P-OGC44 Multi-omic cohort study of Barrett’s oesophagus reveals structural variation and retrotransposon activity to occur early in cancer evolution

Background Barrett’s oesophagus (BE) is the main risk factor for the development of oesophageal adenocarcinoma (OAC), yet few patients ever go on to progress to cancer. The acquisition of events during the metaplasia-dysplasia-cancer sequence is poorly characterised. We present a large, unbiased, multi-omics analysis of a cross-sectional cohort of pre-cancer samples, with the aim of providing a comprehensive insight into the diversity and molecular changes driving the disease to cancer. Methods We generated and integrated the genomic (50x), transcriptomic and epigenomic (850K EPIC array) landscapes of snap-frozen endoscopic biopsies from 146 patients with a range of outcomes (27 long-standing non-dysplastic; 12 prior to progression to dysplasia; 14 low-grade; 25 high-grade; 21 intramucosal carcinoma; 47 cases of BE taken adjacent to OAC) and 642 person years of follow-up. All biopsies were reviewed independently by 3 pathologists and had associated annotation with detailed clinical information. Results The total number of structural variants (SV) captured the most variance between samples. Complex SVs and LINE-1 retrotransposon activity were observed even before dysplasia had developed and increased with progression. Increasing SV burden was associated with chromothripsis (12%, 18/146) and breakage-fusion bridges (BFBs; 8%, 13/146). In more than 50% of these, the BFBs were in chromosome 17, harbouring the oncogenes ERBB2 and CDK12, for which expression was significantly higher. With progression there was increased expression of genes related to cell-cycle checkpoint, DNA repair and chromosomal instability, and the epigenetic silencing of genes in WNT-signalling and cell-cycle pathways. Conclusions Genomic complexity occurs very early in the natural history of BE and increasing genomic instability appears to tip the balance towards cancer. This may inform the potential for progression to cancer beyond the clinically discernible phenotype. Efforts to better understand the triggers for chromosomal breakages and rearrangements that underly progression will aid clinical prediction and prevention strategies.

The extrachromosomal circular DNAs of the rice blast pathogen Magnaporthe oryzae contain a wide variety of LTR retrotransposons, genes, and effectors

One of the ways genomes respond to stress is by shedding extrachromosomal circular DNAs (eccDNAs). EccDNAs can contain genes and dramatically increase their copy number. They can also reinsert into the genome, generating structural variation. They have been shown to provide a source of phenotypic and genotypic plasticity in several species. However, whole-circularome studies have so far been limited to a few model organisms. Fungal plant pathogens are a serious threat to global food security in part because of their rapid adaptation to disease prevention strategies. Understanding the mechanisms fungal pathogens use to escape disease control is paramount to curbing their threat. We present a whole circularome sequencing study of the rice blast pathogen Magnaporthe oryzae. We find that M. oryzae has a highly diverse circularome containing many genes and showing evidence of large LTR retrotransposon activity. We find that genes enriched on eccDNAs in M. oryzae occur in genomic regions prone to presence-absence variation and that disease associated genes are frequently on eccDNAs. Finally, we find that a subset of genes is never present on eccDNAs, which indicates that the presence of these genes on eccDNAs is selected against.

Involvement of non-LTR retrotransposons in the cancer incidence and lifespan of mammals

The natural occurrence of closely related species that show drastic differences in lifespan and cancer incidence raised the interest in finding the particular adaptations and genomic characteristics underlying the evolution of long lifespans. Studies on transposable elements (TEs) have more and more linked them to ageing and cancer development. In this study, we compared the TE content and dynamics in the genomes of four Rodent and six Chiroptera species that show very different lifespans and cancer susceptibility including the long-lived and refractory to cancer naked mole rat (Heterocephalus glaber), the long-lived fruit bats (Pteropus vampyrus, Rousettus aegypticaus) and the short-lived velvety free-tailed bat (Molossus molossus). By analysing the patterns of recent TE accumulation (TEs that are potentially currently active) in high-quality genome assemblies, we found that the shared genomic characteristics between long-lived species that are refractory to cancer, is the strong suppression, or negative selection against the accumulation, of non-LTR retrotransposons. All the short-lived species did show a recent accumulation of these TEs. Non-LTR retrotransposons have been often found to take part in the immune response of the host against viral infections, but their dysregulation can lead to phenomena of "sterile inflammation" and "inflammageing". Therefore, we hypothesise that the uncontrolled non-LTR retrotransposon activity is an important factor explaining the evolution of shorter lifespans in both Rodents and Chiroptera species and potentially in all mammals. Finally, these results suggest that non-LTR retrotransposons can be agents promoting cancer and ageing in mammals thus they may be targets of future oncological therapies.

The Welwitschia genome reveals a unique biology underpinning extreme longevity in deserts

The gymnosperm Welwitschia mirabilis belongs to the ancient, enigmatic gnetophyte lineage. It is a unique desert plant with extreme longevity and two ever-elongating leaves. We present a chromosome-level assembly of its genome (6.8 Gb/1 C) together with methylome and transcriptome data to explore its astonishing biology. We also present a refined, high-quality assembly of Gnetum montanum to enhance our understanding of gnetophyte genome evolution. The Welwitschia genome has been shaped by a lineage-specific ancient, whole genome duplication (~86 million years ago) and more recently (1-2 million years) by bursts of retrotransposon activity. High levels of cytosine methylation (particularly at CHH motifs) are associated with retrotransposons, whilst long-term deamination has resulted in an exceptionally GC-poor genome. Changes in copy number and/or expression of gene families and transcription factors (e.g. R2R3MYB, SAUR) controlling cell growth, differentiation and metabolism underpin the plant’s longevity and tolerance to temperature, nutrient and water stress.

Elevated retrotransposon activity and genomic instability in primed pluripotent stem cells

Background Naïve and primed pluripotent stem cells (PSCs) represent two different pluripotent states. Primed PSCs following in vitro culture exhibit lower developmental potency as evidenced by failure in germline chimera assays, unlike mouse naïve PSCs. However, the molecular mechanisms underlying the lower developmental competency of primed PSCs remain elusive. Results We examine the regulation of telomere maintenance, retrotransposon activity, and genomic stability of primed PSCs and compare them with naïve PSCs. Surprisingly, primed PSCs only minimally maintain telomeres and show fragile telomeres, associated with declined DNA recombination and repair activity, in contrast to naïve PSCs that robustly elongate telomeres. Also, we identify LINE1 family integrant L1Md_T as naïve-specific retrotransposon and ERVK family integrant IAPEz to define primed PSCs, and their transcription is differentially regulated by heterochromatic histones and Dnmt3b. Notably, genomic instability of primed PSCs is increased, in association with aberrant retrotransposon activity. Conclusions Our data suggest that fragile telomere, retrotransposon-associated genomic instability, and declined DNA recombination repair, together with reduced function of cell cycle and mitochondria, increased apoptosis, and differentiation properties may link to compromised developmental potency of primed PSCs, noticeably distinguishable from naïve PSCs.

Genome-wide characterization of LTR retrotransposons in the non-model deep-sea annelid Lamellibrachia luymesi

Background Long Terminal Repeat retrotransposons (LTR retrotransposons) are mobile genetic elements composed of a few genes between terminal repeats and, in some cases, can comprise over half of a genome’s content. Available data on LTR retrotransposons have facilitated comparative studies and provided insight on genome evolution. However, data are biased to model systems and marine organisms, including annelids, have been underrepresented in transposable elements studies. Here, we focus on genome of Lamellibrachia luymesi, a vestimentiferan tubeworm from deep-sea hydrocarbon seeps, to gain knowledge of LTR retrotransposons in a deep-sea annelid. Results We characterized LTR retrotransposons present in the genome of L. luymesi using bioinformatic approaches and found that intact LTR retrotransposons makes up about 0.1% of L. luymesi genome. Previous characterization of the genome has shown that this tubeworm hosts several known LTR-retrotransposons. Here we describe and classify LTR retrotransposons in L. luymesi as within the Gypsy, Copia and Bel-pao superfamilies. Although, many elements fell within already recognized families (e.g., Mag, CSRN1), others formed clades distinct from previously recognized families within these superfamilies. However, approximately 19% (41) of recovered elements could not be classified. Gypsy elements were the most abundant while only 2 Copia and 2 Bel-pao elements were present. In addition, analysis of insertion times indicated that several LTR-retrotransposons were recently transposed into the genome of L. luymesi, these elements had identical LTR’s raising possibility of recent or ongoing retrotransposon activity. Conclusions Our analysis contributes to knowledge on diversity of LTR-retrotransposons in marine settings and also serves as an important step to assist our understanding of the potential role of retroelements in marine organisms. We find that many LTR retrotransposons, which have been inserted in the last few million years, are similar to those found in terrestrial model species. However, several new groups of LTR retrotransposons were discovered suggesting that the representation of LTR retrotransposons may be different in marine settings. Further study would improve understanding of the diversity of retrotransposons across animal groups and environments.

Robust expression of LINE-1 retrotransposon encoded proteins in oral squamous cell carcinoma

Background Oral Squamous Cell Carcinoma (OSCC) results from a series of genetic alteration in squamous cells. This particular type of cancer considers one of the most aggressive malignancies to control because of its frequent local invasions to the regional lymph node. Although several biomarkers have been reported, the key marker used to predict the behavior of the disease is largely unknown. Here we report Long INterpersed Element-1 (LINE1 or L1) retrotransposon activity in post-operative oral cancer samples. L1 is the only active retrotransposon occupying around 17% of the human genome with an estimated 500,000 copies. An active L1 encodes two proteins (L1ORF1p and L1ORF2p); both of which are critical in the process of retrotransposition. Several studies report that the L1 retrotransposon is highly active in many cancers. L1 activity is generally determined by assaying L1ORF1p because of its high expression and availability of the antibody. However, due to its lower expression and unavailability of a robust antibody, detection of L1ORF2p has been limited. L1ORF2p is the crucial protein in the process of retrotransposition as it provides endonuclease and reverse transcriptase (RT) activity. Methods Immunohistochemistry and Western blotting were performed on the post-operative oral cancer samples and murine tissues. Results Using in house novel antibodies against both the L1 proteins (L1ORF1p and L1ORF2p), we found L1 retrotransposon is extremely active in post-operative oral cancer tissues. Here, we report a novel human L1ORF2p antibody generated using an 80-amino-acid stretch from the RT domain, which is highly conserved among different species. The antibody detects significant L1ORF2p expression in human oral squamous cell carcinoma (OSCC) samples and murine germ tissues. Conclusions We report exceptionally high L1ORF1p and L1ORF2p expression in post-operative oral cancer samples. The novel L1ORF2p antibody reported in this study will serve as a useful tool to understand why L1 activity is deregulated in OSCC and how it contributes to the progression of this particular cancer. Cross-species reactivity of L1ORF2p antibody due to the conserved epitope will be useful to study the retrotransposon biology in mice and rat germ tissues.

Genome stability is in the eye of the beholder: recent retrotransposon activity varies significantly across avian diversity

Since the sequencing of the zebra finch genome it has become clear the avian genome, while largely stable in terms of chromosome number and gene synteny, is more dynamic at an intrachromosomal level. A multitude of intrachromosomal rearrangements and significant variation in transposable element content have been noted across the avian tree. Transposable elements (TEs) are a source of genome plasticity, because their high similarity enables chromosomal rearrangements through non-allelic homologous recombination, and they have potential for exaptation as regulatory and coding sequences. Previous studies have investigated the activity of the dominant TE in birds, CR1 retrotransposons, either focusing on their expansion within single orders, or comparing passerines to non-passerines. Here we comprehensively investigate and compare the activity of CR1 expansion across orders of birds, finding levels of CR1 activity vary significantly both between and with orders. We describe high levels of TE expansion in genera which have speciated in the last 10 million years including kiwis, geese and Amazon parrots; low levels of TE expansion in songbirds across their diversification, and near inactivity of TEs in the cassowary and emu for millions of years. CR1s have remained active over long periods of time across most orders of neognaths, with activity at any one time dominated by one or two families of CR1s. Our findings of higher TE activity in species-rich clades and dominant families of TEs within lineages mirror past findings in mammals.Author SummaryTransposable elements (TEs) are mobile, self replicating DNA sequences within a species’ genome, and are ubiquitous sources of mutation. The dominant group of TEs within birds are chicken repeat 1 (CR1) retrotransposons, making up 7-10% of the typical avian genome. Because past research has examined the recent inactivity of CR1s within model birds such as the chicken and the zebra finch, this has fostered an erroneous view that all birds have low or no TE activity on recent timescales. Our analysis of numerous high quality avian genomes across multiple orders identified both similarities and significant differences in how CR1s expanded. Our results challenge the established view that TEs in birds are largely inactive and instead suggest that their variation in recent activity may contribute to lineage-specific changes in genome structure. Many of the patterns we identify in birds have previously been seen in mammals, highlighting parallels between the evolution of birds and mammals.

Long-read assembly of a Great Dane genome highlights the contribution of GC-rich sequence and mobile elements to canine genomes

Technological advances have allowed improvements in genome reference sequence assemblies. Here, we combined long- and short-read sequence resources to assemble the genome of a female Great Dane dog. This assembly has improved continuity compared to the existing Boxer-derived (CanFam3.1) reference genome. Annotation of the Great Dane assembly identified 22,182 protein-coding gene models and 7,049 long noncoding RNAs, including 49 protein-coding genes not present in the CanFam3.1 reference. The Great Dane assembly spans the majority of sequence gaps in the CanFam3.1 reference and illustrates that 2,151 gaps overlap the transcription start site of a predicted protein-coding gene. Moreover, a subset of the resolved gaps, which have an 80.95% median GC content, localize to transcription start sites and recombination hotspots more often than expected by chance, suggesting the stable canine recombinational landscape has shaped genome architecture. Alignment of the Great Dane and CanFam3.1 assemblies identified 16,834 deletions and 15,621 insertions, as well as 2,665 deletions and 3,493 insertions located on secondary contigs. These structural variants are dominated by retrotransposon insertion/deletion polymorphisms and include 16,221 dimorphic canine short interspersed elements (SINECs) and 1,121 dimorphic long interspersed element-1 sequences (LINE-1_Cfs). Analysis of sequences flanking the 3′ end of LINE-1_Cfs (i.e., LINE-1_Cf 3′-transductions) suggests multiple retrotransposition-competent LINE-1_Cfs segregate among dog populations. Consistent with this conclusion, we demonstrate that a canine LINE-1_Cf element with intact open reading frames can retrotranspose its own RNA and that of a SINEC_Cf consensus sequence in cultured human cells, implicating ongoing retrotransposon activity as a driver of canine genetic variation.

MicroRNA retrocopies generated via L1-mediated retrotransposition in placental mammals help to reveal how their parental genes were transcribed

In mammalian genomes, most retrocopies emerged via the L1 retrotransposition machinery. The hallmarks of an L1-mediated retrocopy, i.e., the intronlessness, the presence of a 3′ poly-A tail, and the TSDs at both ends, were frequently used to identify retrotransposition events. However, most previous studies only focused on protein-coding genes as their possible parental sources and thus only a few retrocopies derived from non-coding genes were reported. Remarkably, none of them was from microRNAs. Here in this study, we found several retrocopies generated from the mir-302–367 cluster gene (MIR302CHG), and identified a novel alternatively spliced exon encoding mir-302a. The other recognized microRNA retrotransposition events are primate-specific with mir-373 and mir-498 as their parental genes. The 3′ poly-A tracts of these two retrocopy groups were directly attached to the end of the microRNA precursor homologous regions, which suggests that their parental transcripts might alternatively terminate at the end of mir-373 and mir-498. All the three parental microRNAs are highly expressed in specific tissues with elevated retrotransposon activity, such as the embryonic stem cells and the placenta. This might be the reason that our first microRNA retrocopy findings were derived from these three microRNA genes.