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 employing 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.

Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.

Subcellular location of L1 retrotransposon-encoded ORF1p, reverse transcription products, and DNA sensors in lupus granulocytes.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with an unpredictable course of recurrent exacerbations alternating with more stable disease. SLE is characterized by broad immune activation and autoantibodies against double-stranded DNA and numerous proteins that exist in cells as aggregates with nucleic acids, such as Ro60, MOV10, and the L1 retrotransposon-encoded ORF1p. RESULTS: Here we report that these 3 proteins are co-expressed and co-localized in a subset of SLE granulocytes and are concentrated in cytosolic dots that also contain DNA: RNA heteroduplexes and the DNA sensor ZBP1, but not cGAS. The DNA: RNA heteroduplexes vanished from the neutrophils when they were treated with a selective inhibitor of the L1 reverse transcriptase. We also report that ORF1p granules escape neutrophils during the extrusion of neutrophil extracellular traps (NETs) and, to a lesser degree, from neutrophils dying by pyroptosis, but not apoptosis. CONCLUSIONS: These results bring new insights into the composition of ORF1p granules in SLE neutrophils and may explain, in part, why proteins in these granules become targeted by autoantibodies in this disease.

DNA lesion bypass and the stochastic dynamics of transcription-coupled repair.

DNA base damage is a major source of oncogenic mutations and disruption to gene expression. The stalling of RNA polymerase II (RNAP) at sites of DNA damage and the subsequent triggering of repair processes have major roles in shaping the genome-wide distribution of mutations, clearing barriers to transcription, and minimizing the production of miscoded gene products. Despite its importance for genetic integrity, key mechanistic features of this transcription-coupled repair (TCR) process are controversial or unknown. Here, we exploited a well-powered in vivo mammalian model system to explore the mechanistic properties and parameters of TCR for alkylation damage at fine spatial resolution and with discrimination of the damaged DNA strand. For rigorous interpretation, a generalizable mathematical model of DNA damage and TCR was developed. Fitting experimental data to the model and simulation revealed that RNA polymerases frequently bypass lesions without triggering repair, indicating that small alkylation adducts are unlikely to be an efficient barrier to gene expression. Following a burst of damage, the efficiency of transcription-coupled repair gradually decays through gene bodies with implications for the occurrence and accurate inference of driver mutations in cancer. The reinitation of transcription from the repair site is not a general feature of transcription-coupled repair, and the observed data is consistent with reinitiation never taking place. Collectively, these results reveal how the directional but stochastic activity of TCR shapes the distribution of mutations following DNA damage.

Single-mitosis dissection of acute and chronic DNA mutagenesis and repair.

How chronic mutational processes and punctuated bursts of DNA damage drive evolution of the cancer genome is poorly understood. Here, we demonstrate a strategy to disentangle and quantify distinct mechanisms underlying genome evolution in single cells, during single mitoses and at single-strand resolution. To distinguish between chronic (reactive oxygen species (ROS)) and acute (ultraviolet light (UV)) mutagenesis, we microfluidically separate pairs of sister cells from the first mitosis following burst UV damage. Strikingly, UV mutations manifest as sister-specific events, revealing mirror-image mutation phasing genome-wide. In contrast, ROS mutagenesis in transcribed regions is reduced strand agnostically. Successive rounds of genome replication over persisting UV damage drives multiallelic variation at CC dinucleotides. Finally, we show that mutation phasing can be resolved to single strands across the entire genome of liver tumors from F1 mice. This strategy can be broadly used to distinguish the contributions of overlapping cancer relevant mutational processes.

Convergence of coronary artery disease genes onto endothelial cell programs.

Linking variants from genome-wide association studies (GWAS) to underlying mechanisms of disease remains a challenge1-3. For some diseases, a successful strategy has been to look for cases in which multiple GWAS loci contain genes that act in the same biological pathway1-6. However, our knowledge of which genes act in which pathways is incomplete, particularly for cell-type-specific pathways or understudied genes. Here we introduce a method to connect GWAS variants to functions. This method links variants to genes using epigenomics data, links genes to pathways de novo using Perturb-seq and integrates these data to identify convergence of GWAS loci onto pathways. We apply this approach to study the role of endothelial cells in genetic risk for coronary artery disease (CAD), and discover 43 CAD GWAS signals that converge on the cerebral cavernous malformation (CCM) signalling pathway. Two regulators of this pathway, CCM2 and TLNRD1, are each linked to a CAD risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. These results suggest a model whereby CAD risk is driven in part by the convergence of causal genes onto a particular transcriptional pathway in endothelial cells. They highlight shared genes between common and rare vascular diseases (CAD and CCM), and identify TLNRD1 as a new, previously uncharacterized member of the CCM signalling pathway. This approach will be widely useful for linking variants to functions for other common polygenic diseases.

SOX17 enables immune evasion of early colorectal adenomas and cancers.

A hallmark of cancer is the avoidance of immune destruction. This process has been primarily investigated in locally advanced or metastatic cancer1-3; however, much less is known about how pre-malignant or early invasive tumours evade immune detection. Here, to understand this process in early colorectal cancers (CRCs), we investigated how naive colon cancer organoids that were engineered in vitro to harbour Apc-null, KrasG12D and Trp53-null (AKP) mutations adapted to the in vivo native colonic environment. Comprehensive transcriptomic and chromatin analyses revealed that the endoderm-specifying transcription factor SOX17 became strongly upregulated in vivo. Notably, whereas SOX17 loss did not affect AKP organoid propagation in vitro, its loss markedly reduced the ability of AKP tumours to persist in vivo. The small fraction of SOX17-null tumours that grew displayed notable interferon-γ (IFNγ)-producing effector-like CD8+ T cell infiltrates in contrast to the immune-suppressive microenvironment in wild-type counterparts. Mechanistically, in both endogenous Apc-null pre-malignant adenomas and transplanted organoid-derived AKP CRCs, SOX17 suppresses the ability of tumour cells to sense and respond to IFNγ, preventing anti-tumour T cell responses. Finally, SOX17 engages a fetal intestinal programme that drives differentiation away from LGR5+ tumour cells to produce immune-evasive LGR5- tumour cells with lower expression of major histocompatibility complex class I (MHC-I). We propose that SOX17 is a transcription factor that is engaged during the early steps of colon cancer to orchestrate an immune-evasive programme that permits CRC initiation and progression.

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.

Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains.

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.

Ultrasensitive Detection of Circulating LINE-1 ORF1p as a Specific Multicancer Biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. Although proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible expression in normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 mol/L) ORF1p concentrations in plasma across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multianalyte panel, provides early therapeutic response monitoring in gastroesophageal cancers, and is prognostic for overall survival in gastroesophageal and colorectal cancers. Together, these observations nominate ORF1p as a multicancer biomarker with potential utility for disease detection and monitoring. SIGNIFICANCE: The LINE-1 ORF1p transposon protein is pervasively expressed in many cancers and is a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 µL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring. See related commentary by Doucet and Cristofari, p. 2502. This article is featured in Selected Articles from This Issue, p. 2489.

Expression of L1 retrotransposons in granulocytes from patients with active systemic lupus erythematosus.

BACKGROUND: Patients with systemic lupus erythematosus (SLE) have autoantibodies against the L1-encoded open-reading frame 1 protein (ORF1p). Here, we report (i) which immune cells ORF1p emanates from, (ii) which L1 loci are transcriptionally active, (iii) whether the cells express L1-dependent interferon and interferon-stimulated genes, and (iv) the effect of inhibition of L1 ORF2p by reverse transcriptase inhibitors. RESULTS: L1 ORF1p was detected by flow cytometry primarily in SLE CD66b+CD15+ regular and low-density granulocytes, but much less in other immune cell lineages. The amount of ORF1p was higher in neutrophils from patients with SLE disease activity index (SLEDAI) > 6 (p = 0.011) compared to patients with inactive disease, SLEDAI < 4. Patient neutrophils transcribed seven to twelve human-specific L1 loci (L1Hs), but only 3 that are full-length and with an intact ORF1. Besides serving as a source of detectable ORF1p, the most abundant transcript encoded a truncated ORF2p reverse transcriptase predicted to remain cytosolic, while the two other encoded an intact full-length ORF2p. A number of genes encoding proteins that influence L1 transcription positively or negatively were altered in patients, particularly those with active disease, compared to healthy controls. Components of nucleic acid sensing and interferon induction were also altered. SLE neutrophils also expressed type I interferon-inducible genes and interferon ß, which were substantially reduced after treatment of the cells with drugs known to inhibit ORF2p reverse transcriptase activity. CONCLUSIONS: We identified L1Hs loci that are transcriptionally active in SLE neutrophils, and a reduction in the epigenetic silencing mechanisms that normally counteract L1 transcription. SLE neutrophils contained L1-encoded ORF1p protein, as well as activation of the type I interferon system, which was inhibited by treatment with reverse transcriptase inhibitors. Our findings will enable a deeper analysis of L1 dysregulation and its potential role in SLE pathogenesis.

Ultrasensitive detection of circulating LINE-1 ORF1p as a specific multi-cancer biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. While proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1, L1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible detectable expression in corresponding normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore the potential of ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 M) ORF1p concentrations in patient plasma samples across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multi-analyte panel, and provides early therapeutic response monitoring in gastric and esophageal cancers. Together, these observations nominate ORF1p as a multi-cancer biomarker with potential utility for disease detection and monitoring.

Affinity-Based Interactome Analysis of Endogenous LINE-1 Macromolecules.

During their proliferation and the host's concomitant attempts to suppress it, LINE-1 (L1) retrotransposons give rise to a collection of heterogeneous ribonucleoproteins (RNPs); their protein and RNA compositions remain poorly defined. The constituents of L1-associated macromolecules can differ depending on numerous factors, including, for example, position within the L1 life cycle, whether the macromolecule is productive or under suppression, and the cell type within which the proliferation is occurring. This chapter describes techniques that aid the capture and characterization of protein and RNA components of L1 macromolecules from tissues that natively express them. The protocols described have been applied to embryonal carcinoma cell lines that are popular model systems for L1 molecular biology (e.g., N2102Ep, NTERA-2, and PA-1 cells), as well as colorectal cancer tissues. N2102Ep cells are given as the use case for this chapter; the protocols should be applicable to essentially any tissue exhibiting endogenous L1 expression with minor modifications.

Immunological and Clinico-Molecular Features of Tumor Border Configuration in Colorectal Cancer.

INTRODUCTION: Infiltrating tumor border configuration (ITBC) portends a poor prognosis compared with pushing tumor border configuration (PTBC) in colorectal cancer. The tumor and its surrounding immune microenvironment of tumor border configuration is not well-characterized. We aim to elucidate the differences in expression of molecular markers between the 2 groups using tissue microarray (TMA). STUDY DESIGN: Immunohistochemistry was performed on TMAs of surgical pathology specimens obtained from colorectal cancer patients consecutively operated at our institution from 2004 to 2015. TMAs were stained for immune cells (CD8, FOXP3, LAG3, PU1, CD163, and PDL1); HLA II, beta 2 microglobulin, and HC10 on tumor cells; BRAFV600E mutation; and DNA mismatch repair proteins (MMR) status. Patients who received neoadjuvant therapy were excluded. RESULTS: There were 646 tumors with ITBC and 310 tumors with PTBC. There was a significantly lower expression (p < 0.05) of immune components, namely CD8, FOXP3, LAG3, PU1, PDL1 immune cells, and Beta-2 Microglobulin on tumor cells in the tumors with ITBC compared with PTBC, except CD163 immune cells, and HC10 and HLAII on tumor cells. Tumors with ITBC were less likely to be associated with BRAFV600E mutations and deficient MMR proteins (p < 0.001). On analyzing MMR-proficient tumors separately, we could not find any difference in the expression of any molecular marker (including BRAF), except a lower expression of PDL1 immune cells in tumors with ITBC (p < 0.001). CONCLUSIONS: Colorectal tumors with ITBC are associated with a generalized low immune microenvironment and low rates of BRAFV600E mutation compared with tumors with PTBC. However, the molecular expression of tumor border configuration seems confounded by the MMR molecular signature. MMR-proficient colorectal tumors with ITBC are associated with a lower expression of only PDL1 immune cells among all immune markers examined.

Molecular Basis of Extramural Vascular Invasion (EMVI) in Colorectal Carcinoma.

BACKGROUND: Extramural vascular invasion (EMVI) is a known poor prognostic factor in colorectal carcinoma; however, its molecular basis has not been defined. This study aimed to assess the expression of molecular markers in EMVI positive colorectal carcinoma to understand their tumor microenvironment. METHODS: Immunohistochemistry was performed on tissue microarrays of surgically resected colorectal cancer specimens for immunological markers, and BRAFV600E mutation (and on the tissue blocks for mismatch repair proteins). Automated quantification was used for CD8, LAG3, FOXP3, PU1, and CD163, and manual quantification was used for PDL1, HLA I markers (beta-2 microglobulin, HC10), and HLA II. The Wilcoxon rank-sum test was used to compare EMVI positive and negative tumors. A logistic regression model was fitted to assess the predictive effect of biomarkers on EMVI. RESULTS: There were 340 EMVI positive and 678 EMVI negative chemo naïve tumors. PDL1 was barely expressed on tumor cells (median 0) in the entire cohort. We found a significantly lower expression of CD8, LAG3, FOXP3, PU1 cells, PDL1 positive macrophages, and beta-2 microglobulin on tumor cells in the EMVI positive subset (p ≤ 0.001). There was no association of BRAFV600E or deficient mismatch repair proteins (dMMR) with EMVI. PU1 (OR 0.8, 0.7-0.9) and low PDL1 (OR 1.6, 1.1-2.3) independently predicted EMVI on multivariate logistic regression among all biomarkers examined. CONCLUSION: There is a generalized blunting of immune response in EMVI positive colorectal carcinoma, which may contribute to a worse prognosis. Tumor-associated macrophages seem to play the most significant role in determining EMVI.

The contribution of evolutionarily volatile promoters to molecular phenotypes and human trait variation.

BACKGROUND: Promoters are sites of transcription initiation that harbour a high concentration of phenotype-associated genetic variation. The evolutionary gain and loss of promoters between species (collectively, termed turnover) is pervasive across mammalian genomes and may play a prominent role in driving human phenotypic diversity. RESULTS: We classified human promoters by their evolutionary history during the divergence of mouse and human lineages from a common ancestor. This defined conserved, human-inserted and mouse-deleted promoters, and a class of functional-turnover promoters that align between species but are only active in humans. We show that promoters of all evolutionary categories are hotspots for substitution and often, insertion mutations. Loci with a history of insertion and deletion continue that mode of evolution within contemporary humans. The presence of an evolutionary volatile promoter within a gene is associated with increased expression variance between individuals, but only in the case of human-inserted and mouse-deleted promoters does that correspond to an enrichment of promoter-proximal genetic effects. Despite the enrichment of these molecular quantitative trait loci (QTL) at evolutionarily volatile promoters, this does not translate into a corresponding enrichment of phenotypic traits mapping to these loci. CONCLUSIONS: Promoter turnover is pervasive in the human genome, and these promoters are rich in molecularly quantifiable but phenotypically inconsequential variation in gene expression. However, since evolutionarily volatile promoters show evidence of selection, coupled with high mutation rates and enrichment of QTLs, this implicates them as a source of evolutionary innovation and phenotypic variation, albeit with a high background of selectively neutral expression variation.

Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer.

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements. SIGNIFICANCE: Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397.

Signatures of TOP1 transcription-associated mutagenesis in cancer and germline.

The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4-a cancer insertion-deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions -is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome.