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ASXL transcriptional regulator 1 (ASXL1) is one of the three most frequently mutated genes in age-related clonal hematopoiesis (CH), alongside DNA methyltransferase 3 alpha (DNMT3A) and Tet methylcytosine dioxygenase 2 ( TET2) . CH can progress to myeloid malignancies including chronic monomyelocytic leukemia (CMML) and is also strongly associated with inflammatory cardiovascular disease and all-cause mortality in humans. DNMT3A and TET2 regulate DNA methylation and demethylation pathways, respectively, and loss-of-function mutations in these genes reduce DNA methylation in heterochromatin, allowing derepression of silenced elements in heterochromatin. In contrast, the mechanisms that connect mutant ASXL1 and CH are not yet fully understood. CH/CMML-associated ASXL1 mutations encode C-terminally truncated proteins that enhance the deubiquitinase activity of the ASXL-BAP1 "PR-DUB" deubiquitinase complex, which removes monoubiquitin from H2AK119Ub. Here, we show that ASXL1 mutant proteins interact with the euchromatic histone lysine methyltransferases 1 and 2 (EHMT1-EHMT2) complex, which generates H3K9me1 and me2, the latter a repressive modification in constitutive heterochromatin. Compared to cells from age-matched wild-type mice, we found that expanded myeloid cells from old (≥18-mo-old) Asxl1tm/+ mice, a heterozygous knock-in mouse model of CH, display genome-wide decreases of H3K9me2, H3K9me3, and H2AK119Ub as well as an associated increase in expression of transposable elements (TEs) and satellite repeats. Increased TE expression was also observed in monocytes from ASXL1 -mutant CMML patients compared to monocytes from healthy controls. Our data suggest that mutant ASXL1 proteins compromise the integrity of both constitutive and facultative heterochromatin in an age-dependent manner by reducing the levels of H3K9me2/3 and H2AK119Ub. This increase in TE expression correlated with increased expression of nearby genes, including many interferon-inducible (inflammation-associated) genes (ISGs)., Competing Interests: Competing interests statement:A.R. is a member of the Scientific Advisory Board of biomodal, formerly Cambridge Epigenetix. All other authors declare no competing interests.

ASXL1 is one of the three most frequently mutated genes in age-related clonal hematopoiesis (CH), alongside DNMT3A and TET2 . CH can progress to myeloid malignancies including chronic monomyelocytic leukemia (CMML), and is also strongly associated with inflammatory cardiovascular disease and all-cause mortality in humans. DNMT3A and TET2 regulate DNA methylation and demethylation pathways respectively, and loss-of-function mutations in these genes reduce DNA methylation in heterochromatin, allowing de-repression of silenced elements in heterochromatin. In contrast, the mechanisms that connect mutant ASXL1 and CH are not yet fully understood. CH/CMML-associated ASXL1 mutations encode C-terminally truncated proteins that enhance the deubiquitinase activity of the ASXL-BAP1 "PR-DUB" deubiquitinase complex, which removes mono-ubiquitin from H2AK119Ub. Here we show that ASXL1 mutant proteins interact with the EHMT1-EHMT2 methyltransferase complex, which generates H3K9me1 and me2, the latter a repressive modification in constitutive heterochromatin. Compared to cells from age-matched wildtype mice, we found that expanded myeloid cells from old (≥18-month-old) Asxl1tm/+ mice, a heterozygous knock-in mouse model of CH, display genome-wide decreases of H3K9me2, H3K9me3 and H2AK119Ub as well as an associated increase in expression of transposable elements (TEs) and satellite repeats. Increased TE expression was also observed in monocytes from ASXL1 -mutant CMML patients compared to monocytes from healthy controls. Our data suggest that mutant ASXL1 proteins compromise the integrity of both constitutive and facultative heterochromatin in an age-dependent manner, by reducing the levels of H3K9me2/3 and H2AK119Ub. This increase in TE expression correlated with increased expression of nearby genes, including many interferon-inducible (inflammation-associated) genes (ISGs)., Significance Statement: Age-related clonal hematopoiesis (CH) is a premalignant condition associated with inflammatory cardiovascular disease. ASXL1 mutations are very frequent in CH. We show that ASXL1 interacts with EHMT1 and EHMT2, H3K9 methyltransferases that deposit H3K9me1 and me2. Using a mouse model of mutant ASXL1 to recapitulate CH, we found that old ASXL1-mutant mice showed marked expansion of myeloid cells in bone marrow, with decreased H3K9me2/3 and increased expression of transposable elements (TEs) in heterochromatin. In humans, ASXL1-mutant CH progresses to chronic monomyelocytic leukemia (CMML); CMML patient samples showed striking upregulation of many TE families, suggesting that ASXL1 mutations compromise heterochromatin integrity, hence causing derepression of TEs. Targeting heterochromatin-associated proteins and TEs might counter the progression of CH, CMML and other myeloid malignancies.

Cell types are traditionally thought to be specified and stabilized by gene regulatory networks. Here, we explore how chromatin memory contributes to the specification and stabilization of cell states. Through pervasive, local, feedback loops, chromatin memory enables cell states that were initially unstable to become stable. Deeper appreciation of this self-stabilizing role for chromatin broadens our perspective of Waddington's epigenetic landscape from a static surface with islands of stability shaped by evolution, to a plasticine surface molded by experience. With implications for the evolution of cell types, stabilization of resistant states in cancer, and the widespread plasticity of complex life., Competing Interests: Declarations. Competing interests: G.J.F. is a member of the Genome Biology editorial board. All other authors declare no competing interests., (© 2024. The Author(s).)

In this issue of Molecular Cell, Danac et al. 1 identify a second HUSH complex, HUSH2, that represses interferon-stimulated genes and, by competing for subunits with the canonical HUSH complex, couples LINE-1 (L1) retrotransposon transcription with immune activation., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Retrotransposons are mobile DNA sequences duplicated via transcription and reverse transcription of an RNA intermediate. Cis-regulatory elements encoded by retrotransposons can also promote the transcription of adjacent genes. Somatic LINE-1 (L1) retrotransposon insertions have been detected in mammalian neurons. It is, however, unclear whether L1 sequences are mobile in only some neuronal lineages or therein promote neurodevelopmental gene expression. Here we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development. Mouse PV interneurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters and express full-length L1 mRNAs and proteins. Using nanopore long-read sequencing, we identify unmethylated L1s proximal to PV interneuron genes, including a novel L1 promoter-driven Caps2 transcript isoform that enhances neuron morphological complexity in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV interneuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu., (© 2024. The Author(s).)

Whole genome sequencing of viruses provides high-resolution molecular insights, enhancing our understanding of viral genome function and phylogeny. Beyond fundamental research, viral sequencing is increasingly vital for pathogen surveillance, epidemiology, and clinical applications. As sequencing methods rapidly evolve, the diversity of viral genomics applications and catalogued genomes continues to expand. Advances in long-read, single molecule, real-time sequencing methodologies present opportunities to sequence contiguous, haplotype resolved viral genomes in a range of research and applied settings. Here we present an overview of nucleic acid sequencing methods and their applications in studying viral genomes. We emphasise the advantages of different viral sequencing approaches, with a particular focus on the benefits of third-generation sequencing technologies in elucidating viral evolution, transmission networks, and pathogenesis., (© 2024 The Author(s).)

Eukaryotic transcription factors (TFs) activate gene expression by recruiting cofactors to promoters. However, the relationships between TFs, promoters and their associated cofactors remain poorly understood. Here we combine GAL4-transactivation assays with comparative CRISPR-Cas9 screens to identify the cofactors used by nine different TFs and core promoters in human cells. Using this dataset, we associate TFs with cofactors, classify cofactors as ubiquitous or specific and discover transcriptional co-dependencies. Through a reductionistic, comparative approach, we demonstrate that TFs do not display discrete mechanisms of activation. Instead, each TF depends on a unique combination of cofactors, which influences distinct steps in transcription. By contrast, the influence of core promoters appears relatively discrete. Different promoter classes are constrained by either initiation or pause-release, which influences their dynamic range and compatibility with cofactors. Overall, our comparative cofactor screens characterize the interplay between TFs, cofactors and core promoters, identifying general principles by which they influence transcription., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The O- GlcNAc transferase OGT interacts robustly with all three mammalian TET methylcytosine dioxygenases. We show here that deletion of the Ogt gene in mouse embryonic stem cells (mESC) results in a widespread increase in the TET product 5-hydroxymethylcytosine (5hmC) in both euchromatic and heterochromatic compartments, with concomitant reduction of the TET substrate 5-methylcytosine (5mC) at the same genomic regions. mESC engineered to abolish the TET1-OGT interaction likewise displayed a genome-wide decrease of 5mC. DNA hypomethylation in OGT-deficient cells was accompanied by de-repression of transposable elements (TEs) predominantly located in heterochromatin, and this increase in TE expression was sometimes accompanied by increased cis -expression of genes and exons located 3' of the expressed TE. Thus, the TET-OGT interaction prevents DNA demethylation and TE expression in heterochromatin by restraining TET activity genome-wide. We suggest that OGT protects the genome against DNA hypomethylation and impaired heterochromatin integrity, preventing the aberrant increase in TE expression observed in cancer, autoimmune-inflammatory diseases, cellular senescence and ageing.

Genomic regulation of cardiomyocyte differentiation is central to heart development and function. This study uses genetic loss-of-function human-induced pluripotent stem cell-derived cardiomyocytes to evaluate the genomic regulatory basis of the non-DNA-binding homeodomain protein HOPX. We show that HOPX interacts with and controls cardiac genes and enhancer networks associated with diverse aspects of heart development. Using perturbation studies in vitro, we define how upstream cell growth and proliferation control HOPX transcription to regulate cardiac gene programs. We then use cell, organoid, and zebrafish regeneration models to demonstrate that HOPX-regulated gene programs control cardiomyocyte function in development and disease. Collectively, this study mechanistically links cell signaling pathways as upstream regulators of HOPX transcription to control gene programs underpinning cardiomyocyte identity and function., Competing Interests: Declaration of interests E.P., R.J.M., and J.E.H. are co-inventors on patents relating to cardiac organoid maturation and cardiac therapeutics. J.E.H. is co-inventor on licensed patents for engineered heart muscle. E.P., R.J.M., and J.E.H. are co-founders, scientific advisors, and stockholders in Dynomics. N.J.P. is an inventor on patents relating to stem cells and heart regeneration and is a co-founder and equity holder in Infensa Bioscience., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cholinergic signaling plays a crucial role in the regulation of adult hippocampal neurogenesis; however, the mechanisms by which acetylcholine mediates neurogenic effects are not completely understood. Here, we report the expression of muscarinic acetylcholine receptor subtype M4 (M4 mAChR) on a subpopulation of neural precursor cells (NPCs) in the adult mouse hippocampus, and demonstrate that its pharmacological stimulation promotes their proliferation, thereby enhancing the production of new neurons in vivo. Using a targeted ablation approach, we also show that medial septum (MS) and the diagonal band of Broca (DBB) cholinergic neurons support both the survival and morphological maturation of adult-born neurons in the mouse hippocampus. Although the systemic administration of an M4-selective allosteric potentiator fails to fully rescue the MS/DBB cholinergic lesion-induced decrease in hippocampal neurogenesis, it further exacerbates the impairment in the morphological maturation of adult-born neurons. Collectively, these findings reveal stage-specific roles of M4 mAChRs in regulating adult hippocampal neurogenesis, uncoupling their positive role in enhancing the production of new neurons from the M4-induced inhibition of their morphological maturation, at least in the context of cholinergic signaling dysfunction., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Mice harbor ∼2800 intact copies of the retrotransposon Long Interspersed Element 1 (L1). The in vivo retrotransposition capacity of an L1 copy is defined by both its sequence integrity and epigenetic status, including DNA methylation of the monomeric units constituting young mouse L1 promoters. Locus-specific L1 methylation dynamics during development may therefore elucidate and explain spatiotemporal niches of endogenous retrotransposition but remain unresolved. Here, we interrogate the retrotransposition efficiency and epigenetic fate of source (donor) L1s, identified as mobile in vivo. We show that promoter monomer loss consistently attenuates the relative retrotransposition potential of their offspring (daughter) L1 insertions. We also observe that most donor/daughter L1 pairs are efficiently methylated upon differentiation in vivo and in vitro. We use Oxford Nanopore Technologies (ONT) long-read sequencing to resolve L1 methylation genome-wide and at individual L1 loci, revealing a distinctive "smile" pattern in methylation levels across the L1 promoter region. Using Pacific Biosciences (PacBio) SMRT sequencing of L1 5' RACE products, we then examine DNA methylation dynamics at the mouse L1 promoter in parallel with transcription start site (TSS) distribution at locus-specific resolution. Together, our results offer a novel perspective on the interplay between epigenetic repression, L1 evolution, and genome stability., (© 2023 Gerdes et al.; Published by Cold Spring Harbor Laboratory Press.)

Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function 1-8 . These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory., (© 2023. The Author(s).)

How does neurodegeneration spread in the brain? Leveraging TDP-43 fly models of amyotrophic lateral sclerosis (ALS), Chang and Dubnau recently reported that the endogenous retrovirus (ERV) mdg4 can trigger and transmit TDP-43 proteinopathy in vivo. Their results suggest that human ERVs could be targeted to develop future ALS therapies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Accumulation of somatic mutations in human neurons is associated with aging and neurodegeneration. To shed light on the somatic mutational burden in Alzheimer's disease (AD) neurons and get more insight into the role of somatic mutations in AD pathogenesis, we performed single-neuron whole genome sequencing to detect genome-wide somatic mutations (single nucleotide variants (SNVs) and Indels) in 96 single prefrontal cortex neurons from 8 AD patients and 8 elderly controls. We found that the mutational burden is ∼3000 somatic mutations per neuron genome in elderly subjects. AD patients have increased somatic mutation burden in AD-related annotation categories, including AD risk genes and differentially expressed genes in AD neurons. Mutational signature analysis showed somatic SNVs (sSNVs) primarily caused by aging and oxidative DNA damage processes but no significant difference was detected between AD and controls. Additionally, functional somatic mutations identified in AD patients showed significant enrichment in several AD-related pathways, including AD pathway, Notch-signaling pathway and Calcium-signaling pathway. These findings provide genetic insights into how somatic mutations may alter the function of single neurons and exert their potential roles in the pathogenesis of AD., Competing Interests: Disclosure statement The authors report no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Molecular characterisation of hepatocellular carcinoma (HCC) is central to the development of novel therapeutic strategies for the disease. We have previously demonstrated mutagenic consequences of Long-Interspersed Nuclear Element-1 (LINE1s/L1) retrotransposition. However, the role of L1 in HCC, besides somatic mutagenesis, is not well understood., Methods: We analysed L1 expression in the TCGA-HCC RNAseq dataset (n = 372) and explored potential relationships between L1 expression and clinical features. The findings were confirmed by immunohistochemical (IHC) analysis of an independent human HCC cohort (n = 48) and functional mechanisms explored using in vitro and in vivo model systems., Results: We observed positive associations between L1 and activated TGFβ-signalling, TP53 mutation, alpha-fetoprotein and tumour invasion. IHC confirmed a positive association between pSMAD3, a surrogate for TGFβ-signalling status, and L1 ORF1p (P < 0.0001, n = 32). Experimental modulation of L1 ORF1p levels revealed an influence of L1 ORF1p on key hepatocarcinogenesis-related pathways. Reduction in cell migration and invasive capacity was observed upon L1 ORF1 knockdown, both in vitro and in vivo. In particular, L1 ORF1p increased PIN1 cytoplasmic localisation. Blocking PIN1 activity abrogated L1 ORF1p-induced NF-κB-mediated inflammatory response genes while further activated TGFβ-signalling confirming differential alteration of PIN1 activity in cellular compartments by L1 ORF1p., Discussion: Our data demonstrate a causal link between L1 ORF1p and key oncogenic pathways mediated by PIN1, presenting a novel therapeutic avenue., (© 2023. The Author(s).)

The Central Dogma has been a useful conceptualization of the transfer of genetic information, and our understanding of the detailed mechanisms involved in that transfer continues to evolve. Here, we speak to several scientists about their research, how it influences our understanding of information transfer, and questions for the future., Competing Interests: Declaration of interests T.C. is a member of the scientific advisory boards of Storm Therapeutics, Eikon Therapeutics, and SomaLogic. L.S. sits on the advisory board of Prose Foods. A.E.W. is a member of the scientific advisory board of Molecular Cell., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Over the past 20 years, high-throughput genomic assays have fundamentally changed how transposable elements (TEs) are studied. While short-read DNA sequencing has been at the heart of these efforts, novel technologies that generate longer reads are driving a shift in the field. Long-read sequencing now permits locus-specific approaches to locate individual TE insertions and understand their epigenetic and transcriptional regulation, while still profiling TE activity genome-wide. Here we provide detailed guidelines to implement Oxford Nanopore Technologies (ONT) sequencing to identify polymorphic TE insertions and profile TE epigenetic landscapes. Using human long interspersed element-1 (LINE-1, L1) as an example, we explain the procedures involved, including final visualization, and potential bottlenecks and pitfalls. ONT sequencing will be, in our view, a workhorse technology for the foreseeable future in the TE field., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs., (© 2022. The Author(s).)

The retrotransposon LINE-1 (L1) is central to the recent evolutionary history of the human genome and continues to drive genetic diversity and germline pathogenesis. However, the spatiotemporal extent and biological significance of somatic L1 activity are poorly defined and are virtually unexplored in other primates. From a single L1 lineage active at the divergence of apes and Old World monkeys, successive L1 subfamilies have emerged in each descendant primate germline. As revealed by case studies, the presently active human L1 subfamily can also mobilize during embryonic and brain development in vivo. It is unknown whether nonhuman primate L1s can similarly generate somatic insertions in the brain. Here we applied approximately 40× single-cell whole-genome sequencing (scWGS), as well as retrotransposon capture sequencing (RC-seq), to 20 hippocampal neurons from two rhesus macaques ( Macaca mulatta ). In one animal, we detected and PCR-validated a somatic L1 insertion that generated target site duplications, carried a short 5' transduction, and was present in ∼7% of hippocampal neurons but absent from cerebellum and nonbrain tissues. The corresponding donor L1 allele was exceptionally mobile in vitro and was embedded in PRDM4 , a gene expressed throughout development and in neural stem cells. Nanopore long-read methylome and RNA-seq transcriptome analyses indicated young retrotransposon subfamily activation in the early embryo, followed by repression in adult tissues. These data highlight endogenous macaque L1 retrotransposition potential, provide prototypical evidence of L1-mediated somatic mosaicism in a nonhuman primate, and allude to L1 mobility in the brain over the past 30 million years of human evolution., (© 2022 Billon et al.; Published by Cold Spring Harbor Laboratory Press.)

Garland et al. (2022) discover a physical and functional connection between the HUSH silencing complex and the NEXT nuclear RNA decay complex in mouse embryonic stem cells and conclude HUSH recruits NEXT to degrade prematurely terminated retrotransposon RNAs., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Age is one of the strongest risk factors for the development of neurodegenerative diseases, the majority of which involve misfolded protein aggregates in the brain. These protein aggregates are thought to drive pathology and are attractive targets for the development of new therapies. However, it is unclear how age influences the onset of pathology and the accompanying molecular response. To address this knowledge gap, we used a model of seeded tau pathology to profile the transcriptomic changes in 3 and 12 month old mice in response to developing tau hyperphosphorylation and aggregation. First, we found the burden of hyperphosphorylated tau pathology in mice injected at 12 months of age was moderately reduced compared to animals injected at 3 months. On a molecular level, we found an inflammation-related subset of genes, including C3 and the disease-associated microglia genes Ctsd, Cst7, and Clec7a, were more expressed early in disease in 12 but not 3 month old mice. These findings provide evidence of an early, age-specific response to tau pathology, which could serve as a marker for the severity of downstream pathology., (© 2021 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

Somatic mutations arise randomly or are induced by environmental factors, which may increase the risk of Alzheimer's disease (AD). Identifying somatic mutations in sporadic AD (SAD) may provide new insight of the disease. To evaluate the potential contribution of somatic single nucleotide variations (SNVs), particularly that of well-known AD-candidate genes, we investigated sequencing data sets from four platforms: whole-genome sequencing (WGS), deep whole-exome sequencing (WES) on paired brain and liver samples, RNA sequencing (RNA-seq), and single-cell whole-genome sequencing (scWGS) of brain samples from 16 AD patients and 16 non-AD individuals. We found that the average number, mean variant allele fractions (VAFs) and mutational signatures of somatic SNVs have similar distributions between AD brains and non-AD brains. We did not identify any somatic SNVs within coding regions of the APP, PSEN1, PSEN2, nor in APOE. This study shows that somatic SNVs within the coding region of AD-candidate genes are unlikely to be a common causal factor for SAD., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Processed pseudogenes may serve as a genetic reservoir for evolutionary innovation. Here, we argue that through the activity of long interspersed element-1 retrotransposons, processed pseudogenes disperse coding and noncoding sequences rich with regulatory potential throughout the human genome. While these sequences may appear to be non-functional, a lack of contemporary function does not prohibit future development of biological activity. Here, we discuss the dynamic evolution of certain processed pseudogenes into coding and noncoding genes and regulatory elements, and their implication in wide-ranging biological and pathological processes. Also see the video abstract here: https://youtu.be/iUY&#95;mteVoPI., (© 2021 Wiley Periodicals LLC.)

Hepatitis C virus (HCV) is a common cause of hepatocellular carcinoma (HCC). The activation and mutagenic consequences of L1 retrotransposons in virus-associated-HCC have been documented. However, the direct influence of HCV upon L1 elements is unclear, and is the focus of the present study. L1 transcript expression was evaluated in a publicly available liver tissue RNA-seq dataset from patients with chronic HCV hepatitis (CHC), as well as healthy controls. L1 transcript expression was significantly higher in CHC than in controls. L1orf1p (a L1 encoded protein) expression was observed in six out of 11 CHC livers by immunohistochemistry. To evaluate the influence of HCV on retrotransposition efficiency, in vitro engineered-L1 retrotransposition assays were employed in Huh7 cells in the presence and absence of an HCV replicon. An increased retrotransposition rate was observed in the presence of replicating HCV RNA, and persisted in cells after viral clearance due to sofosbuvir (PSI7977) treatment. Increased retrotransposition could be due to dysregulation of the DNA-damage repair response, including homologous recombination, due to HCV infection. Altogether these data suggest that L1 expression can be activated before oncogenic transformation in CHC patients, with HCV-upregulated retrotransposition potentially contributing to HCC genomic instability and a risk of transformation that persists post-viral clearance.

A recent study proposed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we apply deep (>50×) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2 and do not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolve 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV)-positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions by ONT sequencing. That we find no evidence of SARS-CoV-2 integration suggests that such events are, at most, extremely rare in vivo and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Impaired extinction of conditioned fear is associated with anxiety disorders. Common lifestyle factors, like isolation stress and exercise, may alter the ability to extinguish fear. However, the effect of and interplay between these factors on adolescent fear extinction, and the relevant underlying neural mechanisms are unknown. Here we examined the effects of periadolescent social isolation and physical activity on adolescent fear extinction in rats and explored neurogenesis as a potential mechanism. Isolation stress impaired extinction recall in male adolescents, an effect prevented by exercise. Extinction recall in female adolescents was unaffected by isolation stress. However, exercise disrupted extinction recall in isolated females. Extinction recall in isolated females was positively correlated to the number of immature neurons in the ventral hippocampus, suggesting that exercise affected extinction recall via neurogenesis in females. Pharmacologically suppressing cellular proliferation in isolated adolescents using temozolomide blocked the effect of exercise on extinction recall in both sexes. Together, these findings highlight sex-specific outcomes of isolation stress and exercise on adolescent brain and behavior, and highlights neurogenesis as a potential mechanism underlying lifestyle effects on adolescent fear extinction., Competing Interests: None., (© 2021 The Authors. Published by Elsevier Inc.)

Endogenous retroviruses (ERVs) are emerging as promising therapeutic targets in cancer. As remnants of ancient retroviral infections, ERV-derived regulatory elements coordinate expression from gene networks, including those underpinning embryogenesis and immune cell function. ERV activation can promote an interferon response, a phenomenon termed viral mimicry. Although ERV expression is associated with cancer, and provisionally with autoimmune and neurodegenerative diseases, ERV-mediated inflammation is being explored as a way to sensitize tumors to immunotherapy. Here we review ERV co-option in development and innate immunity, the aberrant contribution of ERVs to tumorigenesis, and the wider biomedical potential of therapies directed at ERVs.

Pseudogenes are gene copies presumed to mainly be functionless relics of evolution due to acquired deleterious mutations or transcriptional silencing. Using deep full-length PacBio cDNA sequencing of normal human tissues and cancer cell lines, we identify here hundreds of novel transcribed pseudogenes expressed in tissue-specific patterns. Some pseudogene transcripts have intact open reading frames and are translated in cultured cells, representing unannotated protein-coding genes. To assess the biological impact of noncoding pseudogenes, we CRISPR-Cas9 delete the nucleus-enriched pseudogene PDCL3P4 and observe hundreds of perturbed genes. This study highlights pseudogenes as a complex and dynamic component of the human transcriptional landscape.

Transposable elements (TEs) drive genome evolution and are a notable source of pathogenesis, including cancer. While CpG methylation regulates TE activity, the locus-specific methylation landscape of mobile human TEs has to date proven largely inaccessible. Here, we apply new computational tools and long-read nanopore sequencing to directly infer CpG methylation of novel and extant TE insertions in hippocampus, heart, and liver, as well as paired tumor and non-tumor liver. As opposed to an indiscriminate stochastic process, we find pronounced demethylation of young long interspersed element 1 (LINE-1) retrotransposons in cancer, often distinct to the adjacent genome and other TEs. SINE-VNTR-Alu (SVA) retrotransposons, including their internal tandem repeat-associated CpG island, are near-universally methylated. We encounter allele-specific TE methylation and demethylation of aberrantly expressed young LINE-1s in normal tissues. Finally, we recover the complete sequences of tumor-specific LINE-1 insertions and their retrotransposition hallmarks, demonstrating how long-read sequencing can simultaneously survey the epigenome and detect somatic TE mobilization., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pseudogenes are defined as regions of the genome that contain defective copies of genes. They exist across almost all forms of life, and in mammalian genomes are annotated in similar numbers to recognized protein-coding genes. Although often presumed to lack function, growing numbers of pseudogenes are being found to play important biological roles. In consideration of their evolutionary origins and inherent limitations in genome annotation practices, we posit that pseudogenes have been classified on a scientifically unsubstantiated basis. We reflect that a broad misunderstanding of pseudogenes, perpetuated in part by the pejorative inference of the 'pseudogene' label, has led to their frequent dismissal from functional assessment and exclusion from genomic analyses. With the advent of technologies that simplify the study of pseudogenes, we propose that an objective reassessment of these genomic elements will reveal valuable insights into genome function and evolution.

RNA-Seq is a powerful transcriptome profiling technology enabling transcript discovery and quantification. Whilst most commonly used for gene-level quantification, the data can be used for the analysis of transcript isoforms. However, when the underlying transcript assemblies are complex, current visualization approaches can be limiting, with splicing events a challenge to interpret. Here, we report on the development of a graph-based visualization method as a complementary approach to understanding transcript diversity from short-read RNA-Seq data. Following the mapping of reads to a reference genome, a read-to-read comparison is performed on all reads mapping to a given gene, producing a weighted similarity matrix between reads. This is used to produce an RNA assembly graph, where nodes represent reads and edges similarity scores between them. The resulting graphs are visualized in 3D space to better appreciate their sometimes large and complex topology, with other information being overlaid on to nodes, e.g. transcript models. Here we demonstrate the utility of this approach, including the unusual structure of these graphs and how they can be used to identify issues in assembly, repetitive sequences within transcripts and splice variants. We believe this approach has the potential to significantly improve our understanding of transcript complexity., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Epigenetic silencing defends against LINE-1 (L1) retrotransposition in mammalian cells. However, the mechanisms that repress young L1 families and how L1 escapes to cause somatic genome mosaicism in the brain remain unclear. Here we report that a conserved Yin Yang 1 (YY1) transcription factor binding site mediates L1 promoter DNA methylation in pluripotent and differentiated cells. By analyzing 24 hippocampal neurons with three distinct single-cell genomic approaches, we characterized and validated a somatic L1 insertion bearing a 3' transduction. The source (donor) L1 for this insertion was slightly 5' truncated, lacked the YY1 binding site, and was highly mobile when tested in vitro. Locus-specific bisulfite sequencing revealed that the donor L1 and other young L1s with mutated YY1 binding sites were hypomethylated in embryonic stem cells, during neurodifferentiation, and in liver and brain tissue. These results explain how L1 can evade repression and retrotranspose in the human body., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The retrotransposon LINE-1 (L1) is a significant source of endogenous mutagenesis in humans. In each individual genome, a few retrotransposition-competent L1s (RC-L1s) can generate new heritable L1 insertions in the early embryo, primordial germ line, and germ cells. L1 retrotransposition can also occur in the neuronal lineage and cause somatic mosaicism. Although DNA methylation mediates L1 promoter repression, the temporal pattern of methylation applied to individual RC-L1s during neurogenesis is unclear. Here, we identified a de novo L1 insertion in a human induced pluripotent stem cell (hiPSC) line via retrotransposon capture sequencing (RC-seq). The L1 insertion was full-length and carried 5' and 3' transductions. The corresponding donor RC-L1 was part of a large and recently active L1 transduction family and was highly mobile in a cultured-cell L1 retrotransposition reporter assay. Notably, we observed distinct and dynamic DNA methylation profiles for the de novo L1 and members of its extended transduction family during neuronal differentiation. These experiments reveal how a de novo L1 insertion in a pluripotent stem cell is rapidly recognized and repressed, albeit incompletely, by the host genome during neurodifferentiation, while retaining potential for further retrotransposition., (Copyright © 2019 Salvador-Palomeque et al.)

This Article contains an error in the author affiliations. The correct affiliation for author Ruchi Shukla is 'MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK', and is not 'Mater Research Institute - University of Queensland, TRI Building, Woolloongabba QLD 4102, Australia'.

Retrotransposons are transposable elements (TEs) capable of "jumping" in germ, embryonic and tumor cells and, as is now clearly established, in the neuronal lineage. Mosaic TE insertions form part of a broader landscape of somatic genome variation and hold significant potential to generate phenotypic diversity, in the brain and elsewhere. At present, the LINE-1 (L1) retrotransposon family appears to be the most active autonomous TE in most mammals, based on experimental data obtained from disease-causing L1 mutations, engineered L1 reporter systems tested in cultured cells and transgenic rodents, and single-cell genomic analyses. However, the biological consequences of almost all somatic L1 insertions identified thus far remain unknown. In this review, we briefly summarize the current state-of-the-art in the field, including estimates of L1 retrotransposition rate in neurons. We bring forward the hypothesis that an extensive subset of retrotransposition-competent L1s may be de-repressed and mobile in the soma but largely inactive in the germline. We discuss recent reports of non-canonical L1-associated sequence variants in the brain and propose that the elevated L1 DNA content reported in several neurological disorders may predominantly comprise accumulated, unintegrated L1 nucleic acids, rather than somatic L1 insertions. Finally, we consider the main objectives and obstacles going forward in elucidating the biological impact of somatic retrotransposition., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Over the past decade, major discoveries in retrotransposon biology have depicted the neural genome as a dynamic structure during life. In particular, the retrotransposon LINE-1 (L1) has been shown to be transcribed and mobilized in the brain. Retrotransposition in the developing brain, as well as during adult neurogenesis, provides a milieu in which neural diversity can arise. Dysregulation of retrotransposon activity may also contribute to neurological disease. Here, we review recent reports of retrotransposon activity in the brain, and discuss the temporal nature of retrotransposition and its regulation in neural cells in response to stimuli. We also put forward hypotheses regarding the significance of retrotransposons for brain development and neurological function, and consider the potential implications of this phenomenon for neuropsychiatric and neurodegenerative conditions., (© 2018 The Authors.)

LINE-1 (L1) retrotransposons are a source of insertional mutagenesis in tumor cells. However, the clinical significance of L1 mobilization during tumorigenesis remains unclear. Here, we applied retrotransposon capture sequencing (RC-seq) to multiple single-cell clones isolated from five ovarian cancer cell lines and HeLa cells and detected endogenous L1 retrotransposition in vitro. We then applied RC-seq to ovarian tumor and matched blood samples from 19 patients and identified 88 tumor-specific L1 insertions. In one tumor, an intronic de novo L1 insertion supplied a novel cis-enhancer to the putative chemoresistance gene STC1. Notably, the tumor subclone carrying the STC1 L1 mutation increased in prevalence after chemotherapy, further increasing STC1 expression. We also identified hypomethylated donor L1s responsible for new L1 insertions in tumors and cultivated cancer cells. These congruent in vitro and in vivo results highlight L1 insertional mutagenesis as a common component of ovarian tumorigenesis and cancer genome heterogeneity., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Mobile Genetic Elements and Genome Plasticity conference was hosted by Keystone Symposia in Santa Fe, NM USA, February 11-15, 2018. The organizers were Marlene Belfort, Evan Eichler, Henry Levin and Lynn Maquat. The goal of this conference was to bring together scientists from around the world to discuss the function of transposable elements and their impact on host species. Central themes of the meeting included recent innovations in genome analysis and the role of mobile DNA in disease and evolution. The conference included 200 scientists who participated in poster presentations, short talks selected from abstracts, and invited talks. A total of 58 talks were organized into eight sessions and two workshops. The topics varied from mechanisms of mobilization, to the structure of genomes and their defense strategies to protect against transposable elements., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The retrotransposon Long Interspersed Element 1 (LINE-1 or L1) has played a major role in shaping the sequence composition of the mammalian genome. In our recent publication, "Heritable L1 retrotransposition in the mouse primordial germline and early embryo," we systematically assessed the rate and developmental timing of de novo, heritable endogenous L1 insertions in mice. Such heritable retrotransposition events allow L1 to exert an ongoing influence upon genome evolution. Here, we place our findings in the context of earlier studies, and highlight how our results corroborate, and depart from, previous research based on human patient samples and transgenic mouse models harboring engineered L1 reporter genes. In parallel, we outline outstanding questions regarding the stage-specificity, regulation, and functional impact of embryonic and germline L1 retrotransposition, and propose avenues for future research in this field., (© 2018 The Authors. BioEssays Published by WILEY Periodicals, Inc.)

The retrotransposon Long Interspersed Element 1 (LINE-1 or L1) is a continuing source of germline and somatic mutagenesis in mammals. Deregulated L1 activity is a hallmark of cancer, and L1 mutagenesis has been described in numerous human malignancies. We previously employed retrotransposon capture sequencing (RC-seq) to analyze hepatocellular carcinoma (HCC) samples from patients infected with hepatitis B or hepatitis C virus and identified L1 variants responsible for activating oncogenic pathways. Here, we have applied RC-seq and whole-genome sequencing (WGS) to an Abcb4 (Mdr2) -/- mouse model of hepatic carcinogenesis and demonstrated for the first time that L1 mobilization occurs in murine tumors. In 12 HCC nodules obtained from 10 animals, we validated four somatic L1 insertions by PCR and capillary sequencing, including T F subfamily elements, and one G F subfamily example. One of the T F insertions carried a 3' transduction, allowing us to identify its donor L1 and to demonstrate that this full-length T F element retained retrotransposition capacity in cultured cancer cells. Using RC-seq, we also identified eight tumor-specific L1 insertions from 25 HCC patients with a history of alcohol abuse. Finally, we used RC-seq and WGS to identify three tumor-specific L1 insertions among 10 intra-hepatic cholangiocarcinoma (ICC) patients, including one insertion traced to a donor L1 on Chromosome 22 known to be highly active in other cancers. This study reveals L1 mobilization as a common feature of hepatocarcinogenesis in mammals, demonstrating that the phenomenon is not restricted to human viral HCC etiologies and is encountered in murine liver tumors., (© 2018 Schauer et al.; Published by Cold Spring Harbor Laboratory Press.)

Genetic variants underlying complex traits, including disease susceptibility, are enriched within the transcriptional regulatory elements, promoters and enhancers. There is emerging evidence that regulatory elements associated with particular traits or diseases share similar patterns of transcriptional activity. Accordingly, shared transcriptional activity (coexpression) may help prioritise loci associated with a given trait, and help to identify underlying biological processes. Using cap analysis of gene expression (CAGE) profiles of promoter- and enhancer-derived RNAs across 1824 human samples, we have analysed coexpression of RNAs originating from trait-associated regulatory regions using a novel quantitative method (network density analysis; NDA). For most traits studied, phenotype-associated variants in regulatory regions were linked to tightly-coexpressed networks that are likely to share important functional characteristics. Coexpression provides a new signal, independent of phenotype association, to enable fine mapping of causative variants. The NDA coexpression approach identifies new genetic variants associated with specific traits, including an association between the regulation of the OCT1 cation transporter and genetic variants underlying circulating cholesterol levels. NDA strongly implicates particular cell types and tissues in disease pathogenesis. For example, distinct groupings of disease-associated regulatory regions implicate two distinct biological processes in the pathogenesis of ulcerative colitis; a further two separate processes are implicated in Crohn's disease. Thus, our functional analysis of genetic predisposition to disease defines new distinct disease endotypes. We predict that patients with a preponderance of susceptibility variants in each group are likely to respond differently to pharmacological therapy. Together, these findings enable a deeper biological understanding of the causal basis of complex traits.

The retrotransposon LINE-1 (long interspersed element 1, L1) is a transposable element that has extensively colonized the mammalian germline. L1 retrotransposition can also occur in somatic cells, causing genomic mosaicism, as well as in cancer. However, the extent of L1-driven mosaicism arising during ontogenesis is unclear. We discuss here recent experimental data which, at a minimum, fully substantiate L1 mosaicism in early embryonic development and neural cells, including post-mitotic neurons. We also consider the possible biological impact of somatic L1 insertions in neurons, the existence of donor L1s that are highly active ('hot') in specific spatiotemporal niches, and the evolutionary selection of donor L1s driving neuronal mosaicism., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

LINE-1 (L1) retrotransposons are a noted source of genetic diversity and disease in mammals. To expand its genomic footprint, L1 must mobilize in cells that will contribute their genetic material to subsequent generations. Heritable L1 insertions may therefore arise in germ cells and in pluripotent embryonic cells, prior to germline specification, yet the frequency and predominant developmental timing of such events remain unclear. Here, we applied mouse retrotransposon capture sequencing (mRC-seq) and whole-genome sequencing (WGS) to pedigrees of C57BL/6J animals, and uncovered an L1 insertion rate of ≥1 event per eight births. We traced heritable L1 insertions to pluripotent embryonic cells and, strikingly, to early primordial germ cells (PGCs). New L1 insertions bore structural hallmarks of target-site primed reverse transcription (TPRT) and mobilized efficiently in a cultured cell retrotransposition assay. Together, our results highlight the rate and evolutionary impact of heritable L1 retrotransposition and reveal retrotransposition-mediated genomic diversification as a fundamental property of pluripotent embryonic cells in vivo., (© 2017 Richardson et al.; Published by Cold Spring Harbor Laboratory Press.)

The ST18 gene has been proposed to act either as a tumor suppressor or as an oncogene in different human cancers, but direct evidence for its role in tumorigenesis has been lacking thus far. Here, we demonstrate that ST18 is critical for tumor progression and maintenance in a mouse model of liver cancer, based on oncogenic transformation and adoptive transfer of primary precursor cells (hepatoblasts). ST18 messenger RNA (mRNA) and protein were detectable neither in normal liver nor in cultured hepatoblasts, but were readily expressed after subcutaneous engraftment and tumor growth. ST18 expression in liver cells was induced by inflammatory cues, including acute or chronic inflammation in vivo, as well as coculture with macrophages in vitro. Knocking down the ST18 mRNA in transplanted hepatoblasts delayed tumor progression. Induction of ST18 knockdown in pre-established tumors caused rapid tumor involution associated with pervasive morphological changes, proliferative arrest, and apoptosis in tumor cells, as well as depletion of tumor-associated macrophages, vascular ectasia, and hemorrhage. Reciprocally, systemic depletion of macrophages in recipient animals had very similar phenotypic consequences, impairing either tumor development or maintenance, and suppressing ST18 expression in hepatoblasts. Finally, RNA sequencing of ST18-depleted tumors before involution revealed down-regulation of inflammatory response genes, pointing to the suppression of nuclear factor kappa B-dependent transcription., Conclusion: ST18 expression in epithelial cells is induced by tumor-associated macrophages, contributing to the reciprocal feed-forward loop between both cell types in liver tumorigenesis. Our findings warrant the exploration of means to interfere with ST18-dependent epithelium-macrophage interactions in a therapeutic setting. (Hepatology 2017;65:1708-1719)., (© 2016 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)