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The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Increasing natural resistance and resilience in plants is key for ensuring food security within a changing climate. Breeders improve these traits by crossing cultivars with their wild relatives and introgressing specific alleles through meiotic recombination. However, some genomic regions are devoid of recombination especially in crosses between divergent genomes, limiting the combinations of desirable alleles. Here, we used pooled-pollen sequencing to build a map of recombinant and non-recombinant regions between tomato and five wild relatives commonly used for introgressive tomato breeding. We detected hybrid-specific recombination coldspots that underscore the role of structural variations in modifying recombination patterns and maintaining genetic linkage in interspecific crosses. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. About two-thirds of the genome are conserved coldspots, located mostly in the pericentromeres and enriched with retrotransposons. The coldspots also harbor genes associated with agronomic traits and stress resistance, revealing undesired consequences of linkage drag and possible barriers to breeding. We presented examples of linkage drag that can potentially be resolved by pairing tomato with other wild species. Overall, this catalogue will help breeders better understand crossover localization and make informed decisions on generating new tomato varieties., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fuentes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Genome evolution is partly driven by the mobility of transposable elements (TEs) which often leads to deleterious effects, but their activity can also facilitate genetic novelty and catalyze local adaptation. We explored how the intraspecific diversity of TE polymorphisms might contribute to the broad geographic success and adaptive capacity of the emerging oil crop Thlaspi arvense (field pennycress). We classified the TE inventory based on a high-quality genome assembly, estimated the age of retrotransposon TE families and comprehensively assessed their mobilization potential. A survey of 280 accessions from 12 regions across the Northern hemisphere allowed us to quantify over 90,000 TE insertion polymorphisms (TIPs). Their distribution mirrored the genetic differentiation as measured by single nucleotide polymorphisms (SNPs). The number and types of mobile TE families vary substantially across populations, but there are also shared patterns common to all accessions. Ty3/Athila elements are the main drivers of TE diversity in T. arvense populations, while a single Ty1/Alesia lineage might be particularly important for transcriptome divergence. The number of retrotransposon TIPs is associated with variation at genes related to epigenetic regulation, including an apparent knockout mutation in BROMODOMAIN AND ATPase DOMAIN-CONTAINING PROTEIN 1 (BRAT1), while DNA transposons are associated with variation at the HSP19 heat shock protein gene. We propose that the high rate of mobilization activity can be harnessed for targeted gene expression diversification, which may ultimately present a toolbox for the potential use of transposition in breeding and domestication of T. arvense., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: D.W. holds equity in Computomics, which advises breeders. D.W. advises KWS SE, a plant breeder and seed producer. All the other authors have declared that no competing interests exist.
, (Copyright: © 2024 Contreras-Garrido et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Retrotransposons have generated about half of the human genome and LINE-1s (L1s) are the only autonomously active retrotransposons. The cell has evolved an arsenal of defense mechanisms to protect against retrotransposition with factors we are only beginning to understand. In this study, we investigate Zinc Finger CCHC-Type Containing 3 (ZCCHC3), a gag-like zinc knuckle protein recently reported to function in the innate immune response to infecting viruses. We show that ZCCHC3 also severely restricts human retrotransposons and associates with the L1 ORF1p ribonucleoprotein particle. We identify ZCCHC3 as a bona fide stress granule protein, and its association with LINE-1 is further supported by colocalization with L1 ORF1 protein in stress granules, dense cytoplasmic aggregations of proteins and RNAs that contain stalled translation pre-initiation complexes and form when the cell is under stress. Our work also draws links between ZCCHC3 and the anti-viral and retrotransposon restriction factors Mov10 RISC Complex RNA Helicase (MOV10) and Zinc Finger CCCH-Type, Antiviral 1 (ZC3HAV1, also called ZAP). Furthermore, collective evidence from subcellular localization, co-immunoprecipitation, and velocity gradient centrifugation connects ZCCHC3 with the RNA exosome, a multi-subunit ribonuclease complex capable of degrading various species of RNA molecules and that has previously been linked with retrotransposon control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Goodier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Retroviruses and closely related LTR retrotransposons export full-length, unspliced genomic RNA (gRNA) for packaging into virions and to serve as the mRNA encoding GAG and POL polyproteins. Because gRNA often includes splice acceptor and donor sequences used to splice viral mRNAs, retroelements must overcome host mechanisms that retain intron-containing RNAs in the nucleus. Here we examine gRNA expression in Cer1, an LTR retrotransposon in C. elegans which somehow avoids silencing and is highly expressed in germ cells. Newly exported Cer1 gRNA associates rapidly with the Cer1 GAG protein, which has structural similarity with retroviral GAG proteins. gRNA export requires CERV (C. elegans regulator of viral expression), a novel protein encoded by a spliced Cer1 mRNA. CERV phosphorylation at S214 is essential for gRNA export, and phosphorylated CERV colocalizes with nuclear gRNA at presumptive sites of transcription. By electron microscopy, tagged CERV proteins surround clusters of distinct, linear fibrils that likely represent gRNA molecules. Single fibrils, or groups of aligned fibrils, also localize near nuclear pores. During the C. elegans self-fertile period, when hermaphrodites fertilize oocytes with their own sperm, CERV concentrates in two nuclear foci that are coincident with gRNA. However, as hermaphrodites cease self-fertilization, and can only produce cross-progeny, CERV undergoes a remarkable transition to form giant nuclear rods or cylinders that can be up to 5 microns in length. We propose a novel mechanism of rod formation, in which stage-specific changes in the nucleolus induce CERV to localize to the nucleolar periphery in flattened streaks of protein and gRNA; these streaks then roll up into cylinders. The rods are a widespread feature of Cer1 in wild strains of C. elegans, but their function is not known and might be limited to cross-progeny. We speculate that the adaptive strategy Cer1 uses for the identical self-progeny of a host hermaphrodite might differ for heterozygous cross-progeny sired by males. For example, mating introduces male chromosomes which can have different, or no, Cer1 elements., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Retroelements (REs) had been considered 'Junk' until the encyclopedia of DNA elements (ENCODE) project demonstrated that most genome is functional. Although the function of retroelements has been reported in diverse cancers including human breast cancer (HBC) and subtypes, only a few studies have suggested the putative functions of REs via their random genome integration. A canine mammary tumor (CMT) has been highlighted due to the similarities in molecular and pathophysiology with HBC. This study investigated the putative roles of REs common in both HBC and CMT. The human LINE and HERV-K sequences harbor many miRNAs responsive elements (MREs) for tumor-suppressive miRNA such as let-7. We also observed that various MREs are exist in the ERV and LINE highly expressed in the transcriptome data of CMT as well as HBC sets. MREs against miR-126 were highly expressed in both HBC and CMT while the levels of miR-126 were down-regulated. Oppositely, the expression of miR-126 target genes was significantly up-regulated in the cancers. Moreover, cancer patients with an increased level of miR-126 showed better overall survival. The expression of ENPP5, a putative miR-126 target gene, was downregulated by miR-126 mimic. Importantly, overexpression of LINE fragment significantly suppressed miR-126 function on the target gene expression. We propose the functional role of REs expression in tumorigenesis as competing endogenous RNAs (ceRNA) against tumor-suppressive miRNAs. This study provided pieces of evidence that LINE expression, even partial and fragmented, have a regulatory function in ENPP5 gene expression via the competition with miR-126., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Transposable elements (TE) are mobile DNA sequences whose excessive proliferation endangers the host. Although animals have evolved robust TE-targeting defenses, including Piwi-interacting (pi)RNAs, retrotransposon LINE-1 (L1) still thrives in humans and mice. To gain insights into L1 endurance, we characterized L1 Bodies (LBs) and ORF1p complexes in germ cells of piRNA-deficient Maelstrom null mice. We report that ORF1p interacts with TE RNAs, genic mRNAs, and stress granule proteins, consistent with earlier studies. We also show that ORF1p associates with the CCR4-NOT deadenylation complex and PRKRA, a Protein Kinase R factor. Despite ORF1p interactions with these negative regulators of RNA expression, the stability and translation of LB-localized mRNAs remain unchanged. To scrutinize these findings, we studied the effects of PRKRA on L1 in cultured cells and showed that it elevates ORF1p levels and L1 retrotransposition. These results suggest that ORF1p-driven condensates promote L1 propagation, without affecting the metabolism of endogenous RNAs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 De Luca et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Transposition of transposable elements affect expression levels, splicing and epigenetic status, and function of genes located in, or near, the inserted/excised locus. For example, in grape, presence of the Gret1 retrotransposon in the promoter region of the VvMYBA1a allele at the VvMYBA1 locus suppress the expression of the VvMYBA1 transcription factor gene for the anthocyanin biosynthesis and this transposon insertion is responsible for the green berry skin color of Vitis labrascana, 'Shine Muscat', a major grape cultivar in Japan. To prove that transposons in grape genome can be removed by genome editing, we focused on Gret1 in the VvMYBA1a allele as a target of CRISPR/Cas9 mediated transposon removal. PCR amplification and sequencing detected Gret1 eliminated cells in 19 of 45 transgenic plants. Although we have not yet confirmed any effects on grape berry skin color, we were successful in demonstrating that cleaving the long terminal repeat (LTR) present at both ends of Gret1 can efficiently eliminate the transposon., Competing Interests: The authors declare that they have no conflict of interest., (Copyright: © 2023 Nakajima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

There are many mechanisms that give rise to genomic change: while point mutations are often emphasized in genomic analyses, evolution acts upon many other types of genetic changes that can result in less subtle perturbations. Changes in chromosome structure, DNA copy number, and novel transposon insertions all create large genomic changes, which can have correspondingly large impacts on phenotypes and fitness. In this study we investigate the spectrum of adaptive mutations that arise in a population under consistently fluctuating nitrogen conditions. We specifically contrast these adaptive alleles and the mutational mechanisms that create them, with mechanisms of adaptation under batch glucose limitation and constant selection in low, non-fluctuating nitrogen conditions to address if and how selection dynamics influence the molecular mechanisms of evolutionary adaptation. We observe that retrotransposon activity accounts for a substantial number of adaptive events, along with microhomology-mediated mechanisms of insertion, deletion, and gene conversion. In addition to loss of function alleles, which are often exploited in genetic screens, we identify putative gain of function alleles and alleles acting through as-of-yet unclear mechanisms. Taken together, our findings emphasize that how selection (fluctuating vs. non-fluctuating) is applied also shapes adaptation, just as the selective pressure (nitrogen vs. glucose) does itself. Fluctuating environments can activate different mutational mechanisms, shaping adaptive events accordingly. Experimental evolution, which allows a wider array of adaptive events to be assessed, is thus a complementary approach to both classical genetic screens and natural variation studies to characterize the genotype-to-phenotype-to-fitness map., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hays et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Repressive KRAB domain-containing zinc-finger proteins (KRAB-ZFPs) are abundant in mammalian genomes and contribute both to the silencing of transposable elements (TEs) and to the regulation of developmental stage- and cell type-specific gene expression. Here we describe studies of zinc finger protein 92 (Zfp92), an X-linked KRAB-ZFP that is highly expressed in pancreatic islets of adult mice, by analyzing global Zfp92 knockout (KO) mice. Physiological, transcriptomic and genome-wide chromatin binding studies indicate that the principal function of ZFP92 in mice is to bind to and suppress the activity of B1/Alu type of SINE elements and modulate the activity of surrounding genomic entities. Deletion of Zfp92 leads to changes in expression of select LINE and LTR retroelements and genes located in the vicinity of ZFP92-bound chromatin. The absence of Zfp92 leads to altered expression of specific genes in islets, adipose and muscle that result in modest sex-specific alterations in blood glucose homeostasis, body mass and fat accumulation. In islets, Zfp92 influences blood glucose concentration in postnatal mice via transcriptional effects on Mafb, whereas in adipose and muscle, it regulates Acacb, a rate-limiting enzyme in fatty acid metabolism. In the absence of Zfp92, a novel TE-Capn11 fusion transcript is overexpressed in islets and several other tissues due to de-repression of an IAPez TE adjacent to ZFP92-bound SINE elements in intron 3 of the Capn11 gene. Together, these studies show that ZFP92 functions both to repress specific TEs and to regulate the transcription of specific genes in discrete tissues., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Osipovich et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Transposable elements constitute nearly half of the mammalian genome and play important roles in genome evolution. While a multitude of both transcriptional and post-transcriptional mechanisms exist to silence transposable elements, control of transposition in vivo remains poorly understood. MOV10, an RNA helicase, is an inhibitor of mobilization of retrotransposons and retroviruses in cell culture assays. Here we report that MOV10 restricts LINE1 retrotransposition in mice. Although MOV10 is broadly expressed, its loss causes only incomplete penetrance of embryonic lethality, and the surviving MOV10-deficient mice are healthy and fertile. Biochemically, MOV10 forms a complex with UPF1, a key component of the nonsense-mediated mRNA decay pathway, and primarily binds to the 3' UTR of somatically expressed transcripts in testis. Consequently, loss of MOV10 results in an altered transcriptome in testis. Analyses using a LINE1 reporter transgene reveal that loss of MOV10 leads to increased LINE1 retrotransposition in somatic and reproductive tissues from both embryos and adult mice. Moreover, the degree of LINE1 retrotransposition inhibition is dependent on the Mov10 gene dosage. Furthermore, MOV10 deficiency reduces reproductive fitness over successive generations. Our findings demonstrate that MOV10 attenuates LINE1 retrotransposition in a dosage-dependent manner in mice., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Guan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Although homologous recombination between transposable elements can drive genomic evolution in yeast by facilitating chromosomal rearrangements, the details of the underlying mechanisms are not fully clarified. In the genome of the yeast Saccharomyces cerevisiae, the most common class of transposon is the retrotransposon Ty1. Here, we explored how Cas9-induced double-strand breaks (DSBs) directed to Ty1 elements produce genomic alterations in this yeast species. Following Cas9 induction, we observed a significant elevation of chromosome rearrangements such as deletions, duplications and translocations. In addition, we found elevated rates of mitotic recombination, resulting in loss of heterozygosity. Using Southern analysis coupled with short- and long-read DNA sequencing, we revealed important features of recombination induced in retrotransposons. Almost all of the chromosomal rearrangements reflect the repair of DSBs at Ty1 elements by non-allelic homologous recombination; clustered Ty elements were hotspots for chromosome rearrangements. In contrast, a large proportion (about three-fourths) of the allelic mitotic recombination events have breakpoints in unique sequences. Our analysis suggests that some of the latter events reflect extensive processing of the broken ends produced in the Ty element that extend into unique sequences resulting in break-induced replication. Finally, we found that haploid and diploid strain have different preferences for the pathways used to repair double-stranded DNA breaks. Our findings demonstrate the importance of DNA lesions in retrotransposons in driving genome evolution., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Qi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT3 family enzymes, demethylation by the TET-mediated conversion of 5mC to 5-hydroxymethylation (5hmC), and maintenance of the remaining DNAme by DNMT1 are actively repeated throughout cell cycles, dynamically forming a constant 5mC profile. Nevertheless, the detailed mechanism and physiological significance of this active cyclic DNA modification in mouse ESCs remain unclear. Here by visualizing the localization of DNA modifications on metaphase chromosomes and comparing whole-genome methylation profiles before and after the mid-S phase in ESCs lacking Dnmt1 (1KO ESCs), we demonstrated that in 1KO ESCs, DNMT3-mediated remethylation was interrupted during and after DNA replication. This results in a marked asymmetry in the distribution of 5hmC between sister chromatids at mitosis, with one chromatid being almost no 5hmC. When introduced in 1KO ESCs, the catalytically inactive form of DNMT1 (DNMT1CI) induced an increase in DNAme in pericentric heterochromatin and the DNAme-independent repression of IAPEz, a retrotransposon family, in 1KO ESCs. However, DNMT1CI could not restore the ability of DNMT3 to methylate unmodified dsDNA de novo in S phase in 1KO ESCs. Furthermore, during in vitro differentiation into epiblasts, 1KO ESCs expressing DNMT1CI showed an even stronger tendency to differentiate into the primitive endoderm than 1KO ESCs and were readily reprogrammed into the primitive streak via an epiblast-like cell state, reconfirming the importance of DNMT1 enzymatic activity at the onset of epiblast differentiation. These results indicate a novel function of DNMT1, in which DNMT1 actively regulates the timing and genomic targets of de novo methylation by DNMT3 in an enzymatic activity-dependent and independent manner, respectively., Competing Interests: The authors have declared that no competing interests exist.

Drosophila chromosomes are elongated by retrotransposon attachment, a process poorly understood. Here we characterized a mutation affecting the HipHop telomere-capping protein. In mutant ovaries and the embryos that they produce, telomere retrotransposons are activated and transposon RNP accumulates. Genetic results are consistent with that this hiphop mutation weakens the efficacy of HP1-mediated silencing while leaving piRNA-based mechanisms largely intact. Remarkably, mutant females display normal fecundity suggesting that telomere de-silencing is compatible with germline development. Moreover, unlike prior mutants with overactive telomeres, the hiphop stock does not over-accumulate transposons for hundreds of generations. This is likely due to the loss of HipHop's abilities both to silence transcription and to recruit transposons to telomeres in the mutant. Furthermore, embryos produced by mutant mothers experience a checkpoint activation, and a further loss of maternal HipHop leads to end-to-end fusion and embryonic arrest. Telomeric retroelements fulfill an essential function yet maintain a potentially conflicting relationship with their Drosophila host. Our study thus showcases a possible intermediate in this arm race in which the host is adapting to over-activated transposons while maintaining genome stability. Our results suggest that the collapse of such a relationship might only occur when the selfish element acquires the ability to target non-telomeric regions of the genome. HipHop is likely part of this machinery restricting the elements to the gene-poor region of telomeres. Lastly, our hiphop mutation behaves as a recessive suppressor of PEV that is mediated by centric heterochromatin, suggesting its broader effect on chromatin not limited to telomeres., Competing Interests: The authors have declared that no competing interests exist.

Telomeres are nucleoprotein complexes that protect the ends of eukaryotic linear chromosomes from degradation and fusions. Telomere dysfunction leads to cell growth arrest, oncogenesis, and premature aging. Telomeric RNAs have been found in all studied species; however, their functions and biogenesis are not clearly understood. We studied the mechanisms of development disorders observed upon overexpression of telomeric repeats in Drosophila. In somatic cells, overexpression of telomeric retrotransposon HeT-A is cytotoxic and leads to the accumulation of HeT-A Gag near centrosomes. We found that RNA and RNA-binding protein Gag encoded by the telomeric retrotransposon HeT-A interact with Polo and Cdk1 mitotic kinases, which are conserved regulators of centrosome biogenesis and cell cycle. The depletion of proteins Spindle E, Ccr4 or Ars2 resulting in HeT-A overexpression in the germline was accompanied by mislocalization of Polo as well as its abnormal stabilization during oogenesis and severe deregulation of centrosome biogenesis leading to maternal-effect embryonic lethality. These data suggest a mechanistic link between telomeric HeT-A ribonucleoproteins and cell cycle regulators that ensures the cell response to telomere dysfunction., Competing Interests: The authors have declared that no competing interests exist.

It has become increasingly clear that retrotransposons (RTEs) are more widely expressed in somatic tissues than previously appreciated. RTE expression has been implicated in a myriad of biological processes ranging from normal development and aging, to age related diseases such as cancer and neurodegeneration. Long Terminal Repeat (LTR)-RTEs are evolutionary ancestors to, and share many features with, exogenous retroviruses. In fact, many organisms contain endogenous retroviruses (ERVs) derived from exogenous retroviruses that integrated into the germ line. These ERVs are inherited in Mendelian fashion like RTEs, and some retain the ability to transmit between cells like viruses, while others develop the ability to act as RTEs. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly. But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Like murine IAP/IAP-E elements, the gypsy family of retroelements in arthropods appear to sit along this evolutionary transition. Indeed, there is some evidence that gypsy may exhibit retroviral properties. Given that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration, this raises the question of whether gypsy replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two. These modes of replication in somatic tissues would have quite different biological implications. Here, we demonstrate that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin. Further, we demonstrate that the ability of gypsy to move between cells is dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene. On the other hand, we also find that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome., Competing Interests: The authors have declared that no competing interests exist.

Piwi-interacting RNAs (piRNAs) play critical roles in protecting germline genome integrity and promoting normal spermiogenic differentiation. In mammals, there are two populations of piRNAs: pre-pachytene and pachytene. Transposon-rich pre-pachytene piRNAs are expressed in fetal and perinatal germ cells and are required for retrotransposon silencing, whereas transposon-poor pachytene piRNAs are expressed in spermatocytes and round spermatids and regulate mRNA transcript levels. MOV10L1, a germ cell-specific RNA helicase, is essential for the production of both populations of piRNAs. Although the requirement of the RNA helicase domain located in the MOV10L1 C-terminal region for piRNA biogenesis is well known, its large N-terminal region remains mysterious. Here we report a novel Mov10l1 mutation, named yama, in the Mov10l1 N-terminal region. The yama mutation results in a single amino acid substitution V229E. The yama mutation causes meiotic arrest, de-repression of transposable elements, and male sterility because of defects in pre-pachytene piRNA biogenesis. Moreover, restricting the Mov10l1 mutation effects to later stages in germ cell development by combining with a postnatal conditional deletion of a complementing wild-type allele causes absence of pachytene piRNAs, accumulation of piRNA precursors, polar conglomeration of piRNA pathway proteins in spermatocytes, and spermiogenic arrest. Mechanistically, the V229E substitution in MOV10L1 reduces its interaction with PLD6, an endonuclease that generates the 5' ends of piRNA intermediates. Our results uncover an important role for the MOV10L1-PLD6 interaction in piRNA biogenesis throughout male germ cell development., Competing Interests: The authors have declared that no competing interests exist.

Ionizing radiation-induced tissue damage recruits monocytes into the exposed area where they are differentiated to macrophages. These implement phagocytic removal of dying cells and elicit an acute inflammatory response, but can also facilitate tumorigenesis due to production of anti-inflammatory cytokines. Using primary human monocyte-derived macrophages (MDMs) and the THP1 monocytic cell line, we demonstrate that gamma radiation triggers monocyte differentiation toward the macrophage phenotype with increased expression of type I interferons (IFN-I) and both pro- and anti-inflammatory macrophage activation markers. We found that these changes correlate with significantly upregulated expression of 622 retroelements from various groups, particularly of several clades of human endogenous retroviruses (HERVs). Elevated transcription was detected in both sense and antisense directions in the HERV subgroups tested, including the most genetically homogeneous clade HML-2. The level of antisense transcription was three- to five-fold higher than of the sense strand levels. Using a proximity ligation assay and immunoprecipitation followed by RNA quantification, we identified an increased amount of the dsRNA receptors MDA-5 and TLR3 bound to an equivalent number of copies of sense and antisense chains of HERVK HML-2 RNA. This binding triggered MAVS-associated signaling pathways resulting in increased expression of IFN-I and inflammation related genes that enhanced the cumulative inflammatory effect of radiation-induced senescence. HML-2 knockdown was accompanied with reduced expression and secretion of IFNα, pro-inflammatory (IL-1β, IL-6, CCL2, CCL3, CCL8, and CCL20) and anti-inflammatory (IL10) modulators in irradiated monocytes and MDMs. Taken together, our data indicate that radiation stress-induced HERV expression enhances the IFN-I and cytokine response and results in increased levels of pro-inflammatory modulators along with expression of anti-inflammatory factors associated with the macrophage tumorigenic phenotype., Competing Interests: The authors have declared that no competing interests exist.

Pleurotus ostreatus, one of the most widely cultivated edible mushrooms, produces high numbers of spores causing severe respiratory health problems for people, clogging of filters and spoilage of produce. A non-sporulating commercial variety (SPOPPO) has been successfully introduced into the market in 2006. This variety was generated by introgression breeding of a natural mutation into a commercial variety. Our cytological studies revealed that meiosis in the natural and derived sporeless strains was blocked in metaphase I, apparently resulting in a loss of spore formation. The gene(s) underlying this phenotype were mapped to an 80 kb region strongly linked to sporelessness and identified by transformation of wild type genes of this region into a sporeless strain. Sporulation was restored by re-introduction of the DNA sequence encoding the P. ostreatus meiotic recombination gene MSH4 homolog (poMSH4). Subsequent molecular analysis showed that poMSH4 in the sporeless P. ostreatus was interrupted by a DNA fragment containing a region encoding a CxC5/CxC6 cysteine cluster associated with Copia-type retrotransposons. The block of meiosis in metaphase I by a poMSH4 null mutant suggests that this protein plays an essential role in both Class I and II crossovers in mushrooms, similar to animals (mice), but unlike in plants. MSH4 was previously shown to be a target for breeding of sporeless varieties in P. pulmonarius, and the null mutant of the MSH4 homolog of S. commune (scMSH4) confers an extremely low level of spore formation. We propose that MSH4 homologs are likely to be a breeding target for sporeless strains both within Pleurotus sp. and in other Agaricales., Competing Interests: The authors have declared that no competing interests exist.

The interactions between transposable elements (TEs) and their hosts constitute one of the most profound co-evolutionary processes found in nature. The population dynamics of TEs depends on factors specific to each TE families, such as the rate of transposition and insertional preference, the demographic history of the host and the genomic landscape. How these factors interact has yet to be investigated holistically. Here we are addressing this question in the green anole (Anolis carolinensis) whose genome contains an extraordinary diversity of TEs (including non-LTR retrotransposons, SINEs, LTR-retrotransposons and DNA transposons). We observed a positive correlation between recombination rate and frequency of TEs and densities for LINEs, SINEs and DNA transposons. For these elements, there was a clear impact of demography on TE frequency and abundance, with a loss of polymorphic elements and skewed frequency spectra in recently expanded populations. On the other hand, some LTR-retrotransposons displayed patterns consistent with a very recent phase of intense amplification. To determine how demography, genomic features and intrinsic properties of TEs interact we ran simulations using SLiM3. We determined that i) short TE insertions are not strongly counter-selected, but long ones are, ii) neutral demographic processes, linked selection and preferential insertion may explain positive correlations between average TE frequency and recombination, iii) TE insertions are unlikely to have been massively recruited in recent adaptation. We demonstrate that deterministic and stochastic processes have different effects on categories of TEs and that a combination of empirical analyses and simulations can disentangle these mechanisms., Competing Interests: The authors have declared that no competing interests exist.

Accounting for continual evolution of deleterious L1 retrotransposon families, which can contain hundreds to thousands of members remains a major issue in mammalian biology. L1 activity generated upwards of 40% of some mammalian genomes, including humans where they remain active, causing genetic defects and rearrangements. L1 encodes a coiled coil-containing protein that is essential for retrotransposition, and the emergence of novel primate L1 families has been correlated with episodes of extensive amino acid substitutions in the coiled coil. These results were interpreted as an adaptive response to maintain L1 activity, however its mechanism remained unknown. Although an adventitious mutation can inactivate coiled coil function, its effect could be buffered by epistatic interactions within the coiled coil, made more likely if the family contains a diverse set of coiled coil sequences-collectively referred to as the coiled coil sequence space. Amino acid substitutions that do not affect coiled coil function (i.e., its phenotype) could be "hidden" from (not subject to) purifying selection. The accumulation of such substitutions, often referred to as cryptic genetic variation, has been documented in various proteins. Here we report that this phenomenon was in effect during the latest episode of primate coiled coil evolution, which occurred 30-10 MYA during the emergence of primate L1Pa7-L1Pa3 families. First, we experimentally demonstrated that while coiled coil function (measured by retrotransposition) can be eliminated by single epistatic mutations, it nonetheless can also withstand extensive amino acid substitutions. Second, principal component and cluster analysis showed that the coiled coil sequence space of each of the L1Pa7-3 families was notably increased by the presence of distinct, coexisting coiled coil sequences. Thus, sampling related networks of functional sequences rather than traversing discrete adaptive states characterized the persistence L1 activity during this evolutionary event., Competing Interests: The authors have declared that no competing interests exist.

Repeat-induced gene silencing (RIGS) establishes the centromere structure, prevents the spread of transposons and silences transgenes, thereby limiting recombinant protein production. We previously isolated a sequence (B-3-31) that alleviates RIGS from the human genome. Here, we developed an assay system for evaluating the influence of repeat sequences on gene expression, based on in vitro ligation followed by our original gene amplification technology in animal cells. Using this assay, we found that the repeat of B-3-31, three core sequences of replication initiation regions (G5, C12, and D8) and two matrix attachment regions (AR1 and 32-3), activated the co-amplified plasmid-encoded d2EGFP gene in both human and hamster cell lines. This upregulation effect persisted for up to 82 days, which was confirmed to be repeat-induced, and was thus designated as a repeat-induced gene activation (RIGA). In clear contrast, the repeat of three bacterial sequences (lambda-phage, Amp, and ColE1) and three human retroposon sequences (Alu, 5'-untranslated region, and ORF1 of a long interspersed nuclear element) suppressed gene expression, thus reflecting RIGS. RIGS was CpG-independent. We suggest that RIGA might be associated with replication initiation. The discovery of RIGS and RIGA has implications for the repeat in mammalian genome, as well as practical value in recombinant production., Competing Interests: The authors have declared that no competing interests exist.

Following allopolyploidization, nascent polyploid wheat species react with massive genomic rearrangements, including deletion of transposable element-containing sequences. While such massive rearrangements are considered to be a prominent process in wheat genome evolution and speciation, their structure, extent, and underlying mechanisms remain poorly understood. In this study, we retrieved ~3500 insertions of a specific variant of Fatima, one of the most dynamic gypsy long-terminal repeat retrotransposons in wheat from the recently available high-quality genome drafts of Triticum aestivum (bread wheat) and Triticum turgidum ssp. dicoccoides or wild emmer, the allotetraploid mother of all modern wheats. The dynamic nature of Fatima facilitated the identification of large (i.e., up to ~ 1 million bases) Fatima-containing insertions/deletions (indels) upon comparison of bread wheat and wild emmer genomes. We characterized 11 such indels using computer-assisted analysis followed by PCR validation, and found that they might have occurred via unequal intra-strand recombination or double-strand break (DSB) events. Additionally, we observed one case of introgression of novel DNA fragments from an unknown source into the wheat genome. Our data thus indicate that massive large-scale DNA rearrangements might play a prominent role in wheat speciation., Competing Interests: The authors have declared that no competing interests exist.

In 1993, Denise Barlow proposed that genomic imprinting might have arisen from a host defense mechanism designed to inactivate retrotransposons. Although there were few examples at hand, she suggested that there should be maternal-specific and paternal-specific factors involved, with cognate imprinting boxes that they recognized; furthermore, the system should build on conserved biochemical factors, including DNA methylation, and maternal control should predominate for imprints. Here, we revisit this hypothesis in the light of recent advances in our understanding of host defense and DNA methylation and in particular, the link with Krüppel-associated box-zinc finger (KRAB-ZF) proteins., Competing Interests: The authors have declared that no competing interests exist.

Transposable elements constitute a large fraction of most eukaryotic genomes. Insertion of mobile DNA sequences typically has deleterious effects on host fitness, and thus diverse mechanisms have evolved to control mobile element proliferation. Mobility of the Ty1 retrotransposon in Saccharomyces yeasts is regulated by copy number control (CNC) mediated by a self-encoded restriction factor derived from the Ty1 gag capsid gene that inhibits virus-like particle function. Here, we survey a panel of wild and human-associated strains of S. cerevisiae and S. paradoxus to investigate how genomic Ty1 content influences variation in Ty1 mobility. We observe high levels of mobility for a tester element with a gag sequence from the canonical Ty1 subfamily in permissive strains that either lack full-length Ty1 elements or only contain full-length copies of the Ty1' subfamily that have a divergent gag sequence. In contrast, low levels of canonical Ty1 mobility are observed in restrictive strains carrying full-length Ty1 elements containing a canonical gag sequence. Phylogenomic analysis of full-length Ty1 elements revealed that Ty1' is the ancestral subfamily present in wild strains of S. cerevisiae, and that canonical Ty1 in S. cerevisiae is a derived subfamily that acquired gag from S. paradoxus by horizontal transfer and recombination. Our results provide evidence that variation in the ability of S. cerevisiae and S. paradoxus strains to repress canonical Ty1 transposition via CNC is regulated by the genomic content of different Ty1 subfamilies, and that self-encoded forms of transposon control can spread across species boundaries by horizontal transfer., Competing Interests: The authors have declared that no competing interests exist.

In most plants, centromeric DNA contains highly repetitive sequences, including tandem repeats and retrotransposons; however, the roles of these sequences in the structure and function of the centromere are unclear. Here, we found that multiple RNA sequences from centromeric retrotransposons (CRMs) were enriched in maize (Zea mays) centromeres, and back-spliced RNAs were generated from CRM1. We identified 3 types of CRM1-derived circular RNAs with the same back-splicing site based on the back-spliced sequences. These circular RNAs bound to the centromere through R-loops. Two R-loop sites inside a single circular RNA promoted the formation of chromatin loops in CRM1 regions. When RNA interference (RNAi) was used to target the back-splicing site of the circular CRM1 RNAs, the levels of R-loops and chromatin loops formed by these circular RNAs decreased, while the levels of R-loops produced by linear RNAs with similar binding sites increased. Linear RNAs with only one R-loop site could not promote chromatin loop formation. Higher levels of R-loops and lower levels of chromatin loops in the CRM1 regions of RNAi plants led to a reduced localization of the centromeric H3 variant (CENH3). Our work reveals centromeric chromatin organization by circular CRM1 RNAs via R-loops and chromatin loops, which suggested that CRM1 elements might help build a suitable chromatin environment during centromere evolution. These results highlight that R-loops are integral components of centromeric chromatin and proper centromere structure is essential for CENH3 localization., Competing Interests: The authors have declared that no competing interests exist.

Transposable elements in crop plants are the powerful drivers of phenotypic variation that has been selected during domestication and breeding programs. In tomato, transpositions of the LTR (long terminal repeat) retrotransposon family Rider have contributed to various phenotypes of agronomical interest, such as fruit shape and colour. However, the mechanisms regulating Rider activity are largely unknown. We have developed a bioinformatics pipeline for the functional annotation of retrotransposons containing LTRs and defined all full-length Rider elements in the tomato genome. Subsequently, we showed that accumulation of Rider transcripts and transposition intermediates in the form of extrachromosomal DNA is triggered by drought stress and relies on abscisic acid signalling. We provide evidence that residual activity of Rider is controlled by epigenetic mechanisms involving siRNAs and the RNA-dependent DNA methylation pathway. Finally, we demonstrate the broad distribution of Rider-like elements in other plant species, including crops. Our work identifies Rider as an environment-responsive element and a potential source of genetic and epigenetic variation in plants., Competing Interests: The authors have declared that no competing interests exist.

Long terminal repeat retrotransposons (LTR-RTs) in plant genomes differ in abundance, structure and genomic distribution, reflecting the large number of evolutionary lineages. Elements within lineages can be considered populations, in which each element is an individual in its genomic environment. In this way, it would be reasonable to apply microevolutionary analyses to understand transposable element (TE) evolution, such as those used to study the genetic structure of natural populations. Here, we applied a Bayesian method to infer genetic structure of populations together with classical phylogenetic and dating tools to analyze LTR-RT evolution using the monocot Setaria italica as a model species. In contrast to a phylogeny, the Bayesian clusterization method identifies populations by assigning individuals to one or more clusters according to the most probabilistic scenario of admixture, based on genetic diversity patterns. In this work, each LTR-RT insertion was considered to be one individual and each LTR-RT lineage was considered to be a single species. Nine evolutionary lineages of LTR-RTs were identified in the S. italica genome that had different genetic structures with variable numbers of clusters and levels of admixture. Comprehensive analysis of the phylogenetic, clusterization and time of insertion data allowed us to hypothesize that admixed elements represent sequences that harbor ancestral polymorphic sequence signatures. In conclusion, application of microevolutionary concepts in genome evolution studies is suitable as a complementary approach to phylogenetic analyses to address the evolutionary history and functional features of TEs., Competing Interests: The authors have declared that no competing interests exist.

Evidence is rapidly mounting that transposable element (TE) expression and replication may impact biology more widely than previously thought. This includes potential effects on normal physiology of somatic tissues and dysfunctional impacts in diseases associated with aging, such as cancer and neurodegeneration. Investigation of the biological impact of mobile elements in somatic cells will be greatly facilitated by the use of donor elements that are engineered to report de novo events in vivo. In multicellular organisms, reporter constructs demonstrating engineered long interspersed nuclear element (LINE-1; L1) mobilization have been in use for quite some time, and strategies similar to L1 retrotransposition reporter assays have been developed to report replication of Ty1 elements in yeast and mouse intracisternal A particle (IAP) long terminal repeat (LTR) retrotransposons in cultivated cells. We describe a novel approach termed cellular labeling of endogenous retrovirus replication (CLEVR), which reports replication of the gypsy element within specific cells in vivo in Drosophila. The gypsy-CLEVR reporter reveals gypsy replication both in cell culture and in individual neurons and glial cells of the aging adult fly. We also demonstrate that the gypsy-CLEVR replication rate is increased when the short interfering RNA (siRNA) silencing system is genetically disrupted. This CLEVR strategy makes use of universally conserved features of retroviruses and should be widely applicable to other LTR retrotransposons, endogenous retroviruses (ERVs), and exogenous retroviruses., Competing Interests: The authors have declared that no competing interests exist.

Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are among the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in our understanding of their functional organization and evolution. Here, we identify the sequence composition and organization of the centromeres of Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, we demonstrate that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. We discover that a specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species D. simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. Our work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA., Competing Interests: The authors have declared that no competing interests exist.

Retrotransposon expression during arbuscular mycorrhizal (AM) fungal colonisation of sunflower roots (Helianthus annuus) was analysed using Illumina RNA-Seq, in order to verify whether mycorrhizal symbiosis can activate retrotransposable elements. Illumina cDNA libraries were produced from RNAs isolated from the roots of sunflower plants at 4 and 16 days after inoculation with the AM fungus Rhizoglomus irregulare and from their respective control plants. Illumina reads were mapped to a library of reverse transcriptase-encoding sequences, putatively belonging to long terminal repeat retrotransposons of Gypsy and Copia superfamilies. Forty-six different reverse transcriptase sequences were transcribed, although at a low rate, in mycorrhizal or control roots and only four were significantly over-expressed at day 16, compared with control roots. Almost all expressed or over-expressed sequences belonged to low-copy elements, mostly, of the Copia superfamily. A meta-analysis, using publicly available Illumina cDNA libraries obtained from sunflower plants treated with different hormones and chemicals, mimicking stimuli produced by abiotic and biotic stresses, was also conducted. Such analyses indicated that the four reverse transcriptase sequences over-expressed in mycorrhizal roots were explicitly induced only by AM symbiosis, showing the specificity of AM stimuli compared to that of other fungal/plant interactions., Competing Interests: The authors have declared that no competing interests exist.

Diversity-generating retroelements (DGRs) are widely distributed in bacteria, archaea, and microbial viruses, and bring about unparalleled levels of sequence variation in target proteins. While DGR variable proteins share low sequence identity, the structures of several such proteins have revealed the C-type lectin (CLec)-fold as a conserved scaffold for accommodating massive sequence variation. This conservation has led to the suggestion that the CLec-fold may be useful in molecular surface display applications. Thermostability is an attractive feature in such applications, and thus we studied the variable protein of a DGR encoded by a prophage of the thermophile Thermus aquaticus. We report here the 2.8 Å resolution crystal structure of the variable protein from the T. aquaticus DGR, called TaqVP, and confirm that it has a CLec-fold. Remarkably, its variable region is nearly identical in structure to those of several other CLec-fold DGR variable proteins despite low sequence identity among these. TaqVP was found to be thermostable, which appears to be a property shared by several CLec-fold DGR variable proteins. These results provide impetus for the pursuit of the DGR variable protein CLec-fold in molecular display applications., Competing Interests: The authors have declared that no competing interests exist.

The sequence drafts of wild emmer and bread wheat facilitated high resolution, genome-wide analysis of transposable elements (TEs), which account for up to 90% of the wheat genome. Despite extensive studies, the role of TEs in reshaping nascent polyploid genomes remains to be fully understood. In this study, we retrieved miniature inverted-repeat transposable elements (MITEs) from the recently published genome drafts of Triticum aestivum, Triticum turgidum ssp. dicoccoides, Aegilops tauschii and the available genome draft of Triticum urartu. Overall, 239,126 MITE insertions were retrieved, including 3,874 insertions of a newly identified, wheat-unique MITE family that we named "Inbar". The Stowaway superfamily accounts for ~80% of the retrieved MITE insertions, while Thalos is the most abundant family. MITE insertions are distributed in the seven homologous chromosomes of the wild emmer and bread wheat genomes. The remarkably high level of insertions in the B sub-genome (~59% of total retrieved MITE insertions in the wild emmer genome draft, and ~41% in the bread wheat genome draft), emphasize its highly repetitive nature. Nearly 52% of all MITE insertions were found within or close (less than 100bp) to coding genes, and ~400 MITE sequences were found in the bread wheat transcriptome, indicating that MITEs might have a strong impact on wheat genome expression. In addition, ~40% of MITE insertions were found within TE sequences, and remarkably, ~90% of Inbar insertions were located in retrotransposon sequences. Our data thus shed new light on the role of MITEs in the diversification of allopolyploid wheat species., Competing Interests: The authors have declared that no competing interests exist.

Pregnancy and parturition are intricately regulated to ensure successful reproductive outcomes. However, the factors that control gestational length in humans and other anthropoid primates remain poorly defined. Here, we show the endogenous retroviral long terminal repeat transposon-like human element 1B (THE1B) selectively controls placental expression of corticotropin-releasing hormone (CRH) that, in turn, influences gestational length and birth timing. Placental expression of CRH and subsequently prolonged gestational length were found in two independent strains of transgenic mice carrying a 180-kb human bacterial artificial chromosome (BAC) DNA that contained the full length of CRH and extended flanking regions, including THE1B. Restricted deletion of THE1B silenced placental CRH expression and normalized birth timing in these transgenic lines. Furthermore, we revealed an interaction at the 5' insertion site of THE1B with distal-less homeobox 3 (DLX3), a transcription factor expressed in placenta. Together, these findings suggest that retroviral insertion of THE1B into the anthropoid primate genome may have initiated expression of CRH in placental syncytiotrophoblasts via DLX3 and that this placental CRH is sufficient to alter the timing of birth., Competing Interests: The authors have declared that no competing interests exist.

Yeasts serve as hosts to several types of genetic parasites. Few studies have addressed the evolutionary trajectory of yeast genes that control the stable co-existence of these parasites with their host cell. In Saccharomyces yeasts, the retrovirus-like Ty retrotransposons must access the nucleus. We show that several genes encoding components of the yeast nuclear pore complex have experienced natural selection for substitutions that change the encoded protein sequence. By replacing these S. cerevisiae genes with orthologs from other Saccharomyces species, we discovered that natural sequence changes have affected the mobility of Ty retrotransposons. Specifically, changing the genetic sequence of NUP84 or NUP82 to match that of other Saccharomyces species alters the mobility of S. cerevisiae Ty1 and Ty3. Importantly, all tested housekeeping functions of NUP84 and NUP82 remained equivalent across species. Signatures of natural selection, resulting in altered interactions with viruses and parasitic genetic elements, are common in host defense proteins. Yet, few instances have been documented in essential housekeeping proteins. The nuclear pore complex is the gatekeeper of the nucleus. This study shows how the evolution of this large, ubiquitous eukaryotic complex can alter the replication of a molecular parasite, but concurrently maintain essential host functionalities regarding nucleocytoplasmic trafficking.

Human Long interspersed element-1 (L1) retrotransposons contain an internal RNA polymerase II promoter within their 5' untranslated region (UTR) and encode two proteins, (ORF1p and ORF2p) required for their mobilization (i.e., retrotransposition). The evolutionary success of L1 relies on the continuous retrotransposition of full-length L1 mRNAs. Previous studies identified functional splice donor (SD), splice acceptor (SA), and polyadenylation sequences in L1 mRNA and provided evidence that a small number of spliced L1 mRNAs retrotransposed in the human genome. Here, we demonstrate that the retrotransposition of intra-5'UTR or 5'UTR/ORF1 spliced L1 mRNAs leads to the generation of spliced integrated retrotransposed elements (SpIREs). We identified a new intra-5'UTR SpIRE that is ten times more abundant than previously identified SpIREs. Functional analyses demonstrated that both intra-5'UTR and 5'UTR/ORF1 SpIREs lack Cis-acting transcription factor binding sites and exhibit reduced promoter activity. The 5'UTR/ORF1 SpIREs also produce nonfunctional ORF1p variants. Finally, we demonstrate that sequence changes within the L1 5'UTR over evolutionary time, which permitted L1 to evade the repressive effects of a host protein, can lead to the generation of new L1 splicing events, which, upon retrotransposition, generates a new SpIRE subfamily. We conclude that splicing inhibits L1 retrotransposition, SpIREs generally represent evolutionary "dead-ends" in the L1 retrotransposition process, mutations within the L1 5'UTR alter L1 splicing dynamics, and that retrotransposition of the resultant spliced transcripts can generate interindividual genomic variation.

The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting non-essential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility do not appear to effect genomic Ty1 RNA levels, Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.

Retroviruses and Long Terminal Repeat (LTR)-retrotransposons have distinct patterns of integration sites. The oncogenic potential of retrovirus-based vectors used in gene therapy is dependent on the selection of integration sites associated with promoters. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is studied as a model for oncogenic retroviruses because it integrates into the promoters of stress response genes. Although integrases (INs) encoded by retroviruses and LTR-retrotransposons are responsible for catalyzing the insertion of cDNA into the host genome, it is thought that distinct host factors are required for the efficiency and specificity of integration. We tested this hypothesis with a genome-wide screen of host factors that promote Tf1 integration. By combining an assay for transposition with a genetic assay that measures cDNA recombination we could identify factors that contribute differentially to integration. We utilized this assay to test a collection of 3,004 S. pombe strains with single gene deletions. Using these screens and immunoblot measures of Tf1 proteins, we identified a total of 61 genes that promote integration. The candidate integration factors participate in a range of processes including nuclear transport, transcription, mRNA processing, vesicle transport, chromatin structure and DNA repair. Two candidates, Rhp18 and the NineTeen complex were tested in two-hybrid assays and were found to interact with Tf1 IN. Surprisingly, a number of pathways we identified were found previously to promote integration of the LTR-retrotransposons Ty1 and Ty3 in Saccharomyces cerevisiae, indicating the contribution of host factors to integration are common in distantly related organisms. The DNA repair factors are of particular interest because they may identify the pathways that repair the single stranded gaps flanking the sites of strand transfer following integration of LTR retroelements.

LINE-1 (L1) retrotransposons can mobilize (retrotranspose) within the human genome, and mutagenic de novo L1 insertions can lead to human diseases, including cancers. As a result, cells are actively engaged in preventing L1 retrotransposition. This work reveals that the human Condensin II complex restricts L1 retrotransposition in both non-transformed and transformed cell lines through inhibition of L1 transcription and translation. Condensin II subunits, CAP-D3 and CAP-H2, interact with members of the Gamma-Interferon Activated Inhibitor of Translation (GAIT) complex including the glutamyl-prolyl-tRNA synthetase (EPRS), the ribosomal protein L13a, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and NS1 associated protein 1 (NSAP1). GAIT has been shown to inhibit translation of mRNAs encoding inflammatory proteins in myeloid cells by preventing the binding of the translation initiation complex, in response to Interferon gamma (IFN-γ). Excitingly, our data show that Condensin II promotes complexation of GAIT subunits. Furthermore, RNA-Immunoprecipitation experiments in epithelial cells demonstrate that Condensin II and GAIT subunits associate with L1 RNA in a co-dependent manner, independent of IFN-γ. These findings suggest that cooperation between the Condensin II and GAIT complexes may facilitate a novel mechanism of L1 repression, thus contributing to the maintenance of genome stability in somatic cells.

Transcriptional silencing by heritable cytosine-5 methylation is an ancient strategy to repress transposable elements. It was previously thought that mammals possess four DNA methyltransferase paralogs-Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l-that establish and maintain cytosine-5 methylation. Here we identify a fifth paralog, Dnmt3c, that is essential for retrotransposon methylation and repression in the mouse male germline. From a phenotype-based forward genetics screen, we isolated a mutant mouse called 'rahu', which displays severe defects in double-strand-break repair and homologous chromosome synapsis during male meiosis, resulting in sterility. rahu is an allele of a transcription unit (Gm14490, renamed Dnmt3c) that was previously mis-annotated as a Dnmt3-family pseudogene. Dnmt3c encodes a cytosine methyltransferase homolog, and Dnmt3crahu mutants harbor a non-synonymous mutation of a conserved residue within one of its cytosine methyltransferase motifs, similar to a mutation in human DNMT3B observed in patients with immunodeficiency, centromeric instability and facial anomalies syndrome. The rahu mutation lies at a potential dimerization interface and near the potential DNA binding interface, suggesting that it compromises protein-protein and/or protein-DNA interactions required for normal DNMT3C function. Dnmt3crahu mutant males fail to establish normal methylation within LINE and LTR retrotransposon sequences in the germline and accumulate higher levels of transposon-derived transcripts and proteins, particularly from distinct L1 and ERVK retrotransposon families. Phylogenetic analysis indicates that Dnmt3c arose during rodent evolution by tandem duplication of Dnmt3b, after the divergence of the Dipodoidea and Muroidea superfamilies. These findings provide insight into the evolutionary dynamics and functional specialization of the transposon suppression machinery critical for mammalian sexual reproduction and epigenetic regulation.

Mammalian genomes harbor millions of retrotransposon copies, some of which are transpositionally active. In mouse prospermatogonia, PIWI-interacting small RNAs (piRNAs) combat retrotransposon activity to maintain the genomic integrity. The piRNA system destroys retrotransposon-derived RNAs and guides de novo DNA methylation at some retrotransposon promoters. However, it remains unclear whether DNA methylation contributes to retrotransposon silencing in prospermatogonia. We have performed comprehensive studies of DNA methylation and polyA(+) RNAs (transcriptome) in developing male germ cells from Pld6/Mitopld and Dnmt3l knockout mice, which are defective in piRNA biogenesis and de novo DNA methylation, respectively. The Dnmt3l mutation greatly reduced DNA methylation levels at most retrotransposons, but its impact on their RNA abundance was limited in prospermatogonia. In Pld6 mutant germ cells, although only a few retrotransposons exhibited reduced DNA methylation, many showed increased expression at the RNA level. More detailed analysis of RNA sequencing, nascent RNA quantification, profiling of cleaved RNA ends, and the results obtained from double knockout mice suggest that PLD6 works mainly at the posttranscriptional level. The increase in retrotransposon expression was larger in Pld6 mutants than it was in Dnmt3l mutants, suggesting that RNA degradation by the piRNA system plays a more important role than does DNA methylation in prospermatogonia. However, DNA methylation had a long-term effect: hypomethylation caused by the Pld6 or Dnmt3l mutation resulted in increased retrotransposon expression in meiotic spermatocytes. Thus, posttranscriptional silencing plays an important role in the early stage of germ cell development, then transcriptional silencing becomes important in later stages. In addition, intergenic and intronic retrotransposon sequences, in particular those containing the antisense L1 promoters, drove ectopic expression of nearby genes in both mutant spermatocytes, suggesting that retrotransposon silencing is important for the maintenance of not only genomic integrity but also transcriptomic integrity.

Retroduplications come from reverse transcription of mRNAs and their insertion back into the genome. Here, we performed comprehensive discovery and analysis of retroduplications in a large cohort of 2,535 individuals from 26 human populations, as part of 1000 Genomes Phase 3. We developed an integrated approach to discover novel retroduplications combining high-coverage exome and low-coverage whole-genome sequencing data, utilizing information from both exon-exon junctions and discordant paired-end reads. We found 503 parent genes having novel retroduplications absent from the reference genome. Based solely on retroduplication variation, we built phylogenetic trees of human populations; these represent superpopulation structure well and indicate that variable retroduplications are effective population markers. We further identified 43 retroduplication parent genes differentiating superpopulations. This group contains several interesting insertion events, including a SLMO2 retroduplication and insertion into CAV3, which has a potential disease association. We also found retroduplications to be associated with a variety of genomic features: (1) Insertion sites were correlated with regular nucleosome positioning. (2) They, predictably, tend to avoid conserved functional regions, such as exons, but, somewhat surprisingly, also avoid introns. (3) Retroduplications tend to be co-inserted with young L1 elements, indicating recent retrotranspositional activity, and (4) they have a weak tendency to originate from highly expressed parent genes. Our investigation provides insight into the functional impact and association with genomic elements of retroduplications. We anticipate our approach and analytical methodology to have application in a more clinical context, where exome sequencing data is abundant and the discovery of retroduplications can potentially improve the accuracy of SNP calling.

The amoeba Dictyostelium discoideum has a haploid genome in which two thirds of the DNA encodes proteins. Consequently, the space available for selfish mobile elements to expand without excess damage to the host genome is limited. The non-long terminal repeat retrotransposon TRE5-A maintains an active population in the D. discoideum genome and apparently adapted to this gene-dense environment by targeting positions ~47 bp upstream of tRNA genes that are devoid of protein-coding regions. Because only ~24% of tRNA genes are associated with a TRE5-A element in the reference genome, we evaluated whether TRE5-A retrotransposition is limited to this subset of tRNA genes. We determined that a tagged TRE5-A element (TRE5-Absr) integrated at 384 of 405 tRNA genes, suggesting that expansion of the current natural TRE5-A population is not limited by the availability of targets. We further observed that TRE5-Absr targets the ribosomal 5S gene on the multicopy extrachromosomal DNA element that carries the ribosomal RNA genes, indicating that TRE5-A integration may extend to the entire RNA polymerase III (Pol III) transcriptome. We determined that both natural TRE5-A and cloned TRE5-Absr retrotranspose to locations on the extrachromosomal rDNA element that contain tRNA gene-typical A/B box promoter motifs without displaying any other tRNA gene context. Based on previous data suggesting that TRE5-A targets tRNA genes by locating Pol III transcription complexes, we propose that A/B box loci reflect Pol III transcription complex assembly sites that possess a function in the biology of the extrachromosomal rDNA element.

The AID / APOBEC genes are a family of cytidine deaminases that have evolved in vertebrates, and particularly mammals, to mutate RNA and DNA at distinct preferred nucleotide contexts (or "hotspots") on foreign genomes such as viruses and retrotransposons. These enzymes play a pivotal role in intrinsic immunity defense mechanisms, often deleteriously mutating invading retroviruses or retrotransposons and, in the case of AID, changing antibody sequences to drive affinity maturation. We investigate the strength of various hotspots on their known biological targets by evaluating the potential impact of mutations on the DNA coding sequences of these targets, and compare these results to hypothetical hotspots that did not evolve. We find that the existing AID / APOBEC hotspots have a large impact on retrotransposons and non-mammalian viruses while having a much smaller effect on vital mammalian genes, suggesting co-evolution with AID / APOBECs may have had an impact on the genomes of the viruses we analyzed. We determine that GC content appears to be a significant, but not sole, factor in resistance to deaminase activity. We discuss possible mechanisms AID and APOBEC viral targets have adopted to escape the impacts of deamination activity, including changing the GC content of the genome.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD.

Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA) forms of active retrotransposons to characterize the repertoire of mobile retrotransposons in plants. This method successfully identified known active retrotransposons in both Arabidopsis and rice material where the epigenome is destabilized. When applying mobilome-seq to developmental stages in wild type rice, we identified PopRice as a highly active retrotransposon producing eccDNA forms in the wild type endosperm. The mobilome-seq strategy opens new routes for the characterization of a yet unexplored fraction of plant genomes.

Promoters are structurally and functionally diverse gene regulatory regions. The presence or absence of sequence motifs and the spacing between the motifs defines the properties of promoters. Recent alternative promoter usage analyses in Drosophila melanogaster revealed that transposable elements significantly contribute to promote diversity. In this work, we analyzed in detail one of the transposable element insertions, named FBti0019985, that has been co-opted to drive expression of CG18446, a candidate stress response gene. We analyzed strains from different natural populations and we found that besides FBti0019985, there are another eight independent transposable elements inserted in the proximal promoter region of CG18446. All nine insertions are solo-LTRs that belong to the roo family. We analyzed the sequence of the nine roo insertions and we investigated whether the different insertions were functionally equivalent by performing 5'-RACE, gene expression, and cold-stress survival experiments. We found that different insertions have different molecular and functional consequences. The exact position where the transposable elements are inserted matters, as they all showed highly conserved sequences but only two of the analyzed insertions provided alternative transcription start sites, and only the FBti0019985 insertion consistently affects CG18446 expression. The phenotypic consequences of the different insertions also vary: only FBti0019985 was associated with cold-stress tolerance. Interestingly, the only previous report of transposable elements inserting repeatedly and independently in a promoter region in D. melanogaster, were also located upstream of a stress response gene. Our results suggest that functional validation of individual structural variants is needed to resolve the complexity of insertion clusters.

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs., Competing Interests: The authors have declared that no competing interests exist.

Transposable elements (TEs) are exceptional contributors to eukaryotic genome diversity. Their ubiquitous presence impacts the genomes of nearly all species and mediates genome evolution by causing mutations and chromosomal rearrangements and by modulating gene expression. We performed an exhaustive analysis of the TE content in 18 fungal genomes, including strains of the same species and species of the same genera. Our results depicted a scenario of exceptional variability, with species having 0.02 to 29.8% of their genome consisting of transposable elements. A detailed analysis performed on two strains of Pleurotus ostreatus uncovered a genome that is populated mainly by Class I elements, especially LTR-retrotransposons amplified in recent bursts from 0 to 2 million years (My) ago. The preferential accumulation of TEs in clusters led to the presence of genomic regions that lacked intra- and inter-specific conservation. In addition, we investigated the effect of TE insertions on the expression of their nearby upstream and downstream genes. Our results showed that an important number of genes under TE influence are significantly repressed, with stronger repression when genes are localized within transposon clusters. Our transcriptional analysis performed in four additional fungal models revealed that this TE-mediated silencing was present only in species with active cytosine methylation machinery. We hypothesize that this phenomenon is related to epigenetic defense mechanisms that are aimed to suppress TE expression and control their proliferation.