LINE-1 retrotransposition and its deregulation in cancers: implications for therapeutic opportunities.

Long interspersed element 1 (LINE-1) is the only protein-coding transposon that is active in humans. LINE-1 propagates in the genome using RNA intermediates via retrotransposition. This activity has resulted in LINE-1 sequences occupying approximately one-fifth of our genome. Although most copies of LINE-1 are immobile, ∼100 copies are retrotransposition-competent. Retrotransposition is normally limited via epigenetic silencing, DNA repair, and other host defense mechanisms. In contrast, LINE-1 overexpression and retrotransposition are hallmarks of cancers. Here, we review mechanisms of LINE-1 regulation and how LINE-1 may promote genetic heterogeneity in tumors. Finally, we discuss therapeutic strategies to exploit LINE-1 biology in cancers.

Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition.

LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts have developed mechanisms to combat retrotransposition's mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here, we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the copurified proteome, identifying 37 high-confidence candidate interactors. These data sets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest that this occurs during or immediately after target-primed reverse transcription.

Human transposon tectonics.

Mobile DNAs have had a central role in shaping our genome. More than half of our DNA is comprised of interspersed repeats resulting from replicative copy and paste events of retrotransposons. Although most are fixed, incapable of templating new copies, there are important exceptions to retrotransposon quiescence. De novo insertions cause genetic diseases and cancers, though reliably detecting these occurrences has been difficult. New technologies aimed at uncovering polymorphic insertions reveal that mobile DNAs provide a substantial and dynamic source of structural variation. Key questions going forward include how and how much new transposition events affect human health and disease.

Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning.

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences single molecule real-time sequencing. By using reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second-strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second-strand synthesis. Deletion and insertion rates increase for all RTs during second-strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.

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.

Transposable elements in human genetic disease.

Transposable elements are abundant in the human genome, and great strides have been made in pinpointing variations in these repetitive sequences using whole-genome sequencing. Now, the focus is shifting to understanding their expression and regulation, and the functional consequences of their insertion and retention in the genome over time. Whereas transposable element insertions have been known to cause human genetic disease since the 1980s, the scope of their contributions to heritable phenotypes is now starting to be uncovered. Here, we review the many ways human retrotransposons contribute to genome function, their dysregulation in diseases including cancer and how they affect genetic disease.

Mobile interspersed repeats are major structural variants in the human genome.

Characterizing structural variants in the human genome is of great importance, but a genome wide analysis to detect interspersed repeats has not been done. Thus, the degree to which mobile DNAs contribute to genetic diversity, heritable disease, and oncogenesis remains speculative. We perform transposon insertion profiling by microarray (TIP-chip) to map human L1(Ta) retrotransposons (LINE-1 s) genome-wide. This identified numerous novel human L1(Ta) insertional polymorphisms with highly variant allelic frequencies. We also explored TIP-chip's usefulness to identify candidate alleles associated with different phenotypes in clinical cohorts. Our data suggest that the occurrence of new insertions is twice as high as previously estimated, and that these repeats are under-recognized as sources of human genomic and phenotypic diversity. We have just begun to probe the universe of human L1(Ta) polymorphisms, and as TIP-chip is applied to other insertions such as Alu SINEs, it will expand the catalog of genomic variants even further.

The landscape of human SVA retrotransposons.

SINE-VNTR-Alu (SVA) retrotransposons are evolutionarily young and still-active transposable elements (TEs) in the human genome. Several pathogenic SVA insertions have been identified that directly mutate host genes to cause neurodegenerative and other types of diseases. However, due to their sequence heterogeneity and complex structures as well as limitations in sequencing techniques and analysis, SVA insertions have been less well studied compared to other mobile element insertions. Here, we identified polymorphic SVA insertions from 3646 whole-genome sequencing (WGS) samples of >150 diverse populations and constructed a polymorphic SVA insertion reference catalog. Using 20 long-read samples, we also assembled reference and polymorphic SVA sequences and characterized the internal hexamer/variable-number-tandem-repeat (VNTR) expansions as well as differing SVA activity for SVA subfamilies and human populations. In addition, we developed a module to annotate both reference and polymorphic SVA copies. By characterizing the landscape of both reference and polymorphic SVA retrotransposons, our study enables more accurate genotyping of these elements and facilitate the discovery of pathogenic SVA insertions.

LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint.

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

Alu insertion variants alter gene transcript levels.

Alu are high copy number interspersed repeats that have accumulated near genes during primate and human evolution. They are a pervasive source of structural variation in modern humans. Impacts that Alu insertions may have on gene expression are not well understood, although some have been associated with expression quantitative trait loci (eQTLs). Here, we directly test regulatory effects of polymorphic Alu insertions in isolation of other variants on the same haplotype. To screen insertion variants for those with such effects, we used ectopic luciferase reporter assays and evaluated 110 Alu insertion variants, including more than 40 with a potential role in disease risk. We observed a continuum of effects with significant outliers that up- or down-regulate luciferase activity. Using a series of reporter constructs, which included genomic context surrounding the Alu, we can distinguish between instances in which the Alu disrupts another regulator and those in which the Alu introduces new regulatory sequence. We next focused on three polymorphic Alu loci associated with breast cancer that display significant effects in the reporter assay. We used CRISPR to modify the endogenous sequences, establishing cell lines varying in the Alu genotype. Our findings indicate that Alu genotype can alter expression of genes implicated in cancer risk, including PTHLH, RANBP9, and MYC These data show that commonly occurring polymorphic Alu elements can alter transcript levels and potentially contribute to disease risk.

Development and validation of the Trust in Multidimensional Healthcare Systems Scale (TIMHSS).

CONTEXT: The COVID-19 pandemic has reignited a commitment from the health policy and health services research communities to rebuilding trust in healthcare and created a renewed appetite for measures of trust for system monitoring and evaluation. The aim of the present paper was to develop a multidimensional measure of trust in healthcare that: (1) Is responsive to the conceptual and methodological limitations of existing measures; (2) Can be used to identify systemic explanations for lower levels of trust in equity-deserving populations; (3) Can be used to design and evaluate interventions aiming to (re)build trust. METHODS: We conducted a 2021 review of existing measures of trust in healthcare, 72 qualitative interviews (Aug-Dec 2021; oversampling for equity-deserving populations), an expert review consensus process (Oct 2021), and factor analyses and validation testing based on two waves of survey data (Nov 2021, n = 694; Jan-Feb 2022, n = 740 respectively). FINDINGS: We present the Trust in Multidimensional Healthcare Systems Scale (TIMHSS); a 38-item correlated three-factor measure of trust in doctors, policies, and the system. Measurement of invariance tests suggest that the TIMHSS can also be reliably administered to diverse populations. CONCLUSIONS: This global measure of trust in healthcare can be used to measure trust over time at a population level, or used within specific subpopulations, to inform interventions to (re)build trust. It can also be used within a clinical setting to provide a stronger evidence base for associations between trust and therapeutic outcomes.

Increasing clinician participation in tobacco cessation by an implementation science-based tobacco cessation champion program.

BACKGROUND: We designed a process to increase tobacco cessation in an academic center and its widely distributed network community sites using clinical champions to overcome referral barriers. METHODS: In 2020 a needs assessment was performed across the City of Hope Medical Center and its 32 community treatment sites. We reviewed information science strategies to choose elements for our expanded tobacco control plan, focusing on distributed leadership with tobacco cessation champions. We analyzed smoking patterns in patients with cancer before and following program implementation. We evaluated the champion experience and measured tobacco abstinence after 6 months of follow-up. RESULTS: Cancer center leadership committed to expanding tobacco control. Funding was obtained through a Cancer Center Cessation Initiative (C3I) grant. Multi-disciplinary leaders developed a comprehensive plan. Disease-focused clinics and community sites named cessation champions (a clinician and nurse) supported by certified tobacco treatment specialists. Patient, staff, clinician, and champion training/education were developed. Roles and responsibilities of the champions were defined. Implementation in pilot sites showed increased tobacco assessment from 80.8 to 96.6%, increased tobacco cessation referral by 367%, and moderate smoking abstinence in both academic (27.2%) and community sites (22.5%). 73% of champions had positive attitudes toward the program. CONCLUSION: An efficient process to expand smoking cessation in the City of Hope network was developed using implementation science strategies and cessation champions. This well-detailed implementation process may be helpful to other cancer centers, particularly those with a tertiary care cancer center and community network.

Informing efforts beyond tailored promotional campaigns by understanding contextual factors shaping vaccine hesitancy among equity-deserving populations in Canada: an exploratory qualitative study.

BACKGROUND: Vaccine hesitancy exists on a continuum ranging between complete adherence and complete refusal due to doubts or concerns within a heterogeneous group of individuals. Despite widespread acknowledgement of the contextual factors influencing attitudes and beliefs shaping COVID-19 vaccine hesitancy, qualitative research with equity-deserving groups, accounting for unique lived experiences, remains a gap in the literature. We aim to identify and begin to understand and document the unique contextual factors shaping hesitancy by equity-deserving groups as it relates to relationships with government and health authorities. METHODS: Participants were recruited and interviewed between Aug-Dec 2021. Semi-structured interviews using a convergent interviewing technique were conducted with individuals from the general population, as well as individuals who identify as First Nations, Métis, or Inuit, members of the LGBT2SQ + community, low-income Canadians, Black Canadians, and newcomers. Interviews were audio recorded and transcribed by a team of researchers. Memos were written following interviews and used to complement the thematic analysis of the interview data. Themes are presented in the results section. RESULTS: The rationale for hesitancy among equity-deserving groups is consistent with literature documenting hesitancy in the general population. Contextual factors surrounding equity-deserving groups' attitudes and beliefs, however, are unique and relate to a history of oppression, discrimination, and genocide. We identified factors unique to subgroups; for example, religious or fatalistic beliefs among participant who identify as FNMI, fear associated with lack of testing and speed of vaccines' production among participants who identify as FNMI, Black, and LGBT2SQ + , distrust of the healthcare system for LGBT2SQ + and Black Canadians, and distrust of the government and opposition to vaccine mandates for participating who identify as LGBT2SQ + , low-income, FNMI, or Black Canadian. Newcomers stood out as very trusting of the government and accepting of COVID-19 vaccination. CONCLUSIONS: While our data on vaccine hesitancy largely mirror concerns reported in the vast body of literature citing rationale for COVID-19 hesitancy in high-income countries, the contextual factors identified in our work point to the need for wider systemic change. Our results may be used to support efforts, beyond tailored promotion campaigns, to support the confident acceptance of vaccines for COVID-19 and the acceptance of novel vaccines as future infectious diseases emerge.

Development and validation of the Trust in Government measure (TGM).

BACKGROUND: Trust in government is associated with health behaviours and is an important consideration in population health interventions. While there is a reported decline in public trust in government across OECD countries, the tools used to measure trust are limited in their use for informing action to (re)build trust, and have limitations related to reliability and validity. To address the limitations of existing measures available to track public trust, the aim of the present work was to develop a new measure of trust in government. METHODS: Fifty-six qualitative interviews (Aug-Oct 2021; oversampling for equity-deserving populations) were conducted to design a national survey, including factor analyses and validation testing (N = 878; June 1-14th 2022) in Canada. RESULTS: The measure demonstrated strong internal consistency (α = 0.96) and test validity (CFI = 0.96, RMSEA = 0.09, SRMR = 0.03), suggesting that trust in government can be measured as a single underlying construct. It also demonstrated strong criterion validity, as measured by significant (p < 0.0001) associations of scores with vaccine hesitancy, vaccine conspiracy beliefs, COVID-19 conspiracy beliefs, trust in public health messaging about COVID-19, and trust in public health advice about COVID-19. We present the Trust in Government Measure (TGM); a 13-item unidimensional measure of trust in Federal government. CONCLUSIONS: This measure can be used within high-income countries, particularly member countries within the OECD already in support of using tools to collect, publish and compare statistics. Our measure should be used by researchers and policy makers to measure trust in government as a key indicator of societal and public health.

Acceptability of an Online Module Addressing Weight Bias: Perspectives and Attitudes of Undergraduate Health Students and Instructors.

Weight bias and discrimination are highly pervasive and harmful to Canadians with higher weights. Researchers and practitioners who deliver, evaluate, and advise on dietary and weight-related interventions may inadvertently perpetuate weight bias through their work; however, trainees in these fields rarely have access to weight bias education within their applied health programs. This study evaluated the acceptability of an online educational weight bias module developed for undergraduate students enrolled in health courses. The intervention included a pre-recorded 20-minute online module with prompts for reflection or discussion, a self-assessment quiz, as well as a separate module and range of resources for instructors. Overall, 211 students from applied health courses and 4 instructors completed an online survey querying the module's delivery, impact, and relevance. Students agreed that the module provided useful information (82%), was easy to understand (97%), and was the right length (75%), but reported wanting more interactivity and engagement with the content. Instructors found the module engaging and useful and expressed interest in additional resources and support for weight bias education. Future research should explore the impact of weight bias education on students' weight-related attitudes and perceptions as well as feasibility and relevance of online features such as multimedia tools.

Autoantibodies targeting LINE-1-encoded ORF1p are associated with systemic lupus erythematosus diagnosis but not disease activity.

OBJECTIVES: Long Interspersed Element 1 (LINE-1) is an endogenous retroelement that constitutes a significant portion of the human genome and has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The LINE-1 RNA chaperone protein ORF1p was recently identified as an SLE autoantigen. Here we analyse ORF1p for qualities underlying SLE autoantigen status, compared anti-ORF1p antibodies to markers of SLE disease activity, and performed screening for antibodies against LINE-1 reverse transcriptase ORF2p. METHODS: ORF1p was examined in epithelial cell lines treated with cytotoxic lymphocyte granules and UV irradiation. Anti-ORF1p and anti-ORF2p antibodies were assayed by ELISA and analysed in two SLE cohorts. RESULTS: We found that ORF1p localises to cytoplasmic RNA-containing blebs in apoptotic cells, and is a substrate of the cytotoxic protease granzyme B (GrB). Anti-ORF1p antibodies were present in 4.2% of healthy controls, compared to 15.8% (p=0.0157) and 15.5% (p=0.036) of subjects in the two SLE cohorts. Anti-ORF1p antibodies were not associated with SLE disease activity nor peripheral blood markers of interferon (IFN) activation. Anti-ORF1p titres demonstrated stability over serial time points. Anti-ORF1p antibodies were not associated with anti-DNA, anti-RNP, or other SLE autoantibodies. There was no difference in anti-ORF2p ELISA results in controls versus SLE patients. CONCLUSIONS: LINE-1 ORF1p is a component of apoptotic blebs and a substrate for GrB. Anti-ORF1p antibodies are enriched in SLE subjects but are not associated with dynamic markers of disease activity. These data support a potential role for LINE-1 dysregulation in SLE pathogenesis.

TypeTE: a tool to genotype mobile element insertions from whole genome resequencing data.

Alu retrotransposons account for more than 10% of the human genome, and insertions of these elements create structural variants segregating in human populations. Such polymorphic Alus are powerful markers to understand population structure, and they represent variants that can greatly impact genome function, including gene expression. Accurate genotyping of Alus and other mobile elements has been challenging. Indeed, we found that Alu genotypes previously called for the 1000 Genomes Project are sometimes erroneous, which poses significant problems for phasing these insertions with other variants that comprise the haplotype. To ameliorate this issue, we introduce a new pipeline - TypeTE - which genotypes Alu insertions from whole-genome sequencing data. Starting from a list of polymorphic Alus, TypeTE identifies the hallmarks (poly-A tail and target site duplication) and orientation of Alu insertions using local re-assembly to reconstruct presence and absence alleles. Genotype likelihoods are then computed after re-mapping sequencing reads to the reconstructed alleles. Using a high-quality set of PCR-based genotyping of >200 loci, we show that TypeTE improves genotype accuracy from 83% to 92% in the 1000 Genomes dataset. TypeTE can be readily adapted to other retrotransposon families and brings a valuable toolbox addition for population genomics.

SQuIRE reveals locus-specific regulation of interspersed repeat expression.

Transposable elements (TEs) are interspersed repeat sequences that make up much of the human genome. Their expression has been implicated in development and disease. However, TE-derived RNA-seq reads are difficult to quantify. Past approaches have excluded these reads or aggregated RNA expression to subfamilies shared by similar TE copies, sacrificing quantitative accuracy or the genomic context necessary to understand the basis of TE transcription. As a result, the effects of TEs on gene expression and associated phenotypes are not well understood. Here, we present Software for Quantifying Interspersed Repeat Expression (SQuIRE), the first RNA-seq analysis pipeline that provides a quantitative and locus-specific picture of TE expression (https://github.com/wyang17/SQuIRE). SQuIRE is an accurate and user-friendly tool that can be used for a variety of species. We applied SQuIRE to RNA-seq from normal mouse tissues and a Drosophila model of amyotrophic lateral sclerosis. In both model organisms, we recapitulated previously reported TE subfamily expression levels and revealed locus-specific TE expression. We also identified differences in TE transcription patterns relating to transcript type, gene expression and RNA splicing that would be lost with other approaches using subfamily-level analyses. Altogether, our findings illustrate the importance of studying TE transcription with locus-level resolution.

Alu insertion variants alter mRNA splicing.

RNA splicing is a highly regulated process dependent on sequences near splice sites. Insertions of Alu retrotransposons can disrupt splice sites or bind splicing regulators. We hypothesized that some common inherited polymorphic Alu insertions are responsible for splicing QTLs (sQTL). We focused on intronic Alu variants mapping within 100 bp of an alternatively used exon and screened for those that alter splicing. We identify five loci, 21.7% of those assayed, where the polymorphic Alu alters splicing. While in most cases the Alu promotes exon skipping, at one locus the Alu increases exon inclusion. Of particular interest is an Alu polymorphism in the CD58 gene. Reduced CD58 expression is associated with risk for developing multiple sclerosis. We show that the Alu insertion promotes skipping of CD58 exon 3 and results in a frameshifted transcript, indicating that the Alu may be the causative variant for increased MS risk at this locus. Using RT-PCR analysis at the endogenous locus, we confirm that the Alu variant is a sQTL for CD58. In summary, altered splicing efficiency is a common functional consequence of Alu polymorphisms including at least one instance where the variant is implicated in disease risk. This work broadens our understanding of splicing regulatory sequences around exons.