and autoradiography or were spotted onto P-81 cationic exchange paper (Whatman), washed in carbonate buffer and quanti®ed by scintillation counting 3. For amino-terminal sequencing, radiolabelled H3 was blotted to polyvinylidene¯uoride and sequenced by Edman degradation (Protein Sequencing Facility, University of Cambridge). We counted fractions for the presence of tritium. Supplementary information (with changes to the original Supplementary Information) is available on Nature's WorldWide Web site (http://www.nature.com).
Condensin I and condensin II are multi-subunit complexes that are known for their individual role... more Condensin I and condensin II are multi-subunit complexes that are known for their individual roles in genome organization and preventing genomic instability. However, interactions between condensin I and condensin II subunits and cooperative roles for condensin I and condensin II, outside of their genome organizing functions, have not been reported. We previously discovered that condensin II cooperates with Gamma Interferon Activated Inhibitor of Translation (GAIT) proteins to associate with Long INterspersed Element-1 (LINE-1 or L1) RNA and repress L1 protein expression and the retrotransposition of engineered L1 retrotransposition in cultured human cells. Here, we report that the L1 3′UTR is required for condensin II and GAIT association with L1 RNA, and deletion of the L1 RNA 3′UTR results in increased L1 protein expression and retrotransposition. Interestingly, like condensin II, we report that condensin I also binds GAIT proteins, associates with the L1 RNA 3′UTR, and represses...
Active retrotransposons in the human genome (L1,Aluand SVA elements) can create genomic mobile el... more Active retrotransposons in the human genome (L1,Aluand SVA elements) can create genomic mobile element insertions (MEIs) in both germline and somatic tissue1. Specific somatic MEIs have been detected at high levels in human cancers2, and at lower to medium levels in human brains3. Dysregulation of somatic retrotransposition in the human brain has been hypothesized to contribute to neuropsychiatric diseases4, 5. However, individual somatic MEIs are present in small proportions of cells at a given anatomical location, and thus standard whole-genome sequencing (WGS) presents a difficult signal-to-noise problem, while single-cell approaches suffer from limited scalability and experimental artifacts introduced by enzymatic whole-genome amplification6. Previous studies produced widely differing estimates for the somatic retrotransposition rates in human brain3, 6–8. Here, we present a highly precise machine learning method (RetroSom) to directly identify somatic L1 andAluinsertions in <...
Genome Sequencing Centres (Listed in order of total genomic sequence contributed, with a partial ... more Genome Sequencing Centres (Listed in order of total genomic sequence contributed, with a partial list of personnel. A full list of contributors at each centre is available as Supplementary Information.
Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; howeve... more Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the ∼400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.
Communicated by Haig H. Kazazian B1 elements are an abundant class of short interspersed elements... more Communicated by Haig H. Kazazian B1 elements are an abundant class of short interspersed elements (SINEs) in the mouse genome and mobilize by a process known as retrotransposition. Here, we report the characterization of a mutagenic B1 insertion into exon 4 of the Atcay gene, which was previously shown to be responsible for the jittery mouse. Mutations in the human ortholog of this gene, ATCAY, are responsible for Cayman ataxia. The B1 insertion is B150-bp long, ends in a 45-50-bp polyadenylic acid (poly A) tail, is flanked by a perfect 13-bp target-site duplication, and is inserted into a sequence that resembles a LINE-1 endonuclease consensus cleavage site. Computational analysis indicates that the mutagenic insertion is most closely related to elements of the B1-C subfamily, and we have identified two possible progenitor B1 sequences on mouse chromosome 19. Together, these data demonstrate that B1 retrotransposition is ongoing in the mouse genome and is consistent with the hypothesis that the reverse transcriptase and endonuclease encoded by LINE-1 elements mediate B1 mobility. Hum Mutat 24:9-13, 2004.
Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genom... more Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ∼10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs.
The human Long Interspersed Element-1 (LINE-1) and the Short Interspersed Element (SINE) Alu comp... more 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 show a different set of biases than those 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.
LINE-1 elements comprise approximately 17% of human DNA and their mobility continues to impact ge... more LINE-1 elements comprise approximately 17% of human DNA and their mobility continues to impact genome evolution. However, little is known about the types of non-transformed cells that can support LINE-1 retrotransposition. Here, we show that human embryonic stem cells express endogenous LINE-1 elements and can accommodate LINE-1 retrotransposition in vitro. The resultant retrotransposition events can occur into genes and can result in the concomitant deletion of genomic DNA at the target site. Thus, these data suggest that LINE-1 retrotransposition events may occur during early stages of human development.
We have found that intron 5a of the COXI gene (al5ao) of yeast mtDNA is a mobile group I intron i... more We have found that intron 5a of the COXI gene (al5ao) of yeast mtDNA is a mobile group I intron in crosses between strains having or lacking the intron. We have demonstrated the following hallmarks of that process: 1) co-conversion of flanking optional intron markers; 2) mutations that truncate the intron open reading frame block intron mobility; and 3) the intron open reading frame encodes an endonuclease activity that is required for intron movement. The endonuclease activity, termed l-Sce IV, cleaves the COXI allele lacking al5a near the site of intron insertion, making a four-base staggered cut with 3' OH overhangs. Three cloned DNAs derived from different forms of the COXI gene, which differ in primary sequence at up to seven nucleotides around the cleavage site, are all good substrates for in vitro l-Sce IV cleavage activity. Two of the strains from which these substrates were derived were tested in crosses and are comparably efficient as al5a recipients. When compared with mobility occurring simultaneously in one cross, al5a is less efficient as a mobile element.
LINE-1 (L1) retrotransposons comprise ∼17% of human DNA, yet little is known about L1 integration... more 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 a...
Proceedings of the National Academy of Sciences, 2019
Long interspersed element-1 (LINE-1 or L1) amplifies via retrotransposition. Active L1s encode 2 ... more Long interspersed element-1 (LINE-1 or L1) amplifies via retrotransposition. Active L1s encode 2 proteins (ORF1p and ORF2p) that bind their encoding transcript to promote retrotransposition in cis. The L1-encoded proteins also promote the retrotransposition of small-interspersed element RNAs, noncoding RNAs, and messenger RNAs in trans. Some L1-mediated retrotransposition events consist of a copy of U6 RNA conjoined to a variably 5′-truncated L1, but how U6/L1 chimeras are formed requires elucidation. Here, we report the following: The RNA ligase RtcB can join U6 RNAs ending in a 2′,3′-cyclic phosphate to L1 RNAs containing a 5′-OH in vitro; depletion of endogenous RtcB in HeLa cell extracts reduces U6/L1 RNA ligation efficiency; retrotransposition of U6/L1 RNAs leads to U6/L1 pseudogene formation; and a unique cohort of U6/L1 chimeric RNAs are present in multiple human cell lines. Thus, these data suggest that U6 small nuclear RNA (snRNA) and RtcB participate in the formation of ch...
Thank you, Haig, for the poignant introduction and kindwords.IwouldalsoliketothankJeffMurray,TheA... more Thank you, Haig, for the poignant introduction and kindwords.IwouldalsoliketothankJeffMurray,TheAmericanSociety of Human Genetics (ASHG), the members of theAwards Committee, and especially Christopher Pearson.IfirstlearnedthatIwastherecipientofthe2013CurtSternAward from Christopher when I was on vacation with myfamily in San Francisco. I was pleasantly surprised and,frankly, was rendered speechless. For those of you whoknow me, you probably know that it takes a lot to renderme speechless. I am truly honored and humbled to bethe recipient of this prestigious honor.As Haig so elegantly outlined, Curt Stern was anoutstanding scientist who conducted genetic studies inmodel organisms to gain fundamental insights aboutbasic cellular processes. Namely, he used the fruit fly,Drosophila melanogaster, to demonstrate crossing-overbetween homologous chromosomes
The third international conference on the genomic impact of eukaryotic transposable elements (TEs... more The third international conference on the genomic impact of eukaryotic transposable elements (TEs) was held 24 to 28 February 2012 at the Asilomar Conference Center, Pacific Grove, CA, USA. Sponsored in part by the National Institutes of Health grant 5 P41 LM006252, the goal of the conference was to bring together researchers from around the world who study the impact and mechanisms of TEs using multiple computational and experimental approaches. The meeting drew close to 170 attendees and included invited floor presentations on the biology of TEs and their genomic impact, as well as numerous talks contributed by young scientists. The workshop talks were devoted to computational analysis of TEs with additional time for discussion of unresolved issues. Also, there was ample opportunity for poster presentations and informal evening discussions. The success of the meeting reflects the important role of Repbase in comparative genomic studies, and emphasizes the need for close interactions between experimental and computational biologists in the years to come.
Malformations of cortical development (MCD) are neurological conditions displaying focal disrupti... more Malformations of cortical development (MCD) are neurological conditions displaying focal disruption of cortical architecture and cellular organization arising during embryogenesis, largely from somatic mosaic mutations. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat epilepsy. Here, we report a genetic atlas from 317 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation and single-cell sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associating distinct pathophysiological and clinical phenotypes. The unique spatiotemporal expression patterns identified by comparing single-nucleus transcriptional sequences of mutated genes in control and patient brains implicate critical roles in excitatory neurogenic pools during brain development, a...
In this article the affiliation 'Laboratory for Retrotransposon Dynamics, RIKEN Center for Integr... more In this article the affiliation 'Laboratory for Retrotransposon Dynamics, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan' for Tomoichiro Miyoshi was missing. The original article has been corrected.
Enabling the genomic revolution in Africa: H3Africa is developing capacity for health-related gen... more Enabling the genomic revolution in Africa: H3Africa is developing capacity for health-related genomics research in Africa The H3Africa Consortium *, † * H3Africa Consortium research investigators, funding agencies, and institutions appear in the supplementary materials Our understanding of genome biology, genomics, and disease, and even human history, has advanced tremendously with the completion of the Human Genome Project. Technological advances coupled with significant cost reductions in genomic research have yielded novel insights into disease etiology, diagnosis, and therapy for some of the world's most intractable and devastating diseases-including malaria, HIV/AIDS, tuberculosis, cancer, and diabetes. Yet, despite the burden of infectious diseases and, more recently, noncommunicable diseases (NCDs) in Africa, Africans have only participated minimally in genomics research. Of the thousands of genome-wide association studies (GWASs) that have been conducted globally, only seven (for HIV susceptibility, malaria, tuberculosis, and podoconiosis) have been conducted exclusively on African participants; four others (for prostate cancer, obsessive compulsive disorder, and anthropometry) included some African participants (www.genome.gov/gwastudies/). As discussed in 2011 (www.h3africa.org), if the dearth of genomics research involving Africans persists, the potential health and economic benefits emanating from genomic science may elude an entire continent.
Retrotransposition of LINEs and other retroelements increases repetition in mammalian genomes and... more Retrotransposition of LINEs and other retroelements increases repetition in mammalian genomes and can cause deleterious mutations. Recent insertions of two full-length L1s, L1 spa and L1 Orl , caused the disease phenotypes of the spastic and Orleans reeler mice respectively. Here we show that these two recently retrotransposed L1s are nearly identical in sequence, have two open reading frames and belong to a novel subfamily related to the ancient F subfamily. We have named this new subfamily T F (for transposable) and show that many full-length members of this family are present in the mouse genome. The T F 5Ј untranslated region has promoter activity, and T F-type RNA is abundant in cytoplasmic ribonucleoprotein particles, which are likely intermediates in retrotransposition. Both L1 spa and L1 Orl have reverse transcriptase activity in a yeast-based assay and retrotranspose at high frequency in cultured cells. Together, our data indicate that the T F subfamily of L1s contains a major class of mobile elements that is expanding in the mouse genome.
Some interferon stimulated genes (ISGs) encode proteins that inhibit LINE-1 (L1) retrotranspositi... more Some interferon stimulated genes (ISGs) encode proteins that inhibit LINE-1 (L1) retrotransposition. Here, we used immunoprecipitation followed by liquid chromatography-tandem mass spectrometry to identify proteins that associate with the L1 ORF1-encoded protein (ORF1p) in ribonucleoprotein particles. Three ISG proteins that interact with ORF1p inhibit retrotransposition: HECT and RLD domain containing E3 ubiquitin-protein ligase 5 (HERC5); 2’-5’-oligoadenylate synthetase-like (OASL); and helicase with zinc finger 2 (HELZ2). HERC5 destabilizes ORF1p, but does not affect its cellular localization. OASL impairs ORF1p cytoplasmic foci formation. HELZ2 recognizes sequences and/or structures within the L1 5’UTR to reduce L1 RNA, ORF1p, and ORF1p cytoplasmic foci levels. Overexpression of WT or reverse transcriptase-deficient L1s led to a modest induction of IFN-α expression, which was abrogated upon HELZ2 overexpression. Notably, IFN-α expression was enhanced upon overexpression of an OR...
transfection, cells were collected and processed for CAT or luciferase activity using standard te... more transfection, cells were collected and processed for CAT or luciferase activity using standard techniques 14. GST pull downs and immunoprecipitations GST±Rb (wild type and mutants 15) and other GST fusion proteins were expressed and puri®ed from Escherichia coli XA90 (ref. 16). GST fusion proteins that were immobilized on glutathione-sepharose (Pharmacia), or H3-derived peptides 3 bound to Sulfolink beads (Pierce), were incubated with extract in IPH buffer 16. Complexes were washed four times in IPH buffer before processing for methylase assays or western blotting. Antibodies against HA (12CA5, Roche), Gal4±DBD (DNA-binding domain; sc-510, Santa Cruz), SUV39H1 (M. Cleary), Rb (G3-245; XZ55, PharMingen) or HP1 (ref. 3) were used. For immunoprecipitations antibodies were incubated with HeLa nuclear extract (Cell Culture Center) or U2OS nuclear extract in IPH buffer at 4 8C (ref. 17). After 2 h a 50:50 mixture of protein A/G-sepharose beads (Pharmacia) was added. To avoid the possibility that DNA mediates the interaction between SUV39H1 and Rb, the immunoprecipitations were probed for the presence of histones with negative results. Histone methylase assays and protein sequencing Precipitations from pull downs or immunoprecipitations were incubated with 20 mg histones (Sigma) and 1 ml [3H-Me]-S-adenosyl methionine (NEN, 80 Ci mmol-1) in PBS at 30 8C for 1 h. Assays were analysed by SDS±PAGE followed by western blotting and autoradiography or were spotted onto P-81 cationic exchange paper (Whatman), washed in carbonate buffer and quanti®ed by scintillation counting 3. For amino-terminal sequencing, radiolabelled H3 was blotted to polyvinylidene¯uoride and sequenced by Edman degradation (Protein Sequencing Facility, University of Cambridge). We counted fractions for the presence of tritium. Supplementary information (with changes to the original Supplementary Information) is available on Nature's WorldWide Web site (http://www.nature.com).
Condensin I and condensin II are multi-subunit complexes that are known for their individual role... more Condensin I and condensin II are multi-subunit complexes that are known for their individual roles in genome organization and preventing genomic instability. However, interactions between condensin I and condensin II subunits and cooperative roles for condensin I and condensin II, outside of their genome organizing functions, have not been reported. We previously discovered that condensin II cooperates with Gamma Interferon Activated Inhibitor of Translation (GAIT) proteins to associate with Long INterspersed Element-1 (LINE-1 or L1) RNA and repress L1 protein expression and the retrotransposition of engineered L1 retrotransposition in cultured human cells. Here, we report that the L1 3′UTR is required for condensin II and GAIT association with L1 RNA, and deletion of the L1 RNA 3′UTR results in increased L1 protein expression and retrotransposition. Interestingly, like condensin II, we report that condensin I also binds GAIT proteins, associates with the L1 RNA 3′UTR, and represses...
Active retrotransposons in the human genome (L1,Aluand SVA elements) can create genomic mobile el... more Active retrotransposons in the human genome (L1,Aluand SVA elements) can create genomic mobile element insertions (MEIs) in both germline and somatic tissue1. Specific somatic MEIs have been detected at high levels in human cancers2, and at lower to medium levels in human brains3. Dysregulation of somatic retrotransposition in the human brain has been hypothesized to contribute to neuropsychiatric diseases4, 5. However, individual somatic MEIs are present in small proportions of cells at a given anatomical location, and thus standard whole-genome sequencing (WGS) presents a difficult signal-to-noise problem, while single-cell approaches suffer from limited scalability and experimental artifacts introduced by enzymatic whole-genome amplification6. Previous studies produced widely differing estimates for the somatic retrotransposition rates in human brain3, 6–8. Here, we present a highly precise machine learning method (RetroSom) to directly identify somatic L1 andAluinsertions in <...
Genome Sequencing Centres (Listed in order of total genomic sequence contributed, with a partial ... more Genome Sequencing Centres (Listed in order of total genomic sequence contributed, with a partial list of personnel. A full list of contributors at each centre is available as Supplementary Information.
Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; howeve... more Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the ∼400,000 L1s are mobile. Using a retrotransposition assay in cultured human cells, we demonstrate that L1-encoded proteins predominantly mobilize the RNA that encodes them. At much lower levels, L1-encoded proteins can act in trans to promote retrotransposition of mutant L1s and other cellular mRNAs, creating processed pseudogenes. Mutant L1 RNAs are mobilized at 0.2 to 0.9% of the retrotransposition frequency of wild-type L1s, whereas cellular RNAs are mobilized at much lower frequencies (ca. 0.01 to 0.05% of wild-type levels). Thus, we conclude that L1-encoded proteins demonstrate a profound cis preference for their encoding RNA. This mechanism could enable L1 to remain retrotransposition competent in the presence of the overwhelming number of nonfunctional L1s present in human DNA.
Communicated by Haig H. Kazazian B1 elements are an abundant class of short interspersed elements... more Communicated by Haig H. Kazazian B1 elements are an abundant class of short interspersed elements (SINEs) in the mouse genome and mobilize by a process known as retrotransposition. Here, we report the characterization of a mutagenic B1 insertion into exon 4 of the Atcay gene, which was previously shown to be responsible for the jittery mouse. Mutations in the human ortholog of this gene, ATCAY, are responsible for Cayman ataxia. The B1 insertion is B150-bp long, ends in a 45-50-bp polyadenylic acid (poly A) tail, is flanked by a perfect 13-bp target-site duplication, and is inserted into a sequence that resembles a LINE-1 endonuclease consensus cleavage site. Computational analysis indicates that the mutagenic insertion is most closely related to elements of the B1-C subfamily, and we have identified two possible progenitor B1 sequences on mouse chromosome 19. Together, these data demonstrate that B1 retrotransposition is ongoing in the mouse genome and is consistent with the hypothesis that the reverse transcriptase and endonuclease encoded by LINE-1 elements mediate B1 mobility. Hum Mutat 24:9-13, 2004.
Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genom... more Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ∼10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs.
The human Long Interspersed Element-1 (LINE-1) and the Short Interspersed Element (SINE) Alu comp... more 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 show a different set of biases than those 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.
LINE-1 elements comprise approximately 17% of human DNA and their mobility continues to impact ge... more LINE-1 elements comprise approximately 17% of human DNA and their mobility continues to impact genome evolution. However, little is known about the types of non-transformed cells that can support LINE-1 retrotransposition. Here, we show that human embryonic stem cells express endogenous LINE-1 elements and can accommodate LINE-1 retrotransposition in vitro. The resultant retrotransposition events can occur into genes and can result in the concomitant deletion of genomic DNA at the target site. Thus, these data suggest that LINE-1 retrotransposition events may occur during early stages of human development.
We have found that intron 5a of the COXI gene (al5ao) of yeast mtDNA is a mobile group I intron i... more We have found that intron 5a of the COXI gene (al5ao) of yeast mtDNA is a mobile group I intron in crosses between strains having or lacking the intron. We have demonstrated the following hallmarks of that process: 1) co-conversion of flanking optional intron markers; 2) mutations that truncate the intron open reading frame block intron mobility; and 3) the intron open reading frame encodes an endonuclease activity that is required for intron movement. The endonuclease activity, termed l-Sce IV, cleaves the COXI allele lacking al5a near the site of intron insertion, making a four-base staggered cut with 3' OH overhangs. Three cloned DNAs derived from different forms of the COXI gene, which differ in primary sequence at up to seven nucleotides around the cleavage site, are all good substrates for in vitro l-Sce IV cleavage activity. Two of the strains from which these substrates were derived were tested in crosses and are comparably efficient as al5a recipients. When compared with mobility occurring simultaneously in one cross, al5a is less efficient as a mobile element.
LINE-1 (L1) retrotransposons comprise ∼17% of human DNA, yet little is known about L1 integration... more 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 a...
Proceedings of the National Academy of Sciences, 2019
Long interspersed element-1 (LINE-1 or L1) amplifies via retrotransposition. Active L1s encode 2 ... more Long interspersed element-1 (LINE-1 or L1) amplifies via retrotransposition. Active L1s encode 2 proteins (ORF1p and ORF2p) that bind their encoding transcript to promote retrotransposition in cis. The L1-encoded proteins also promote the retrotransposition of small-interspersed element RNAs, noncoding RNAs, and messenger RNAs in trans. Some L1-mediated retrotransposition events consist of a copy of U6 RNA conjoined to a variably 5′-truncated L1, but how U6/L1 chimeras are formed requires elucidation. Here, we report the following: The RNA ligase RtcB can join U6 RNAs ending in a 2′,3′-cyclic phosphate to L1 RNAs containing a 5′-OH in vitro; depletion of endogenous RtcB in HeLa cell extracts reduces U6/L1 RNA ligation efficiency; retrotransposition of U6/L1 RNAs leads to U6/L1 pseudogene formation; and a unique cohort of U6/L1 chimeric RNAs are present in multiple human cell lines. Thus, these data suggest that U6 small nuclear RNA (snRNA) and RtcB participate in the formation of ch...
Thank you, Haig, for the poignant introduction and kindwords.IwouldalsoliketothankJeffMurray,TheA... more Thank you, Haig, for the poignant introduction and kindwords.IwouldalsoliketothankJeffMurray,TheAmericanSociety of Human Genetics (ASHG), the members of theAwards Committee, and especially Christopher Pearson.IfirstlearnedthatIwastherecipientofthe2013CurtSternAward from Christopher when I was on vacation with myfamily in San Francisco. I was pleasantly surprised and,frankly, was rendered speechless. For those of you whoknow me, you probably know that it takes a lot to renderme speechless. I am truly honored and humbled to bethe recipient of this prestigious honor.As Haig so elegantly outlined, Curt Stern was anoutstanding scientist who conducted genetic studies inmodel organisms to gain fundamental insights aboutbasic cellular processes. Namely, he used the fruit fly,Drosophila melanogaster, to demonstrate crossing-overbetween homologous chromosomes
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