Flip-flop genomics: Charting inversions in the human population.

Detecting large genomic inversions has long been challenging. In a new study, Porubsky et al. resolve these complex rearrangements in 41 individuals and discover wide regions that undergo recurrent inversions, some of which even toggle back and forth (Porubsky et al., 2022). Many of these regions are associated with genomic disorders.

The Landscape of L1 Retrotransposons in the Human Genome Is Shaped by Pre-insertion Sequence Biases and Post-insertion Selection.

L1 retrotransposons are transposable elements and major contributors of genetic variation in humans. Where L1 integrates into the genome can directly impact human evolution and disease. Here, we experimentally induced L1 retrotransposition in cells and mapped integration sites at nucleotide resolution. At local scales, L1 integration is mostly restricted by genome sequence biases and the specificity of the L1 machinery. At regional scales, L1 shows a broad capacity for integration into all chromatin states, in contrast to other known mobile genetic elements. However, integration is influenced by the replication timing of target regions, suggesting a link to host DNA replication. The distribution of new L1 integrations differs from those of preexisting L1 copies, which are significantly reshaped by natural selection. Our findings reveal that the L1 machinery has evolved to efficiently target all genomic regions and underline a predominant role for post-integrative processes on the distribution of endogenous L1 elements.

Measuring and interpreting transposable element expression.

Transposable elements (TEs) are insertional mutagens that contribute greatly to the plasticity of eukaryotic genomes, influencing the evolution and adaptation of species as well as physiology or disease in individuals. Measuring TE expression helps to understand not only when and where TE mobilization can occur but also how this process alters gene expression, chromatin accessibility or cellular signalling pathways. Although genome-wide gene expression assays such as RNA sequencing include transposon-derived transcripts, most computational analytical tools discard or misinterpret TE-derived reads. Emerging approaches are improving the identification of expressed TE loci and helping to discriminate TE transcripts that permit TE mobilization from chimeric gene-TE transcripts or pervasive transcription. Here we review the main challenges associated with the detection of TE expression, including mappability, insertional and internal sequence polymorphisms, and the diversity of the TE transcriptional landscape, as well as the different experimental and computational strategies to solve them.

RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila.

How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.

A genetic screen identifies BEND3 as a regulator of bivalent gene expression and global DNA methylation.

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elements and higher-order chromatin structures. Consequently, these chromatin marks are indispensable for mammalian development and alterations often lead to disease, such as cancer. Bivalent promoters are especially important during differentiation and development. Here we used a genetic screen to identify new regulators of a bivalent repressed gene. We identify BEND3 as a regulator of hundreds of bivalent promoters, some of which it represses, and some of which it activates. We show that BEND3 is recruited to a CpG-containg consensus site that is present in multiple copies in many bivalent promoters. Besides having direct effect on the promoters it binds, the loss of BEND3 leads to genome-wide gains of DNA methylation, which are especially marked at regions normally protected by the TET enzymes. DNA hydroxymethylation is reduced in Bend3 mutant cells, possibly as consequence of altered gene expression leading to diminished alpha-ketoglutarate production, thus lowering TET activity. Our results clarify the direct and indirect roles of an important chromatin regulator, BEND3, and, more broadly, they shed light on the regulation of bivalent promoters.

Locus-specific chromatin profiling of evolutionarily young transposable elements.

Despite a vast expansion in the availability of epigenomic data, our knowledge of the chromatin landscape at interspersed repeats remains highly limited by difficulties in mapping short-read sequencing data to these regions. In particular, little is known about the locus-specific regulation of evolutionarily young transposable elements (TEs), which have been implicated in genome stability, gene regulation and innate immunity in a variety of developmental and disease contexts. Here we propose an approach for generating locus-specific protein-DNA binding profiles at interspersed repeats, which leverages information on the spatial proximity between repetitive and non-repetitive genomic regions. We demonstrate that the combination of HiChIP and a newly developed mapping tool (PAtChER) yields accurate protein enrichment profiles at individual repetitive loci. Using this approach, we reveal previously unappreciated variation in the epigenetic profiles of young TE loci in mouse and human cells. Insights gained using our method will be invaluable for dissecting the molecular determinants of TE regulation and their impact on the genome.

Integration site selection by retroviruses and transposable elements in eukaryotes.

Transposable elements and retroviruses are found in most genomes, can be pathogenic and are widely used as gene-delivery and functional genomics tools. Exploring whether these genetic elements target specific genomic sites for integration and how this preference is achieved is crucial to our understanding of genome evolution, somatic genome plasticity in cancer and ageing, host-parasite interactions and genome engineering applications. High-throughput profiling of integration sites by next-generation sequencing, combined with large-scale genomic data mining and cellular or biochemical approaches, has revealed that the insertions are usually non-random. The DNA sequence, chromatin and nuclear context, and cellular proteins cooperate in guiding integration in eukaryotic genomes, leading to a remarkable diversity of insertion site distribution and evolutionary strategies.

Inflammatory facioscapulohumeral muscular dystrophy type 2 in 18p deletion syndrome.

Facioscapulohumeral muscular dystrophy (FSHD) has been shown to be related to genetic and epigenetic derepression of DUX4 (mapping to chromosome 4), a gene located within a repeat array of D4Z4 sequences of polymorphic length. FSHD type 1 (FSHD1) is associated with pathogenic D4Z4 repeat array contraction, while FSHD type 2 (FSHD2) is associated with SMCHD1 variants (a chromatin modifier gene that maps to the short arm of chromosome 18). Both FSHD types require permissive polyadenylation signal (4qA) downstream of the D4Z4 array.

euL1db: the European database of L1HS retrotransposon insertions in humans.

Retrotransposons account for almost half of our genome. They are mobile genetics elements-also known as jumping genes--but only the L1HS subfamily of Long Interspersed Nuclear Elements (LINEs) has retained the ability to jump autonomously in modern humans. Their mobilization in germline--but also some somatic tissues--contributes to human genetic diversity and to diseases, such as cancer. Here, we present euL1db, the European database of L1HS retrotransposon insertions in humans (available at http://euL1db.unice.fr). euL1db provides a curated and comprehensive summary of L1HS insertion polymorphisms identified in healthy or pathological human samples and published in peer-reviewed journals. A key feature of euL1db is its sample--wise organization. Hence L1HS insertion polymorphisms are connected to samples, individuals, families and clinical conditions. The current version of euL1db centralizes results obtained in 32 studies. It contains >900 samples, >140,000 sample-wise insertions and almost 9000 distinct merged insertions. euL1db will help understanding the link between L1 retrotransposon insertion polymorphisms and phenotype or disease.

The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

Dismantling papillary renal cell carcinoma classification: The heterogeneity of genetic profiles suggests several independent diseases.

Papillary renal cell carcinoma (pRCC) is the second most frequent renal cell carcinoma (RCC) after clear cell RCC. In contrast to clear cell RCC, there is no consensual protocol using targeted therapy for metastatic pRCC. Moreover, diagnosis of some pRCC, especially pRCC of type 2 (pRCC2) may be challenging. Our aim was to identify molecular biomarkers that could be helpful for the diagnosis and treatment of pRCC. We studied the clinical, histological, immunohistological, and comprehensive genetic features of a series of 31 pRCC including 15 pRCC1 and 16 pRCC2. We aimed to determine whether pRCC represents a unique entity or several diseases. In addition, we compared the genetic features of pRCC2 to those of eight RCC showing various degrees of tubulo-papillary architecture, including three TFE-translocation RCC and five unclassified RCC. We demonstrate that pRCC is a heterogeneous group of tumors with distinct evolution. While most pRCC2 had genetic profiles similar to pRCC1, some shared genomic features, such as loss of 3p and loss of chromosome 14, with clear cell RCC, TFE-translocation RCC, and unclassified RCC. We identified variants of the MET gene in three pRCC1. A mutation in the BRAF gene was also identified in one pRCC1. In addition, using next-generation sequencing (NGS), we identified several variant genes. Genomic profiling completed by NGS allowed us to classify pRCC2 in several groups and to identify novel mutations. Our findings provide novel information on the pathogenesis of pRCC that allow insights for personalized treatment.

A single zinc finger optimizes the DNA interactions of the nucleocapsid protein of the yeast retrotransposon Ty3.

Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which drive the rearrangement of nucleic acid conformation. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein have been extensively studied, and nucleic acid aggregation, duplex destabilization and rapid binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as retrotransposon Ty3 NC, a likely ancestor of HIV-1 NC, are not well understood. In addition, it is unclear whether a single zinc finger is sufficient to optimize the properties characteristic of HIV-1 NC. We used single-molecule DNA stretching as a method for detailed characterization of Ty3 NC chaperone activity. We found that wild type Ty3 NC aggregates single- and double-stranded DNA, weakly stabilizes dsDNA, and exhibits rapid binding kinetics. Single-molecule studies in the presence of Ty3 NC mutants show that the N-terminal basic residues and the unique zinc finger at the C-terminus are required for optimum chaperone activity in this system. While the single zinc finger is capable of optimizing Ty3 NC's DNA interaction kinetics, two zinc fingers may be necessary in order to facilitate the DNA destabilization exhibited by HIV-1 NC.

Locus-level L1 DNA methylation profiling reveals the epigenetic and transcriptional interplay between L1s and their integration sites.

Long interspersed element 1 (L1) retrotransposons are implicated in human disease and evolution. Their global activity is repressed by DNA methylation, but deciphering the regulation of individual copies has been challenging. Here, we combine short- and long-read sequencing to unveil L1 methylation heterogeneity across cell types, families, and individual loci and elucidate key principles involved. We find that the youngest primate L1 families are specifically hypomethylated in pluripotent stem cells and the placenta but not in most tumors. Locally, intronic L1 methylation is intimately associated with gene transcription. Conversely, the L1 methylation state can propagate to the proximal region up to 300 bp. This phenomenon is accompanied by the binding of specific transcription factors, which drive the expression of L1 and chimeric transcripts. Finally, L1 hypomethylation alone is typically insufficient to trigger L1 expression due to redundant silencing pathways. Our results illuminate the epigenetic and transcriptional interplay between retrotransposons and their host genome.

A novel bioinformatic approach reveals cooperation between Cancer/Testis genes in basal-like breast tumors.

Breast cancer is the most prevalent type of cancer in women worldwide. Within breast tumors, the basal-like subtype has the worst prognosis, prompting the need for new tools to understand, detect, and treat these tumors. Certain germline-restricted genes show aberrant expression in tumors and are known as Cancer/Testis genes; their misexpression has diagnostic and therapeutic applications. Here we designed a new bioinformatic approach to examine Cancer/Testis gene misexpression in breast tumors. We identify several new markers in Luminal and HER-2 positive tumors, some of which predict response to chemotherapy. We then use machine learning to identify the two Cancer/Testis genes most associated with basal-like breast tumors: HORMAD1 and CT83. We show that these genes are expressed by tumor cells and not by the microenvironment, and that they are not expressed by normal breast progenitors; in other words, their activation occurs de novo. We find these genes are epigenetically repressed by DNA methylation, and that their activation upon DNA demethylation is irreversible, providing a memory of past epigenetic disturbances. Simultaneous expression of both genes in breast cells in vitro has a synergistic effect that increases stemness and activates a transcriptional profile also observed in double-positive tumors. Therefore, we reveal a functional cooperation between Cancer/Testis genes in basal breast tumors; these findings have consequences for the understanding, diagnosis, and therapy of the breast tumors with the worst outcomes.

The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts.

Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

Genome-Wide Young L1 Methylation Profiling by bs-ATLAS-seq.

By silencing L1 retrotransposons, DNA methylation protects mammalian genomes from potent endogenous mutagens. However, some loci can escape this repressive mechanism and become active, particularly in carcinomas. Alterations of L1 DNA methylation can also locally influence gene expression. Comprehensive measurement of L1 DNA methylation at the locus level remains challenging. Here, we present bs-ATLAS-seq, a genome-wide approach to locate full-length L1 elements in the human genome, and assess their methylation levels at single-base and single-locus resolutions. This strategy targets the youngest, and only retrotransposition-competent family, L1HS, but also detects a significant fraction of older elements (L1PA2 to L1PA8). Bs-ATLAS-seq evaluates methylation at the first 15 CpGs of L1 5' UTR, which corresponds to the first half of the sense promoter. It relies on random fragmentation of the genomic DNA, adapter ligation, bisulfite treatment and suppression PCR, and ends by asymmetrical paired-end sequencing. A dedicated pipeline provides the location of L1 elements and their methylation status, including for non-reference loci, as well as their single-molecule DNA profiles.

Noninvasive and Multicancer Biomarkers: The Promise of LINE-1 Retrotransposons.

SUMMARY: LINE-1 retrotransposons are frequently active in epithelial tumors. In a new study, Taylor, Wu and colleagues now describe that one of the proteins encoded by LINE-1 elements, ORF1p, is detected in the bloodstream of patients with cancer, and can be used as a noninvasive and multicancer biomarker for diagnosis or treatment monitoring. See related article by Taylor, Wu et al., p. 2532 (7).

Targeted Nanopore Resequencing and Methylation Analysis of LINE-1 Retrotransposons.

Retrotransposition of LINE-1 (L1) elements represents a major source of insertional polymorphisms in mammals, and their mutagenic activity is restricted by silencing mechanisms, such as DNA methylation. Despite a very high level of sequence identity between copies, their internal sequence contains small nucleotide polymorphisms (SNPs) that can alter their activity. Such internal SNPs can also appear in different alleles of a given L1 locus. Given their repetitive nature and relatively long size, short-read sequencing approaches have limited access to L1 internal sequence or DNA methylation state. Here, we describe a targeted method to specifically sequence more than a hundred L1-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. Our protocol, modified from the nanopore Cas9 targeted sequencing (nCATS) strategy, provides a full and haplotype-resolved L1 sequence and DNA methylation levels. It introduces a streamlined and multiplex approach to synthesize guide RNAs and a quantitative PCR (qPCR)-based quality check during library preparation for cost-effective L1 sequencing. More generally, this method can be applied to any type of transposable elements and organisms.