Sequencing,identification and mapping of primed L1 elements (SIMPLE) reveals significant variation in full length L1 elements between individuals

Background

There are over a half a million copies of L1 retroelements in the human genome which are responsible for as much as 0.5% of new human genetic diseases. Most new L1 inserts arise from young source elements that are polymorphic in the human genome. Highly active polymorphic “hot” L1 source elements have been shown to be capable of extremely high levels of mobilization and result in numerous instances of disease. Additionally, hot polymorphic L1s have been described to be highly active within numerous cancer genomes. These hot L1s result in mutagenesis by insertion of new L1 copies elsewhere in the genome, but also have been shown to generate additional full length L1 insertions which are also hot and able to further retrotranspose. Through this mechanism, hot L1s may amplify within a tumor and result in a continued cycle of mutagenesis.

Results and conclusions

We have developed a method to detect full-length, polymorphic L1 elements using a targeted next generation sequencing approach, Sequencing Identification and Mapping of Primed L1 Elements (SIMPLE). SIMPLE has 94% sensitivity and detects nearly all full-length L1 elements in a genome. SIMPLE will allow researchers to identify hot mutagenic full-length L1s as potential drivers of genome instability. Using SIMPLE we find that the typical individual has approximately 100 non-reference, polymorphic L1 elements in their genome. These elements are at relatively low population frequencies relative to previously identified polymorphic L1 elements and demonstrate the tremendous diversity in potentially active L1 elements in the human population.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1374-y) contains supplementary material, which is available to authorized users.     

L1 (LINE-1) retrotransposable elements provide a "fossil" record of the phylogenetic history of murid rodents

The single most difficult problem in phylogenetic analysis is deciding whether a shared taxonomic character is due to common ancestry or one that appeared independently due to convergence, parallelism, or reversion to an ancestral state. Mammalian L1 retrotransposons undergo periodic amplifications in which multiple copies of the elements are interspersed in the genome. Because these elements apparently are transmitted only by inheritance and are retained in the genome, a shared L1 amplification event can only be an inherited ancestral character. We propose that L1 amplification events can be an excellent tool for analyzing mammalian evolution and demonstrate here how we addressed several refractory problems in rodent systematics using L1 DNA as a taxonomic character.      

Reconstruction of full-length LINE-1 progenitors from ancestral genomes

Sequences derived from the Long INterspersed Element-1 (L1) family of retrotransposons occupy at least 17% of the human genome, with 67 distinct subfamilies representing successive waves of expansion and extinction in mammalian lineages. L1s contribute extensively to gene regulation, but their molecular history is difficult to trace, because most are present only as truncated and highly mutated fossils. Consequently, L1 entries in current databases of repeat sequences are composed mainly of short diagnostic subsequences, rather than full functional progenitor sequences for each subfamily. Here, we have coupled 2 levels of sequence reconstruction (at the level of whole genomes and L1 subfamilies) to reconstruct progenitor sequences for all human L1 subfamilies that are more functionally and phylogenetically plausible than existing models. Most of the reconstructed sequences are at or near the canonical length of L1s and encode uninterrupted ORFs with expected protein domains. We also show that the presence or absence of binding sites for KRAB-C2H2 Zinc Finger Proteins, even in ancient-reconstructed progenitor L1s, mirrors binding observed in human ChIP-exo experiments, thus extending the arms race and domestication model. RepeatMasker searches of the modern human genome suggest that the new models may be able to assign subfamily resolution identities to previously ambiguous L1 instances. The reconstructed L1 sequences will be useful for genome annotation and functional study of both L1 evolution and L1 contributions to host regulatory networks.     

Recently integrated human Alu repeats: finding needles in the haystack

Alu elements undergo amplification through retroposition and integration into new locations throughout primate genomes. Over 500,000 Alu elements reside in the human genome, making the identification of newly inserted Alu repeats the genomic equivalent of finding needles in the haystack. Here, we present two complementary methods for rapid detection of newly integrated Alu elements. In the first approach we employ computational biology to mine the human genomic DNA sequence databases in order to identify recently integrated Alu elements. The second method is based on an anchor-PCR technique which we term Allele-Specific Alu PCR (ASAP). In this approach, Alu elements are selectively amplified from anchored DNA generating a display or 'fingerprint' of recently integrated Alu elements. Alu insertion polymorphisms are then detected by comparison of the DNA fingerprints generated from different samples. Here, we explore the utility of these methods by applying them to the identification of members of the smallest previously identified subfamily of Alu repeats in the human genome termed Ya8. This subfamily of Alu repeats is composed of about 50 elements within the human genome. Approximately 50% of the Ya8 Alu family members have inserted in the human genome so recently that they are polymorphic, making them useful markers for the study of human evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Allelic heterogeneity in LINE-1 retrotransposition activity

De novo LINE-1 (long interspersed element-1, or L1) retrotransposition events are responsible for approximately 1/1,000 disease-causing mutations in humans. Previously, L1.2 was identified as the likely progenitor of a mutagenic insertion in the factor VIII gene in a patient with hemophilia A. It subsequently was shown to be one of a small number of active L1s in the human genome. Here, we demonstrate that L1.2 is present at an intermediate insertion allele frequency in worldwide human populations and that common alleles (L1.2A and L1.2B) exhibit an approximately 16-fold difference in their ability to retrotranspose in cultured human HeLa cells. Chimera analysis revealed that two amino acid substitutions (S1259L and I1220M) downstream of the conserved cysteine-rich motif in L1 open reading frame 2 are largely responsible for the observed reduction in L1.2A retrotransposition efficiency. Thus, common L1 alleles can vary widely in their retrotransposition potential. We propose that such allelic heterogeneity can influence the potential L1 mutational load present in an individual genome.     

LINE-1 ORF1 protein enhances Alu SINE retrotransposition

Retroelements have contributed over one third of the human genome mass. The currently active LINE-1 (L1) codes for two proteins (ORF1p and ORF2p), both strictly required for retrotransposition. In contrast, the non-coding parasitic SINE (Alu) only appears to need the L1 ORF2p for its own amplification. This requirement was previously determined using a tissue culture assay system in human cells (HeLa). Because HeLa are likely to express functional L1 proteins, it is possible that low levels of endogenous ORF1p are necessary for the observed tagged Alu mobilization. By individually expressing ORF1 and ORF2 proteins from both human (L1RP and LRE3) and rodent (L1A102 and L1spa) L1 sources, we demonstrate that increasing amounts of ORF1 expressing vector enhances tagged Alu mobilization in HeLa cells. In addition, using chicken fibroblast cells as an alternate cell culture source, we confirmed that ORF1p is not strictly required for Alu mobilization in our assay. Supporting our observations in HeLa cells, we find that tagged Alu retrotransposition is improved by supplementation of ORF1p in the cultured chicken cells. We postulate that L1 ORF1p plays either a direct or indirect role in enhancing the interaction between the Alu RNA and the required factors needed for its retrotransposition.     

Sequencing intractable DNA to close microbial genomes

Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.     

Replication of an Autonomous Human Parvovirus in Non-dividing Human Airway Epithelium Is Facilitated through the DNA Damage and Repair Pathways

Human bocavirus 1 (HBoV1) belongs to the genus Bocaparvovirus of the Parvoviridae family, and is an emerging human pathogenic respiratory virus. In vitro, HBoV1 infects well-differentiated/polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). Although it is well known that autonomous parvovirus replication depends on the S phase of the host cells, we demonstrate here that the HBoV1 genome amplifies efficiently in mitotically quiescent airway epithelial cells of HAE-ALI cultures. Analysis of HBoV1 DNA in infected HAE-ALI revealed that HBoV1 amplifies its ssDNA genome following a typical parvovirus rolling-hairpin DNA replication mechanism. Notably, HBoV1 infection of HAE-ALI initiates a DNA damage response (DDR) with activation of all three phosphatidylinositol 3-kinase–related kinases (PI3KKs). We found that the activation of the three PI3KKs is required for HBoV1 genome amplification; and, more importantly, we identified that two Y-family DNA polymerases, Pol η and Pol κ, are involved in HBoV1 genome amplification. Overall, we have provided an example of de novo DNA synthesis (genome amplification) of an autonomous parvovirus in non-dividing cells, which is dependent on the cellular DNA damage and repair pathways.     

Identifying related L1 retrotransposons by analyzing 3' transduced sequences

Background  

A large fraction of the human genome is attributable to L1 retrotransposon sequences. Not only do L1s themselves make up a significant portion of the genome, but L1-encoded proteins are thought to be responsible for the transposition of other repetitive elements and processed pseudogenes. In addition, L1s can mobilize non-L1, 3'-flanking DNA in a process called 3' transduction. Using computational methods, we collected DNA sequences from the human genome for which we have high confidence of their mobilization through L1-mediated 3' transduction.     

Genomic deletions created upon LINE-1 retrotransposition

LINE-1 (L1) retrotransposition continues to impact the human genome, yet little is known about how L1 integrates into DNA. Here, we developed a plasmid-based rescue system and have used it to recover 37 new L1 retrotransposition events from cultured human cells. Sequencing of the insertions revealed the usual L1 structural hallmarks; however, in four instances, retrotransposition generated large target site deletions. Remarkably, three of those resulted in the formation of chimeric L1s, containing the 5' end of an endogenous L1 fused precisely to our engineered L1. Thus, our data demonstrate multiple pathways for L1 integration in cultured cells, and show that L1 is not simply an insertional mutagen, but that its retrotransposition can result in significant deletions of genomic sequence.     

Human L1 retrotransposition: cis preference versus trans complementation

Long interspersed nuclear elements (LINEs or L1s) comprise approximately 17% of human DNA; however, only about 60 of the approximately 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.     

Identification of a novel Bardet-Biedl syndrome protein,BBS7, that shares structural features with BBS1 and BBS2

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder, the primary features of which include obesity, retinal dystrophy, polydactyly, hypogenitalism, learning difficulties, and renal malformations. Conventional linkage and positional cloning have led to the mapping of six BBS loci in the human genome, four of which (BBS1, BBS2, BBS4, and BBS6) have been cloned. Despite these advances, the protein sequences of the known BBS genes have provided little or no insight into their function. To delineate functionally important regions in BBS2, we performed phylogenetic and genomic studies in which we used the human and zebrafish BBS2 peptide sequences to search dbEST and the translation of the draft human genome. We identified two novel genes that we initially named "BBS2L1" and "BBS2L2" and that exhibit modest similarity with two discrete, overlapping regions of BBS2. In the present study, we demonstrate that BBS2L1 mutations cause BBS, thereby defining a novel locus for this syndrome, BBS7, whereas BBS2L2 has been shown independently to be BBS1. The motif-based identification of a novel BBS locus has enabled us to define a potential functional domain that is present in three of the five known BBS proteins and, therefore, is likely to be important in the pathogenesis of this complex syndrome.