The Ty1 retrotransposon harbors a DNA region that performs dual functions as both a gene silencing and chromatin insulator.

In various eukaryotic kingdoms, long terminal repeat (LTR) retrotransposons repress transcription by infiltrating heterochromatin generated within their elements. In contrast, the budding yeast LTR retrotransposon Ty1 does not itself undergo transcriptional repression, although it is capable of repressing the transcription of the inserted genes within it. In this study, we identified a DNA region within Ty1 that exerts its silencing effect via sequence orientation. We identified a DNA region within the Ty1 group-specific antigen (GAG) gene that causes gene silencing, termed GAG silencing (GAGsi), in which the silent chromatin in the GAGsi region is created by euchromatin-specific histone modifications. A characteristic inverted repeat (IR) sequence is present at the 5' end of this region, forming a chromatin boundary between promoter-specific chromatin upstream of the IR sequence and silent chromatin downstream of the IR sequence. In addition, Esc2 and Rad57, which are involved in DNA repair, were required for GAGsi silencing. Finally, the chromatin boundary was required for the transcription of Ty1 itself. Thus, the GAGsi sequence contributes to the creation of a chromatin environment that promotes Ty1 transcription.

Comprehensive analysis of the Xya riparia genome uncovers the dominance of DNA transposons, LTR/Gypsy elements, and their evolutionary dynamics.

Transposable elements (TEs) are DNA sequences that can move or replicate within a genome, and their study has become increasingly important in understanding genome evolution and function. The Tridactylidae family, including Xya riparia (pygmy mole cricket), harbors a variety of transposable elements (TEs) that have been insufficiently investigated. Further research is required to fully understand their diversity and evolutionary characteristics. Hence, we conducted a comprehensive repeatome analysis of X. riparia species using the chromosome-level assembled genome. The study aimed to comprehensively analyze the abundance, distribution, and age of transposable elements (TEs) in the genome. The results indicated that the genome was 1.67 Gb, with 731.63 Mb of repetitive sequences, comprising 27% of Class II (443.25 Mb), 16% of Class I (268.45 Mb), and 1% of unknown TEs (19.92 Mb). The study found that DNA transposons dominate the genome, accounting for approximately 60% of the total repeat size, with retrotransposons and unknown elements accounting for 37% and 3% of the genome, respectively. The members of the Gypsy superfamily were the most abundant amongst retrotransposons, accounting for 63% of them. The transposable superfamilies (LTR/Gypsy, DNA/nMITE, DNA/hAT, and DNA/Helitron) collectively constituted almost 70% of the total repeat size of all six chromosomes. The study further unveiled a significant linear correlation (Pearson correlation: r = 0.99, p-value = 0.00003) between the size of the chromosomes and the repetitive sequences. The average age of DNA transposon and retrotransposon insertions ranges from 25 My (million years) to 5 My. The satellitome analysis discovered 13 satellite DNA families that comprise about 0.15% of the entire genome. In addition, the transcriptional analysis of TEs found that DNA transposons were more transcriptionally active than retrotransposons. Overall, the study suggests that the genome of X. riparia is complex, characterized by a substantial portion of repetitive elements. These findings not only enhance our understanding of TE evolution within the Tridactylidae family but also provide a foundation for future investigations into the genomic intricacies of related species.

Comprehensive Identification and Characterization of HML-9 Group in Chimpanzee Genome.

Endogenous retroviruses (ERVs) are related to long terminal repeat (LTR) retrotransposons, comprising gene sequences of exogenous retroviruses integrated into the host genome and inherited according to Mendelian law. They are considered to have contributed greatly to the evolution of host genome structure and function. We previously characterized HERV-K HML-9 in the human genome. However, the biological function of this type of element in the genome of the chimpanzee, which is the closest living relative of humans, largely remains elusive. Therefore, the current study aims to characterize HML-9 in the chimpanzee genome and to compare the results with those in the human genome. Firstly, we report the distribution and genetic structural characterization of the 26 proviral elements and 38 solo LTR elements of HML-9 in the chimpanzee genome. The results showed that the distribution of these elements displayed a non-random integration pattern, and only six elements maintained a relatively complete structure. Then, we analyze their phylogeny and reveal that the identified elements all cluster together with HML-9 references and with those identified in the human genome. The HML-9 integration time was estimated based on the 2-LTR approach, and the results showed that HML-9 elements were integrated into the chimpanzee genome between 14 and 36 million years ago and into the human genome between 18 and 49 mya. In addition, conserved motifs, cis-regulatory regions, and enriched PBS sequence features in the chimpanzee genome were predicted based on bioinformatics. The results show that pathways significantly enriched for ERV LTR-regulated genes found in the chimpanzee genome are closely associated with disease development, including neurological and neurodevelopmental psychiatric disorders. In summary, the identification, characterization, and genomics of HML-9 presented here not only contribute to our understanding of the role of ERVs in primate evolution but also to our understanding of their biofunctional significance.

The Families of Non-LTR Transposable Elements within Neritimorpha and Other Gastropoda.

Repeated sequences, especially transposable elements (TEs), are known to be abundant in some members of the important invertebrate class Gastropoda. TEs that do not have long terminal repeated sequences (non-LTR TEs) are frequently the most abundant type but have not been well characterised in any gastropod. Despite this, sequences in draft gastropod genomes are often described as non-LTR TEs, but without identification to family type. This study was conducted to characterise non-LTR TEs in neritimorph snails, using genomic skimming surveys of three species and the recently published draft genome of Theodoxus fluviatilis. Multiple families of non-LTR TEs from the I, Jockey, L1, R2 and RTE superfamilies were found, although there were notably few representatives of the first of these, which is nevertheless abundant in other Gastropoda. Phylogenetic analyses of amino acid sequences of the reverse transcriptase domain from the elements ORF2 regions found considerable interspersion of representatives of the four neritimorph taxa within non-LTR families and sub-families. In contrast, phylogenetic analyses of sequences from the elements' ORF1 region resolved the representatives from individual species as monophyletic. However, using either region, members of the two species of the Neritidae were closely related, suggesting their potential for investigation of phyletic evolution at the family level.

NF-κB1 deficiency promotes macrophage-derived adrenal tumors but decreases neurofibromas in HTLV-I LTR-Tax transgenic mice.

Human T-cell leukemia virus type I (HTLV-I) is an oncogenic virus whose infection can cause diverse diseases, most notably adult T-cell leukemia/lymphoma (ATL or ATLL), an aggressive and fatal malignancy of CD4 T cells. The oncogenic ability of HTLV-I is mostly attributed to the viral transcriptional transactivator Tax. Tax alone is sufficient to induce specific tumors in mice depending on the promotor used to drive Tax expression, thereby being used to understand HTLV-I tumorigenesis and model the tumor types developed in Tax transgenic mice. Tax exerts its oncogenic role predominantly by activating the cellular transcription factor NF-κB. Here, we report that genetic deletion of NF-κB1, the prototypic member of the NF-κB family, promotes adrenal medullary tumors but suppresses neurofibromas in mice with transgenic Tax driven by the HTLV-I Long Terminal Repeat (LTR) promoter. The adrenal tumors are derived from macrophages. Neoplastic macrophages also infiltrate the spleen and lymph nodes, causing splenomegaly and lymphadenopathy in mice. Nevertheless, the findings could be human relevant, because macrophages are important target cells of HTLV-I infection and serve as a virus reservoir in vivo. Moreover, the spleen, lymph nodes and adrenal glands are the most common sites of tumor cell infiltration in HTLV-I-infected patients. These data provide new mechanistic insights into the complex interaction between Tax and NF-κB, therefore improving our understanding of HTLV-I oncogenic pathogenesis. They also expand our knowledge and establish a new animal model of macrophage neoplasms and adrenal tumors.

rAAV capsid mutants eliminate leaky expression from DNA donor template for homologous recombination.

Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor templates represents a powerful approach. However, the challenge of effectively suppressing leaky transcription from the rAAV vector, a phenomenon associated to cytotoxicity, persists. In this study, we demonstrated substantial promoter activities of various homology arms and inverted terminal repeats (ITR). To address this issue, we identified a novel rAAV variant, Y704T, which not only yields high-vector quantities but also effectively suppresses in cis mRNA transcription driven by a robust promoter. The Y704T variant maintains normal functionality in receptor interaction, intracellular trafficking, nuclear entry, uncoating, and second-strand synthesis, while specifically exhibiting defects in transcription. Importantly, this inhibitory effect is found to be independent of ITR, promoter types, and RNA polymerases. Mechanistic studies unveiled the involvement of Valosin Containing Protein (VCP/p97) in capsid-mediated transcription repression. Remarkably, the Y704T variant delivers HDR donor templates without compromising DNA replication ability and homologous recombination efficiency. In summary, our findings enhance the understanding of capsid-regulated transcription and introduce novel avenues for the application of the rAAV-CRISPR/Cas9 system in human gene therapy.

KSHV genome harbors both constitutive and lytically induced enhancers.

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition.

Anaerobic fungi contain abundant, diverse, and transcriptionally active Long Terminal Repeat retrotransposons.

Long Terminal Repeat (LTR) retrotransposons are a class of repetitive elements that are widespread in the genomes of plants and many fungi. LTR retrotransposons have been associated with rapidly evolving gene clusters in plants and virulence factor transfer in fungal-plant parasite-host interactions. We report here the abundance and transcriptional activity of LTR retrotransposons across several species of the early-branching Neocallimastigomycota, otherwise known as the anaerobic gut fungi (AGF). The ubiquity of LTR retrotransposons in these genomes suggests key evolutionary roles in these rumen-dwelling biomass degraders, whose genomes also contain many enzymes that are horizontally transferred from other rumen-dwelling prokaryotes. Up to 10% of anaerobic fungal genomes consist of LTR retrotransposons, and the mapping of sequences from LTR retrotransposons to transcriptomes shows that the majority of clusters are transcribed, with some exhibiting expression greater than 104 reads per kilobase million mapped reads (rpkm). Many LTR retrotransposons are strongly differentially expressed upon heat stress during fungal cultivation, with several exhibiting a nearly three-log10 fold increase in expression, whereas growth substrate variation modulated transcription to a lesser extent. We show that some LTR retrotransposons contain carbohydrate-active enzymes (CAZymes), and the expansion of CAZymes within genomes and among anaerobic fungal species may be linked to retrotransposon activity. We further discuss how these widespread sequences may be a source of promoters and other parts towards the bioengineering of anaerobic fungi.

Clinical Value of Human Endogenous Retrovirus-H Long Terminal Repeat Associating 2 (HHLA2) in Small Cell Lung Cancer.

Objective: Human endogenous retrovirus-H long terminal repeat associating 2 (HHLA2) is a new immune checkpoint in the B7 family, and the value of HHLA2 in small cell lung cancer (SCLC) is unknown. Methods: We retrospectively detected HHLA2 expression by immunohistochemistry in SCLC patients. Moreover, plasma biomarkers of SCLC were detected retrospectively. Results: Seventy-four percent of SCLC patients exhibited HHLA2 expression. HHLA2 staining was localised within the nucleus of SCLC cells, while no staining was detected in normal lung tissue specimens. The correlation between HHLA2 expression and clinical factors was also analysed. Limited stage (LS) SCLC was more common than extensive stage (ES) SCLC among patients with HHLA2 staining. SCLC patients without metastasis had higher HHLA2 expression than SCLC patients with metastasis. HHLA2 expression was more frequently detected in the group with a tumour size greater than 5 cm than in the group with a tumour size less than 5 cm. The proportion of patients with HHLA2-positive staining was greater in the stage III and IV SCLC groups than in the stage I and II SCLC groups. A high proportion of SCLC patients with HHLA2-positive staining had a survival time <2 years. Neuron-specific enolase (NSE), CEA and Ki-67 levels were measured. The NSE level in the HHLA2-positive group was significantly greater than that in the HHLA2-negative group. The CEA and Ki-67 levels did not significantly differ between the HHLA2-positive and HHLA2-negative patients, nor were age, sex, smoking status, nodal metastasis status, Karnofsky Performance Scale (KPS) score, or Ki-67 expression score. HHLA2-positive SCLC patients had higher tumour stages and shorter 2-year survival times than HHLA2-negative patients did. Conclusion: The new immune molecule HHLA2 may be an ideal clinical biomarker for predicting SCLC progression and could serve as a new immunotherapy target in SCLC.

Karyotype and LTR-RTs analysis provide insights into oak genomic evolution.

BACKGROUND: Whole-genome duplication and long terminal repeat retrotransposons (LTR-RTs) amplification in organisms are essential factors that affect speciation, local adaptation, and diversification of organisms. Understanding the karyotype projection and LTR-RTs amplification could contribute to untangling evolutionary history. This study compared the karyotype and LTR-RTs evolution in the genomes of eight oaks, a dominant lineage in Northern Hemisphere forests. RESULTS: Karyotype projections showed that chromosomal evolution was relatively conservative in oaks, especially on chromosomes 1 and 7. Modern oak chromosomes formed through multiple fusions, fissions, and rearrangements after an ancestral triplication event. Species-specific chromosomal rearrangements revealed fragments preserved through natural selection and adaptive evolution. A total of 441,449 full-length LTR-RTs were identified from eight oak genomes, and the number of LTR-RTs for oaks from section Cyclobalanopsis was larger than in other sections. Recent amplification of the species-specific LTR-RTs lineages resulted in significant variation in the abundance and composition of LTR-RTs among oaks. The LTR-RTs insertion suppresses gene expression, and the suppressed intensity in gene regions was larger than in promoter regions. Some centromere and rearrangement regions indicated high-density peaks of LTR/Copia and LTR/Gypsy. Different centromeric regional repeat units (32, 78, 79 bp) were detected on different Q. glauca chromosomes. CONCLUSION: Chromosome fusions and arm exchanges contribute to the formation of oak karyotypes. The composition and abundance of LTR-RTs are affected by its recent amplification. LTR-RTs random retrotransposition suppresses gene expression and is enriched in centromere and chromosomal rearrangement regions. This study provides novel insights into the evolutionary history of oak karyotypes and the organization, amplification, and function of LTR-RTs.

Long-read sequencing of extrachromosomal circular DNA and genome assembly of a Solanum lycopersicum breeding line revealed active LTR retrotransposons originating from S. Peruvianum L. introgressions.

Transposable elements (TEs) are a major force in the evolution of plant genomes. Differences in the transposition activities and landscapes of TEs can vary substantially, even in closely related species. Interspecific hybridization, a widely employed technique in tomato breeding, results in the creation of novel combinations of TEs from distinct species. The implications of this process for TE transposition activity have not been studied in modern cultivars. In this study, we used nanopore sequencing of extrachromosomal circular DNA (eccDNA) and identified two highly active Ty1/Copia LTR retrotransposon families of tomato (Solanum lycopersicum), called Salsa and Ketchup. Elements of these families produce thousands of eccDNAs under controlled conditions and epigenetic stress. EccDNA sequence analysis revealed that the major parts of eccDNA produced by Ketchup and Salsa exhibited low similarity to the S. lycopersicum genomic sequence. To trace the origin of these TEs, whole-genome nanopore sequencing and de novo genome assembly were performed. We found that these TEs occurred in a tomato breeding line via interspecific introgression from S. peruvianum. Our findings collectively show that interspecific introgressions can contribute to both genetic and phenotypic diversity not only by introducing novel genetic variants, but also by importing active transposable elements from other species.

Adaptive expansion of ERVK solo-LTRs is associated with Passeriformes speciation events.

Endogenous retroviruses (ERVs) are ancient retroviral remnants integrated in host genomes, and commonly deleted through unequal homologous recombination, leaving solitary long terminal repeats (solo-LTRs). This study, analysing the genomes of 362 bird species and their reptilian and mammalian outgroups, reveals an unusually higher level of solo-LTRs formation in birds, indicating evolutionary forces might have purged ERVs during evolution. Strikingly in the order Passeriformes, and especially the parvorder Passerida, endogenous retrovirus K (ERVK) solo-LTRs showed bursts of formation and recurrent accumulations coinciding with speciation events over past 22 million years. Moreover, our results indicate that the ongoing expansion of ERVK solo-LTRs in these bird species, marked by high transcriptional activity of ERVK retroviral genes in reproductive organs, caused variation of solo-LTRs between individual zebra finches. We experimentally demonstrated that cis-regulatory activity of recently evolved ERVK solo-LTRs may significantly increase the expression level of ITGA2 in the brain of zebra finches compared to chickens. These findings suggest that ERVK solo-LTRs expansion may introduce novel genomic sequences acting as cis-regulatory elements and contribute to adaptive evolution. Overall, our results underscore that the residual sequences of ancient retroviruses could influence the adaptive diversification of species by regulating host gene expression.

Chromosome-level genome assembly and characterization of the Calophaca sinica genome.

Calophaca sinica is a rare plant endemic to northern China which belongs to the Fabaceae family and possesses rich nutritional value. To support the preservation of the genetic resources of this plant, we have successfully generated a high-quality genome of C. sinica (1.06 Gb). Notably, transposable elements (TEs) constituted ~73% of the genome, with long terminal repeat retrotransposons (LTR-RTs) dominating this group of elements (~54% of the genome). The average intron length of the C. sinica genome was noticeably longer than what has been observed for closely related species. The expansion of LTR-RTs and elongated introns emerged had the largest influence on the enlarged genome size of C. sinica in comparison to other Fabaceae species. The proliferation of TEs could be explained by certain modes of gene duplication, namely, whole genome duplication (WGD) and dispersed duplication (DSD). Gene family expansion, which was found to enhance genes associated with metabolism, genetic maintenance, and environmental stress resistance, was a result of transposed duplicated genes (TRD) and WGD. The presented genomic analysis sheds light on the genetic architecture of C. sinica, as well as provides a starting point for future evolutionary biology, ecology, and functional genomics studies centred around C. sinica and closely related species.

Molecular characterization of Marek's Disease virus reveals reticuloendotheliosis virus-long terminal repeat integration in the genome of the field isolates in Egypt.

The highly contagious, immunosuppressive, and cancer-causing Marek's disease virus (MDV) infects chickens. The financial costs of Marek's disease (MD) are significant for the chicken industry. In this study, a total of 180 samples from chicken farms suspected to be MDV-infected were collected. The chickens were sampled during the period between the months of October 2016 and February 2018 at Dakahlia and Damietta Governorates, Egypt. A total of 36 pooled samples were created. The prepared samples were inoculated into embryonated chicken eggs (ECEs). Indirect fluorescent antibody technique (IFAT) and ICP4 gene-based polymerase chain reaction (PCR) were used for MDV identification. For the genetic characterization of the identified virus, The ICP4 gene sequence was identified and compared with the sequences available from various regions of the world. Furthermore, the genomes of all detected MDVs were screened for the long terminal repeat (LTR) region of reticuloendotheliosis (REV) in their genomes. The results showed that 31 out of 36 pooled samples (86.1%) inoculated into ECEs displayed the characteristic pock lesions. By using IFAT and PCR to identify MDV in ECEs, positive results were found in 27 samples (75%). The Egyptian virus is thought to be genetically closely related to MDVs circulating in Ethiopia, China, and India. REV-LTR was amplified from 6 out of 27 field isolates genomes (22.2 %) while MDV vaccine strains were free from REV-LTR insertion. The integrated REV-LTRs depicted a close genetic relationship with those integrated in fowl poxvirus (FWPV) circulating in Egypt as well as those integrated in FWPVs and MDVs from China, USA, South Africa, and Australia. To the best of our knowledge, this investigation represents the first identification and characterization of REV-LTR insertions in Egyptian MDV field isolates. Given the findings above, additional research in the future seems crucial to determine how the REV-LTR insertions affect MDV pathogenesis, virulence, and insufficient vaccination protection.

Species-Specific Transcription Factors Associated with Long Terminal Repeat Promoters of Endogenous Retroviruses: A Comprehensive Review.

Endogenous retroviruses (ERVs) became a part of the eukaryotic genome through endogenization millions of years ago. Moreover, they have lost their innate capability of virulence or replication. Nevertheless, in eukaryotic cells, they actively engage in various activities that may be advantageous or disadvantageous to the cells. The mechanisms by which transcription is triggered and implicated in cellular processes are complex. Owing to the diversity in the expression of transcription factors (TFs) in cells and the TF-binding motifs of viruses, the comprehensibility of ERV initiation and its impact on cellular functions are unclear. Currently, several factors are known to be related to their initiation. TFs that bind to the viral long-terminal repeat (LTR) are critical initiators. This review discusses the TFs shown to actively associate with ERV stimulation across species such as humans, mice, pigs, monkeys, zebrafish, Drosophila, and yeast. A comprehensive summary of the expression of previously reported TFs may aid in identifying similarities between animal species and endogenous viruses. Moreover, an in-depth understanding of ERV expression will assist in elucidating their physiological roles in eukaryotic cell development and in clarifying their relationship with endogenous retrovirus-associated diseases.

Transposition of HOPPLA in siRNA-deficient plants suggests a limited effect of the environment on retrotransposon mobility in Brachypodium distachyon.

Long terminal repeat retrotransposons (LTR-RTs) are powerful mutagens regarded as a major source of genetic novelty and important drivers of evolution. Yet, the uncontrolled and potentially selfish proliferation of LTR-RTs can lead to deleterious mutations and genome instability, with large fitness costs for their host. While population genomics data suggest that an ongoing LTR-RT mobility is common in many species, the understanding of their dual role in evolution is limited. Here, we harness the genetic diversity of 320 sequenced natural accessions of the Mediterranean grass Brachypodium distachyon to characterize how genetic and environmental factors influence plant LTR-RT dynamics in the wild. When combining a coverage-based approach to estimate global LTR-RT copy number variations with mobilome-sequencing of nine accessions exposed to eight different stresses, we find little evidence for a major role of environmental factors in LTR-RT accumulations in B. distachyon natural accessions. Instead, we show that loss of RNA polymerase IV (Pol IV), which mediates RNA-directed DNA methylation in plants, results in high transcriptional and transpositional activities of RLC_BdisC024 (HOPPLA) LTR-RT family elements, and that these effects are not stress-specific. This work supports findings indicating an ongoing mobility in B. distachyon and reveals that host RNA-directed DNA methylation rather than environmental factors controls their mobility in this wild grass model.

Endogenous feline leukemia virus long terminal repeat integration site diversity is highly variable in related and unrelated domestic cats.

Endogenous retroviruses (ERV) are indicators of vertebrate evolutionary history and play important roles as homeostatic regulators. ERV long terminal repeat (LTR) elements may act as cis-activating promoters or trans-activating enhancer elements modifying gene transcription distant from LTR insertion sites. We previously documented that endogenous feline leukemia virus (FeLV)-LTR copy number variation in individual cats tracks inversely with susceptibility to virulent FeLV disease. To evaluate FeLV-LTR insertion characteristics, we assessed enFeLV-LTR integration site diversity in 20 cats from three genetically distinct populations using a baited linker-mediated PCR approach. We documented 765 individual integration sites unequally represented among individuals. Only three LTR integration sites were shared among all individuals, while 412 sites were unique to a single individual. When primary fibroblast cultures were challenged with exogenous FeLV, we found significantly increased expression of both exogenous and endogenous FeLV orthologs, supporting previous findings of potential exFeLV-enFeLV interactions; however, viral challenge did not elicit transcriptional changes in genes associated with the vast majority of integration sites. This study assesses FeLV-LTR integration sites in individual animals, providing unique transposome genotypes. Further, we document substantial individual variation in LTR integration site locations, even in a highly inbred population, and provide a framework for understanding potential endogenous retroviral element position influence on host gene transcription.

Systematic evaluation of retroviral LTRs as cis-regulatory elements in mouse embryos.

In mammals, many retrotransposons are de-repressed during zygotic genome activation (ZGA). However, their functions in early development remain elusive largely due to the challenge to simultaneously manipulate thousands of retrotransposon insertions in embryos. Here, we applied CRISPR interference (CRISPRi) to perturb the long terminal repeat (LTR) MT2_Mm, a well-known ZGA and totipotency marker that exists in ∼2,667 insertions throughout the mouse genome. CRISPRi robustly perturbed 2,485 (∼93%) MT2_Mm insertions and 1,090 (∼55%) insertions of the closely related MT2C_Mm in 2-cell embryos. Remarkably, such perturbation caused downregulation of hundreds of ZGA genes and embryonic arrest mostly at the morula stage. Mechanistically, MT2 LTRs are globally enriched for open chromatin and H3K27ac and function as promoters/enhancers downstream of OBOX/DUX proteins. Thus, we not only provide direct evidence to support the functional importance of MT2 activation in development but also systematically define cis-regulatory function of MT2 in embryos by integrating functional perturbation and multi-omic analyses.

Optimizing protocols for monitoring in vivo replication of a novel chimeric Marek's disease vaccine with an insertion of the long terminal repeat of reticuloendotheliosis virus in the CVI988 strain genome (CVI-LTR).

Monitoring Marek's disease (MD) vaccination is routinely done by evaluating the load of MD vaccine in the feather pulp (FP) between 7 and 10 days of age. However, attempts in our laboratory to detect a novel CVI-LTR vaccine in the FP samples from commercial flocks failed. The objective of this study was to evaluate the most suitable tissue and age to monitor CVI-LTR vaccination. We used two different commercial CVI988 vaccines as controls. One hundred and sixty 1-day-old commercial brown layers were vaccinated with either CVI-LTR, CVI988-A, CVI988-B or remained unvaccinated. Samples of the spleen, thymus, and bursa were collected at 3, 4, 5, and 6 days of age and samples of FP were collected at 7 and 21 days for DNA isolation. Our results showed that CVI-LTR replicated earlier than CVI988 vaccines in the lymphoid organs but was not detected in the FP at either 7 or at 21 days of age. We also confirmed that either the spleen or thymus collected at 4-6 days was a suitable sample to monitor CVI-LTR vaccination in commercial flocks. Finally, we evaluated the load of oncogenic MDV DNA in five commercial flocks that were vaccinated with either CVI-LTR + rHVT or CVI988-A + rHVT. The load of oncogenic MDV DNA was evaluated at 21 days in the FP in 20 chickens per group. Our results demonstrated that CVI-LTR was more successful in reducing oncogenic MDV DNA at 21 days of age than the CVI988-A strain.RESEARCH HIGHLIGHTSCVI-LTR replicates in the thymus and spleen earlier than CVI988.CVI-LTR replicates in lymphoid organs but it cannot be detected in feather pulp.CVI-LTR reduced the load of oncogenic MDV DNA more efficiently than CVI988.

Transposon Removal Reveals Their Adaptive Fitness Contribution.

Transposable elements are molecular parasites that persist in their host genome by generating new copies to outpace natural selection. Transposable elements exert a large influence on host genome evolution, in some cases providing adaptive changes. Here we measure the fitness effect of the transposable element insertions in the fission yeast Schizosaccharomyces pombe type strain by removing all insertions of its only native transposable element family, the long terminal repeat retrotransposon Tf2. We show that Tf2 elements provide a positive fitness contribution to its host. Tf2 ablation results in changes to the regulation of a mitochondrial gene and, consistently, the fitness effect are sensitive to growth conditions. We propose that Tf2 influences host fitness in a directed manner by dynamically rewiring the transcriptional response to metabolic stress.