Meeting report: transposable elements at the crossroads of evolution, health and disease 2023.

The conference "Transposable Elements at the Crossroads of Evolution, Health and Disease" was hosted by Keystone Symposia in Whistler, British Columbia, Canada, on September 3-6, 2023, and was organized by Kathleen Burns, Harmit Malik and Irina Arkhipova. The central theme of the meeting was the incredible diversity of ways in which transposable elements (TEs) interact with the host, from disrupting the existing genes and pathways to creating novel gene products and expression patterns, enhancing the repertoire of host functions, and ultimately driving host evolution. The meeting was organized into six plenary sessions and two afternoon workshops with a total of 50 invited and contributed talks, two poster sessions, and a career roundtable. The topics ranged from TE roles in normal and pathological processes to restricting and harnessing TE activity based on mechanistic insights gained from genetic, structural, and biochemical studies.

Retrotransposon insertions associated with risk of neurologic and psychiatric diseases.

Transposable elements (TEs) are active in neuronal cells raising the question whether TE insertions contribute to risk of neuropsychiatric disease. While genome-wide association studies (GWAS) serve as a tool to discover genetic loci associated with neuropsychiatric diseases, unfortunately GWAS do not directly detect structural variants such as TEs. To examine the role of TEs in psychiatric and neurologic disease, we evaluated 17,000 polymorphic TEs and find 76 are in linkage disequilibrium with disease haplotypes (P < 10-6 ) defined by GWAS. From these 76 polymorphic TEs, we identify potentially causal candidates based on having insertions in genomic regions of regulatory chromatin and on having associations with altered gene expression in brain tissues. We show that lead candidate insertions have regulatory effects on gene expression in human neural stem cells altering the activity of a minimal promoter. Taken together, we identify 10 polymorphic TE insertions that are potential candidates on par with other variants for having a causal role in neurologic and psychiatric disorders.

Identification of an integrase-independent pathway of retrotransposition.

Retroviruses and long terminal repeat retrotransposons rely on integrase (IN) to insert their complementary DNA (cDNA) into the genome of host cells. Nevertheless, in the absence of IN, retroelements can retain notable levels of insertion activity. We have characterized the IN-independent pathway of Tf1 and found that insertion sites had homology to the primers of reverse transcription, which form single-stranded DNAs at the termini of the cDNA. In the absence of IN activity, a similar bias was observed with HIV-1. Our studies showed that the Tf1 insertions result from single-strand annealing, a noncanonical form of homologous recombination mediated by Rad52. By expanding our analysis of insertions to include repeat sequences, we found most formed tandem elements by inserting at preexisting copies of a related transposable element. Unexpectedly, we found that wild-type Tf1 uses the IN-independent pathway as an alternative mode of insertion.

A protocol for transposon insertion sequencing in Schizosaccharomyces pombe to identify factors that maintain heterochromatin.

Transposon insertion sequencing (TIS) is a highly effective method used with bacteria to identify genes important for growth in any condition of interest. Previously, we adapted this method to identify essential genes of the yeast Schizosaccharomyces pombe. Here, we describe modifications used to identify genes necessary for the formation of centromeric heterochromatin. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020).

Transposon Insertion Sequencing, a Global Measure of Gene Function.

The goal of genomics and systems biology is to understand how complex systems of factors assemble into pathways and structures that combine to form living organisms. Great advances in understanding biological processes result from determining the function of individual genes, a process that has classically relied on characterizing single mutations. Advances in DNA sequencing has made available the complete set of genetic instructions for an astonishing and growing number of species. To understand the function of this ever-increasing number of genes, a high-throughput method was developed that in a single experiment can measure the function of genes across the genome of an organism. This occurred approximately 10 years ago, when high-throughput DNA sequencing was combined with advances in transposon-mediated mutagenesis in a method termed transposon insertion sequencing (TIS). In the subsequent years, TIS succeeded in addressing fundamental questions regarding the genes of bacteria, many of which have been shown to play central roles in bacterial infections that result in major human diseases. The field of TIS has matured and resulted in studies of hundreds of species that include significant innovations with a number of transposons. Here, we summarize a number of TIS experiments to provide an understanding of the method and explanation of approaches that are instructive when designing a study. Importantly, we emphasize critical aspects of a TIS experiment and highlight the extension and applicability of TIS into nonbacterial species such as yeast.

Dense Transposon Integration Reveals Essential Cleavage and Polyadenylation Factors Promote Heterochromatin Formation.

Heterochromatin functions as a scaffold for factors responsible for gene silencing and chromosome segregation. Heterochromatin can be assembled by multiple pathways, including RNAi and RNA surveillance. We identified factors that form heterochromatin using dense profiles of transposable element integration in Schizosaccharomyces pombe. The candidates include a large number of essential proteins such as four canonical mRNA cleavage and polyadenylation factors. We find that Iss1, a subunit of the poly(A) polymerase module, plays a role in forming heterochromatin in centromere repeats that is independent of RNAi. Genome-wide maps reveal that Iss1 accumulates at genes regulated by RNA surveillance. Iss1 interacts with RNA surveillance factors Mmi1 and Rrp6, and importantly, Iss1 contributes to RNA elimination that forms heterochromatin at meiosis genes. Our profile of transposable element integration supports the model that a network of mRNA cleavage and polyadenylation factors coordinates RNA surveillance, including the mechanism that forms heterochromatin at meiotic genes.

Fitness Landscape of the Fission Yeast Genome.

The relationship between DNA sequence, biochemical function, and molecular evolution is relatively well-described for protein-coding regions of genomes, but far less clear in noncoding regions, particularly, in eukaryote genomes. In part, this is because we lack a complete description of the essential noncoding elements in a eukaryote genome. To contribute to this challenge, we used saturating transposon mutagenesis to interrogate the Schizosaccharomyces pombe genome. We generated 31 million transposon insertions, a theoretical coverage of 2.4 insertions per genomic site. We applied a five-state hidden Markov model (HMM) to distinguish insertion-depleted regions from insertion biases. Both raw insertion-density and HMM-defined fitness estimates showed significant quantitative relationships to gene knockout fitness, genetic diversity, divergence, and expected functional regions based on transcription and gene annotations. Through several analyses, we conclude that transposon insertions produced fitness effects in 66-90% of the genome, including substantial portions of the noncoding regions. Based on the HMM, we estimate that 10% of the insertion depleted sites in the genome showed no signal of conservation between species and were weakly transcribed, demonstrating limitations of comparative genomics and transcriptomics to detect functional units. In this species, 3'- and 5'-untranslated regions were the most prominent insertion-depleted regions that were not represented in measures of constraint from comparative genomics. We conclude that the combination of transposon mutagenesis, evolutionary, and biochemical data can provide new insights into the relationship between genome function and molecular evolution.

Transposable element insertions in fission yeast drive adaptation to environmental stress.

Cells are regularly exposed to a range of naturally occurring stress that can restrict growth or cause lethality. In response, cells activate expression networks with hundreds of genes that together increase resistance to common environmental insults. However, stress response networks can be insufficient to ensure survival, which raises the question of whether cells possess genetic programs that can promote adaptation to novel forms of stress. We found transposable element (TE) mobility in Schizosaccharomyces pombe was greatly increased when cells were exposed to unusual forms of stress such as heavy metals, caffeine, and the plasticizer phthalate. By subjecting TE-tagged cells to CoCl2, we found the TE integration provided the major path to resistance. Groups of insertions that provided resistance were linked to TOR regulation and metal response genes. We extended our study of adaptation by analyzing TE positions in 57 genetically distinct wild strains. The genomic positions of 1048 polymorphic LTRs were strongly associated with a range of stress response genes, indicating TE integration promotes adaptation in natural conditions. These data provide strong support for the idea, first proposed by Barbara McClintock, that TEs provide a system to modify the genome in response to stress.

Ten things you should know about transposable elements.

Transposable elements (TEs) are major components of eukaryotic genomes. However, the extent of their impact on genome evolution, function, and disease remain a matter of intense interrogation. The rise of genomics and large-scale functional assays has shed new light on the multi-faceted activities of TEs and implies that they should no longer be marginalized. Here, we introduce the fundamental properties of TEs and their complex interactions with their cellular environment, which are crucial to understanding their impact and manifold consequences for organismal biology. While we draw examples primarily from mammalian systems, the core concepts outlined here are relevant to a broad range of organisms.

Meeting report: mobile genetic elements and genome plasticity 2018.

The Mobile Genetic Elements and Genome Plasticity conference was hosted by Keystone Symposia in Santa Fe, NM USA, February 11-15, 2018. The organizers were Marlene Belfort, Evan Eichler, Henry Levin and Lynn Maquat. The goal of this conference was to bring together scientists from around the world to discuss the function of transposable elements and their impact on host species. Central themes of the meeting included recent innovations in genome analysis and the role of mobile DNA in disease and evolution. The conference included 200 scientists who participated in poster presentations, short talks selected from abstracts, and invited talks. A total of 58 talks were organized into eight sessions and two workshops. The topics varied from mechanisms of mobilization, to the structure of genomes and their defense strategies to protect against transposable elements.

High-Frequency Lithium Acetate Transformation of Schizosaccharomyces pombe.

The introduction of ectopic DNA, such as plasmids, into yeast cells has for decades been a critical protocol for the study of this eukaryotic model system. We describe here an efficient transformation procedure for use in the fission yeast Schizosaccharomyces pombe. This method relies on chemical agents (lithium acetate, and polyethylene glycol) and temperature stresses, which ultimately facilitate transfer of the genetic material through the cell wall and plasma membrane without significant impact on the transferred DNA or the recipient cell. Using this protocol, we consistently see transformation efficiencies between 1.0 × 103 and 1.0 × 104 transformants per microgram of the plasmid with 108 S. pombe cells. The principal benefits and advantages of this method are its simplicity, efficiency, and relative speed of completion.

Duplication and Transformation of the Schizosaccharomyces pombe Collection of Deletion Strains.

We present an efficient and organized method of lithium acetate and polyethylene glycol-based transformation of plasmid DNA into the commercially available collection of Schizosaccharomyces pombe with single-gene deletions. We also describe how to prepare a duplicate collection of the deletion strains in order to preserve the longevity of the master set. These protocols are adapted to the 96-well format of the 3004 strains of the Version 2.0 Bioneer set but can also be used for later releases of the collection. This transformation method typically yields efficiencies in the range between 1.0 × 103 and 1.0 × 104 transformants per microgram of plasmid DNA. However, some deletion strains transformed with significantly lower efficiencies. We provide a list of these difficult-to-transform strains. Applications for this methodology include the transformation of the deletion set with plasmids necessary for genetic screens.

Host factors that promote retrotransposon integration are similar in distantly related eukaryotes.

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.

Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes.

The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced.

A long terminal repeat retrotransposon of Schizosaccharomyces japonicus integrates upstream of RNA pol III transcribed genes.

BACKGROUND: Transposable elements (TEs) are common constituents of centromeres. However, it is not known what causes this relationship. Schizosaccharomyces japonicus contains 10 families of Long Terminal Repeat (LTR)-retrotransposons and these elements cluster in centromeres and telomeres. In the related yeast, Schizosaccharomyces pombe LTR-retrotransposons Tf1 and Tf2 are distributed in the promoter regions of RNA pol II transcribed genes. Sequence analysis of TEs indicates that Tj1 of S. japonicus is related to Tf1 and Tf2, and uses the same mechanism of self-primed reverse transcription. Thus, we wondered why these related retrotransposons localized in different regions of the genome. RESULTS: To characterize the integration behavior of Tj1 we expressed it in S. pombe. We found Tj1 was active and capable of generating de novo integration in the chromosomes of S. pombe. The expression of Tj1 is similar to Type C retroviruses in that a stop codon at the end of Gag must be present for efficient integration. 17 inserts were sequenced, 13 occurred within 12 bp upstream of tRNA genes and 3 occurred at other RNA pol III transcribed genes. The link between Tj1 integration and RNA pol III transcription is reminiscent of Ty3, an LTR-retrotransposon of Saccharomyces cerevisiae that interacts with TFIIIB and integrates upstream of tRNA genes. CONCLUSION: The integration of Tj1 upstream of tRNA genes and the centromeric clustering of tRNA genes in S. japonicus demonstrate that the clustering of this TE in centromere sequences is due to a unique pattern of integration.

The Long Terminal Repeat Retrotransposons Tf1 and Tf2 of Schizosaccharomyces pombe.

The long terminal repeat (LTR) retrotransposons Tf1 and Tf2 of Schizosaccharomyces pombe are active mobile elements of the Ty3/gypsy family. The mobilization of these retrotransposons depends on particle formation, reverse transcription and integration, processes typical of other LTR retrotransposons. However, Tf1 and Tf2 are distinct from other LTR elements in that they assemble virus-like particles from a single primary translation product, initiate reverse transcription with an unusual self-priming mechanism, and, in the case of Tf1, integrate with a pattern that favors specific promoters of RNA pol II-transcribed genes. To avoid the chromosome instability and genome damage that results from increased copy number, S. pombe applies a variety of defense mechanisms that restrict Tf1 and Tf2 activity. The mRNA of the Tf elements is eliminated by an exosome-based pathway when cells are in favorable conditions whereas nutrient deprivation triggers an RNA interference-dependent pathway that results in the heterochromatization of the elements. Interestingly, Tf1 integrates into the promoters of stress-induced genes and these insertions are capable of increasing the expression of adjacent genes. These properties of Tf1 transposition raise the possibility that Tf1 benefits cells with specific insertions by providing resistance to environmental stress.

Single-Nucleotide-Specific Targeting of the Tf1 Retrotransposon Promoted by the DNA-Binding Protein Sap1 of Schizosaccharomyces pombe.

Transposable elements (TEs) constitute a substantial fraction of the eukaryotic genome and, as a result, have a complex relationship with their host that is both adversarial and dependent. To minimize damage to cellular genes, TEs possess mechanisms that target integration to sequences of low importance. However, the retrotransposon Tf1 of Schizosaccharomyces pombe integrates with a surprising bias for promoter sequences of stress-response genes. The clustering of integration in specific promoters suggests that Tf1 possesses a targeting mechanism that is important for evolutionary adaptation to changes in environment. We report here that Sap1, an essential DNA-binding protein, plays an important role in Tf1 integration. A mutation in Sap1 resulted in a 10-fold drop in Tf1 transposition, and measures of transposon intermediates support the argument that the defect occurred in the process of integration. Published ChIP-Seq data on Sap1 binding combined with high-density maps of Tf1 integration that measure independent insertions at single-nucleotide positions show that 73.4% of all integration occurs at genomic sequences bound by Sap1. This represents high selectivity because Sap1 binds just 6.8% of the genome. A genome-wide analysis of promoter sequences revealed that Sap1 binding and amounts of integration correlate strongly. More important, an alignment of the DNA-binding motif of Sap1 revealed integration clustered on both sides of the motif and showed high levels specifically at positions +19 and -9. These data indicate that Sap1 contributes to the efficiency and position of Tf1 integration.

Host and viral determinants for MxB restriction of HIV-1 infection.

BACKGROUND: Interferon-induced cellular proteins play important roles in the host response against viral infection. The Mx family of dynamin-like GTPases, which include MxA and MxB, target a wide variety of viruses. Despite considerable evidence demonstrating the breadth of antiviral activity of MxA, human MxB was only recently discovered to specifically inhibit lentiviruses. Here we assess both host and viral determinants that underlie MxB restriction of HIV-1 infection. RESULTS: Heterologous expression of MxB in human osteosarcoma cells potently inhibited HIV-1 infection (~12-fold), yet had little to no effect on divergent retroviruses. The anti-HIV effect manifested as a partial block in the formation of 2-long terminal repeat circle DNA and hence nuclear import, and we accordingly found evidence for an additional post-nuclear entry block. A large number of previously characterized capsid mutations, as well as mutations that abrogated integrase activity, counteracted MxB restriction. MxB expression suppressed integration into gene-enriched regions of chromosomes, similar to affects observed previously when cells were depleted for nuclear transport factors such as transportin 3. MxB activity did not require predicted GTPase active site residues or a series of unstructured loops within the stalk domain that confer functional oligomerization to related dynamin family proteins. In contrast, we observed an N-terminal stretch of residues in MxB to harbor key determinants. Protein localization conferred by a nuclear localization signal (NLS) within the N-terminal 25 residues, which was critical, was fully rescuable by a heterologous NLS. Consistent with this observation, a heterologous nuclear export sequence (NES) abolished full-length MxB activity. We additionally mapped sub-regions within amino acids 26-90 that contribute to MxB activity, finding sequences present within residues 27-50 particularly important. CONCLUSIONS: MxB inhibits HIV-1 by interfering with minimally two steps of infection, nuclear entry and post-nuclear trafficking and/or integration, without destabilizing the inherent catalytic activity of viral preintegration complexes. Putative MxB GTPase active site residues and stalk domain Loop 4 -- both previously shown to be necessary for MxA function -- were dispensable for MxB antiviral activity. Instead, we highlight subcellular localization and a yet-determined function(s) present in the unique MxB N-terminal region to be required for HIV-1 restriction.

Serial number tagging reveals a prominent sequence preference of retrotransposon integration.

Transposable elements (TE) have both negative and positive impact on the biology of their host. As a result, a balance is struck between the host and the TE that relies on directing integration to specific genome territories. The extraordinary capacity of DNA sequencing can create ultra dense maps of integration that are being used to study the mechanisms that position integration. Unfortunately, the great increase in the numbers of insertion sites detected comes with the cost of not knowing which positions are rare targets and which sustain high numbers of insertions. To address this problem we developed the serial number system, a TE tagging method that measures the frequency of integration at single nucleotide positions. We sequenced 1 million insertions of retrotransposon Tf1 in the genome of Schizosaccharomyces pombe and obtained the first profile of integration with frequencies for each individual position. Integration levels at individual nucleotides varied over two orders of magnitude and revealed that sequence recognition plays a key role in positioning integration. The serial number system is a general method that can be applied to determine precise integration maps for retroviruses and gene therapy vectors.