Endogenous Long Terminal Repeat Research Articles

The LORE1 insertion mutant resource.

Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity canbe used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research.

Endogenous retroviral long terminal repeats as host gene promoters in normal and cancer cells

The human genome contains nearly 400,000 sequences related to retroviruses that have accumulated due to ancient infections of the germ line and subsequent fixation during evolution. Most of these endogenous retroviral sequences (ERVs) currently exist as solitary long terminal repeats (LTRs), the product of recombination between LTRs of the integrated proviral form. Since retroviral LTRs naturally contain transcriptional promoters and enhancers, these sequences have great potential to impact regulation of individual genes and gene regulatory networks. Numerous cases of LTRs serving as promoters for human genes have been described by our group and others, and some examples will be presented. While such co-option of LTRs as regulatory gene modules indeed occurs in normal cells, particularly in placenta or in early development, transcriptional activity of most endogenous LTRs is epigenetically suppressed in somatic tissues, likely as a host defense against unregulated transcription. Cancer cells represent an abnormal epigenetic environment where LTRs and other classes of transposable elements (TEs) are often hypomethylated, leading to their transcriptional activation. This activation could result in abnormal, cancer-specific expression of nearby genes. To study this phenomenon, we are analyzing whole transcriptome data of cancers specifically to identify gene deregulation due to transcriptional activity of LTRs or other TEs. Our results suggest that the regulatory potential of these sequences is often used by cancer cells, providing one avenue to up-regulate genes. Thus, while some LTRs/TEs have been co-opted to serve in normal gene expression, the same regulatory qualities can be exploited in carcinogenesis.

Drosophila errantiviruses

Retroelements with long-terminal repeats (LTRs) inhabit nearly all eukaryotic genomes. During the time of their rich evolutionary history they have developed highly diverse forms, ranging from ordinary retrotransposons to complex pathogenic retroviruses such as HIV-I. Errantiviruses are a group of insect endogenous LTR elements that share structural and functional features with vertebrate endogenous retroviruses. The errantiviruses illustrate one of the evolutionary strategies of retrotransposons to become infective, which together with their similarities to vertebrate retroviruses make them an attractive object of research promising to shed more light on the evolution of retroviruses.

Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma

Mammalian genomes contain many repetitive elements, including long terminal repeats (LTRs), which have long been suspected to have a role in tumorigenesis. Here we present evidence that aberrant LTR activation contributes to lineage-inappropriate gene expression in transformed human cells and that such gene expression is central for tumor cell survival. We show that B cell-derived Hodgkin's lymphoma cells depend on the activity of the non-B, myeloid-specific proto-oncogene colony-stimulating factor 1 receptor (CSF1R). In these cells, CSF1R transcription initiates at an aberrantly activated endogenous LTR of the MaLR family (THE1B). Derepression of the THE1 subfamily of MaLR LTRs is widespread in the genome of Hodgkin's lymphoma cells and is associated with impaired epigenetic control due to loss of expression of the corepressor CBFA2T3. Furthermore, we detect LTR-driven CSF1R transcripts in anaplastic large cell lymphoma, in which CSF1R is known to be expressed aberrantly. We conclude that LTR derepression is involved in the pathogenesis of human lymphomas, a finding that might have diagnostic, prognostic and therapeutic implications.

Differential gene expression in a murine model of cancer cachexia.

Murine adenocarcinoma 16 (MAC16) tumors and cell lines induce cachexia in NMRI nude mice, whereas histologically similar MAC13 tumors do not. After confirming these findings in BALB/c nude mice, we demonstrated that this tissue wasting was not related to decreased food intake or increased total body oxidative metabolism. Previous studies have suggested that MAC16's cachexigenic properties may involve the production of tumor-specific factors. We therefore screened for genes having increased expression in the MAC16 compared with the MAC13 cell line by performing hybridization to a murine cDNA expression array, by generation and comparison of cDNA libraries from each cell line, and by PCR-based subtractive hybridization. Northern blot hybridization was performed to confirm differences in transcript expression. Transcripts encoding insulin-like growth factor binding protein-4, cathepsin B, ferritin light and heavy chain, endogenous long-terminal repeat sequences, and a viral envelope glycoprotein demonstrated increased expression in the MAC16 cell line. The roles of a number of these genes in known metabolic pathways identify them as potential participants in the induction of cachexia.

Splicing of a human endogenous retrovirus to a novel phospholipase A2 related gene.

As part of an investigation into the effects of endogenous retroviruses on adjacent genes, we have isolated a cDNA clone derived from the human teratocarcinoma cell line NTera2D1 representing a chimeric transcript in which an endogenous retrovirus-like element, RTVL-H, has been spliced to downstream cellular sequences. The 5' terminus of this clone, termed AF-5, occurs one bp downstream of the predicted transcriptional start site in the RTVL-H long terminal repeat (LTR). AF-5 contains an open reading frame of 689 amino acids beginning within RTVL-H sequences that has two domains of homology with phospholipase A2 (PLA2). These domains, of approximately 120 amino acids each, are 30-38% identical to secreted PLA2s and contain sequence features of both group I and II enzymes. The corresponding AF-5 transcript is 2.5 kb and is derived from a single copy novel gene termed PLA2L. Southern analysis indicates that the RTVL-H element is normally present in human DNA upstream of the PLA2L gene. RTVL-H/PLA2L chimeric transcripts were detected in two independent teratocarcinoma cell lines but not in several other cell lines or primary human tissues. Characterization of additional cDNA clones and PCR analysis indicates that multiple RTVL-H/PLA2L alternatively spliced transcripts are expressed. No evidence has been found for transcription from a non-LTR promoter. These findings strongly suggest that the endogenous LTR promotes expression of the human PLA2L gene in teratocarcinoma cells.

The long terminal repeat of feline endogenous RD-114 retroviral DNAs: analysis of transcription regulatory activity and nucleotide sequence

Six cloned 5' long terminal repeat (LTR) and adjoining cellular DNA regions of partially deleted feline endogenous RD-114 proviral loci were linked to the chloramphenicol acetyltransferase (CAT) gene and assayed for their ability to promote transient CAT expression. One endogenous LTR (clone CRL-3) and the LTR from an infectious RD-114 provirus, EX-LTR, were capable of actively expressing the CAT gene. DNA sequence comparison of these LTRs with an inactive endogenous LTR (CR-1) revealed extensive homology in all regions except in the 5' half of U3. The homologous portion contained transcriptional regulatory sequences including CAT, TATA, polyadenylation signal boxes and an octamer enhancer, which is rarely seen in retroviruses. Variations in the 5' half of U3 were primarily due to insertions and deletions. A major difference was in number of copies and integrity of tandemly repeated sequences. EX-LTR contained two pairs of tandem direct repeats, while the two endogenous LTRs contained different deletions of repeated sequences. DNA sequence data also revealed that the primer binding site for RD-114 loci was complementary to a glycine tRNA isotype, the use of which is distinct from any other known retrovirus. An analysis of the steady state RNA levels in T-lymphoid cell lines showed that at least three different incomplete proviral transcripts and their spliced products made up the majority of expressed RD-114 mRNA, and further demonstrated that partially deleted proviral loci have the potential to be transcriptionally vigorous in certain feline cell types.

Selective shedding and congenital transmission of endogenous avian leukosis viruses

Shedding and congenital transmission of endogenous avian leukosis viruses were studied in viremic White Leghorn hens exogenously infected with viruses with endogenous long terminal repeats (LTRs) and in four semicongenic lines of hens that naturally express infectious endogenous viruses (EVs). Relatively high titers of infectious virus EV7 (encoded at locus ev7), Rous-associated virus-0 (RAV-0), and recombinant 882/-16 RAV-0 were detected in blood cells and sera from exogenously infected hens, but marked differences were noted in the incidence of congenitally infected progeny. In enzyme immunoassays that detect viral group-specific antigen, little or no p27 was detected in albumens from dams infected with RAV-0. However, hatchmates infected with either EV7 or recombinant 882/-16 RAV-0, which was constructed with an RAV-0 LTR, shed high titers of p27. Similarly, semicongenic hens that expressed RAV-0 (EV2) (encoded at locus ev2) shed little or no p27 into albumens, but hens that harbored ev10, ev11, and ev12 shed high titers of p27. A slower electrophoretic mobility of p27, considered to be characteristic of EVs that are restricted in congenital transmission, was not associated with low levels of shedding or congenital transmission; p27 from other EVs and p27 from an avian leukosis virus field strain, all of which are shed at high levels, had mobilities identical to that of p27 from RAV-0. Although shedding and congenital transmission appear to be controlled by the viral genome, there was no correlation between low efficiency of shedding or congenital transmission and endogenous LTR or p27 sequences.

Endogenous murine leukemia proviral long terminal repeats contain a unique 190-base-pair insert.

We have determined the nucleotide sequence in the U3-R regions of the long terminal repeat (LTR) associated with NFS-Th-1 xenotropic murine leukemia virus (MuLV) DNA and the LTR components of five endogenous proviruses cloned from BALB/c mouse chromosomal DNA. The five endogenous MuLV LTRs contained the regulatory signals thought to be important in viral transcription, such as "TATAA" and CCAAT-like boxes. A unique feature in four of the endogenous LTRs was the presence of a highly conserved 190-base-pair (bp) insert bounded by 6-bp direct repeats located 48 bp upstream from the C-C-A-A-T sequence. This segment was absent from LTRs associated with ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. All five endogenous LTR segments also contained a 14-bp duplication of a sequence located near the 5' end of the first component of the long (greater than 72-bp) direct repeat of ecotropic and MCF MuLV LTRs. An evolutionary scheme relating LTRs associated with endogenous MuLV proviral DNAs to those found in ecotropic or xenotropic proviruses is presented. Nucleotide sequence analysis also suggested that the U3 region of the MCF247 MuLV LTR is derived from an NFS xenotropic related MuLV.

Cloning of endogenous murine leukemia virus-related sequences from chromosomal DNA of BALB/c and AKR/J mice: identification of an env progenitor of AKR-247 mink cell focus-forming proviral DNA.

Recombinant phages containing murine leukemia virus (MuLV)-reactive DNA sequences were isolated after screening of a BALB/c mouse embryo DNA library and from shotgun cloning of EcoRI-restricted AKR/J mouse liver DNA. Twelve different clones were isolated which contained incomplete MuLV proviral DNA sequences extending various distances from either the 5' or 3' long terminal repeat (LTR) into the viral genome. Restriction maps indicated that the endogenous MuLV DNAs were related to xenotropic MuLVs, but they shared several unique restriction sites among themselves which were not present in known MuLV proviral DNAs. Analyses of internal restriction fragments of the endogenous LTRs suggested the existence of at least two size classes, both of which were larger than the LTRs of known ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. Five of the six cloned endogenous MuLV proviral DNAs which contained envelope (env) DNA sequences annealed to a xenotropic MuLV env-specific DNA probe; in addition, four of these five also hybridized to an ecotropic MuLV-specific env DNA probe. Cloned MCF 247 proviral DNA also contained such dual-reactive env sequences. One of the dual-reactive cloned endogenous MuLV DNAs contained an env region that was indistinguishable by AluI and HpaII digestion from the analogous segment in MCF 247 proviral DNA and may therefore represent a progenitor for the env gene of this recombinant MuLV. In addition, the endogenous MuLV DNAs were highly related by AluI cleavage to the Moloney MuLV provirus in the gag and pol regions.