Sensing of transposable elements by the antiviral innate immune system

We thank Dr. Francisco Sanchez-Luque, Dr. Martin Reijns, and Priscilla Chin for helpful discussions and comments on the manuscript. This work was funded by grants from the Wellcome Trust 107665/Z/15/Z and Royal Society grant RGS\R1\191368 to S.M., and from MINECO SAF2015-71589-P and Ramon y Cajal grant RYC-2016-21395 to S.R.H. Figures were designed using Smart Servier Medical Art (https://smart.servier.com).Around half of the genomes in mammals are composed of transposable elements (TEs) such as DNA transposons and retrotransposons. Several mechanisms have evolved to prevent their activity and the detrimental impact of their insertional mutagenesis. Despite these potentially negative effects, TEs are essential drivers of evolution, and in certain settings, beneficial to their hosts. For instance, TEs have rewired the antiviral gene regulatory network and are required for early embryonic development. However, due to structural similarities between TE-derived and viral nucleic acids, cells can misidentify TEs as invading viruses and trigger the major antiviral innate immune pathway, the type I interferon (IFN) response. This review will focus on the different settings in which the role of TE-mediated IFN activation has been documented, including cancer and senescence. Importantly, TEs may also play a causative role in the development of complex autoimmune diseases characterized by constitutive type I IFN activation. All these observations suggest the presence of strong but opposing forces driving the coevolution of TEs and antiviral defense. A better biological understanding of the TE replicative cycle as well as of the antiviral nucleic acid sensing mechanisms will provide insights into how these two biological processes interact and will help to design better strategies to treat human diseases characterized by aberrant TE expression and/ or type I IFN activation.Wellcome TrustEuropean Commission 107665/Z/15/ZRoyal Society of London RGS\R1\191368MINECO SAF2015-71589-PSpanish Government RYC-2016-2139

Control of mammalian retrotransposons by cellular RNA processing activities

Retrotransposons make up roughly 50% of the mammalian genome and have played an important role in genome evolution. A small fraction of non-LTR retrotransposons, LINE-1 and SINE elements, is currently active in the human genome. These elements move in our genome using an intermediate RNA and a reverse transcriptase activity by a copy and paste mechanism. Their ongoing mobilization can impact the human genome leading to several human disorders. However, how the cell controls the activity of these elements minimizing their mutagenic effect is not fully understood. Recent studies have highlighted that the intermediate RNA of retrotransposons is a target of different mechanisms that limit the mobilization of endogenous retrotransposons in mammals. Here, we provide an overview of recent discoveries that show how RNA processing events can act to control the activity of mammalian retrotransposons and discuss several arising questions that remain to be answered

Amidated and ibuprofen-conjugated kyotorphins promote neuronal rescue and memory recovery in cerebral hypoperfusion dementia model

Copyright © 2016 Sá Santos, Santos, Pinto, Ramu, Heras, Bardaji, Tavares and Castanho. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Chronic brain ischemia is a prominent risk factor for neurological dysfunction and progression for dementias, including Alzheimer's disease (AD). In rats, permanent bilateral common carotid artery occlusion (2VO) causes a progressive neurodegeneration in the hippocampus, learning deficits and memory loss as it occurs in AD. Kyotorphin (KTP) is an endogenous antinociceptive dipeptide whose role as neuromodulator/neuroprotector has been suggested. Recently, we designed two analgesic KTP-derivatives, KTP-amide (KTP-NH2) and KTP-NH2 linked to ibuprofen (IbKTP-NH2) to improve KTP brain targeting. This study investigated the effects of KTP-derivatives on cognitive/behavioral functions (motor/spatial memory/nociception) and hippocampal pathology of female rats in chronic cerebral hypoperfusion (2VO-rat model). 2VO-animals were treated with KTP-NH2 or IbKTP-NH2 for 7 days at weeks 2 and 5 post-surgery. After behavioral testing (week 6), coronal sections of hippocampus were H&E-stained or immunolabeled for the cellular markers GFAP (astrocytes) and NFL (neurons). Our findings show that KTP-derivatives, mainly IbKTP-NH2, enhanced cognitive impairment of 2VO-animals and prevented neuronal damage in hippocampal CA1 subfield, suggesting their potential usefulness for the treatment of dementia.Funding was provided by the Portuguese Agency Fundação para a Ciência e a Tecnologia SFRH/BPD/79542/2011 fellowship)info:eu-repo/semantics/publishedVersio

Engineered LINE-1 retrotransposition in nondividing human neurons

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought.We thank current members of the J.L.G.-P. laboratory for helpful discussions. We also thank Drs. Geoffrey Faulkner (Mater Research, Australia) and John V. Moran (University of Michigan) for sharing unpublished data and for critical input during the project; Ms. Raquel Marrero (Microscopy Unit, Genyo) for technical support; Simon Mendez-Ferrer (CNIC, Spain) for providing total RNA isolated from human mesenspheres; Dr. Oliver Weichenrieder (Max-Planck, Tubingen, Germany) for providing a polyclonal L1-ORF1p antibody; and Dr. Aurelien Doucet (IRCAN, Nice, France) for providing a plasmid containing an UBC-driven EGFP retrotransposition indicator cassette. J.L.G. was funded by the US Department of Defense, Breast Cancer Research Program (award #BC051386), the National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (1R03NS087290-01), and the ALS Therapy Alliance (2013-F-067). A.M. has been partially funded by a Marie Curie IRG project (FP7-PEOPLE-2007-4-3-IRG: SOMATIC LINE-1). J.L.G.-P's laboratory is supported by CICE-FEDER-P09-CTS-4980, CICE-FEDER-P12-CTS-2256, Plan Nacional de I+D+I 2008–2011 and 2013–2016 (FIS-FEDER-PI11/01489 and FIS-FEDER-PI14/02152), PCIN-2014-115-ERA-NET NEURON II, the European Research Council (ERC-Consolidator ERC-STG-2012-233764), by an International Early Career Scientist grant from the Howard Hughes Medical Institute (IECS-55007420), and by The Wellcome Trust–University of Edinburgh Institutional Strategic Support Fund (ISFF2).S

Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells

Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of gene therapy approaches. Generally, inducible ON systems require a chimeric transcription factor (transactivator) that becomes activated by an inductor, which is not optimal for clinical translation due to their toxicity. We generated previously the first all-in-one, transactivator-free, doxycycline (Dox)-responsive (Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control transgene expression in human stem cells. Here, we have generated new versions of the LOP LVs and have analyzed their applicability for the generation of inducible advanced therapy medicinal products (ATMPs) with special focus on primary human T cells. We have shown that, contrary to all other cell types analyzed, an Is2 insulator must be inserted into the 30 long terminal repeat of the LOP LVs in order to control transgene expression in human primary T cells. Importantly, inducible primary T cells generated by the LOPIs2 LVs are responsive to ultralow doses of Dox and have no changes in phenotype or function compared with untransduced T cells. We validated the LOPIs2 system by generating inducible CAR-T cells that selectively kill CD19+ cells in the presence of Dox. In summary, we describe here the first transactivatorfree, all-one-one system capable of generating Dox-inducible ATMPs.Spanish ISCIII Health Research FundEuropean Union (EU) PI18/00337 PI21/00298 RD21/0017/0004 PI18/00330 PI17/00672Red TerAvJunta de Andalucia FEDER/European Cohesion Fund (FSE) for AndalusiaSpanish Government PI18/00337 PI21/00298European Union-NextGenerationEU - Maria Zambrano Senior Program RD21/0017/0004 PI18/00330 PI17/00672Ministry of Health 2016000073332-TRA PI-57069 CARTPI-0001-201 PE-CART-0031-2020 PI-0014-2016 PECART-0027-2020 ProyExcel_00875 PEER-0286-2019European Cooperation in Science and Technology (COST) 00123009/SNEO-20191072MINECO - European Regional Development Fund PLEC2021-008094Spanish Government 0006/2018FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades CA21113Spanish Government SAF2015-71589-PMCI RYC-2016-21395German Research Foundation (DFG) PY20_00619 y A-CTS-28_UGR20Biomedicine Program of the University of Granada (Spain) FPU16/05467 FPU17/02268 FPU17/04327 DIN2018-010180 DIN2020-011550 PEJ-2018-001760-

RNase H2, mutated in Aicardi-Goutières syndrome, promotes LINE-1 retrotransposition

Long INterspersed Element class 1 (LINE-1) elements are a type of abundant retrotransposons active in mammalian genomes. An average human genome contains ~100 retrotransposition-competent LINE-1s, whose activity is influenced by the combined action of cellular repressors and activators. TREX1, SAMHD1 and ADAR1 are known LINE-1 repressors and when mutated cause the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). Mutations in RNase H2 are the most common cause of AGS, and its activity was proposed to similarly control LINE-1 retrotransposition. It has therefore been suggested that increased LINE-1 activity may be the cause of aberrant innate immune activation in AGS. Here, we establish that, contrary to expectations, RNase H2 is required for efficient LINE-1 retrotransposition. As RNase H1 overexpression partially rescues the defect in RNase H2 null cells, we propose a model in which RNase H2 degrades the LINE-1 RNA after reverse transcription, allowing retrotransposition to be completed. This also explains how LINE-1 elements can retrotranspose efficiently without their own RNase H activity. Our findings appear to be at odds with LINE-1-derived nucleic acids driving autoinflammation in AGS.M.B.-G. is funded by a “Formacion Profesorado Universitario” (FPU) PhD fellowship from the Government of Spain (MINECO, Ref FPU15/03294), and this paper is part of her thesis project (“Epigenetic control of the mobility of a human retrotransposon”). R.V.-A. is funded by a PFIS Fellowship from the Government of Spain (ISCiii, FI16/00413). O.M. is funded by an EMBO Long-Term Fellowship (ALTF 7-2015), the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409) and the Swiss National Science Foundation (P2ZHP3_158709). S.R.H. is funded by the Government of Spain (MINECO, RYC-2016-21395 and SAF2015-71589-P). A.P.J’s laboratory is supported by the UK Medical Research Council (MRC University Unit grant U127527202). J.L.G.P’s laboratory is supported by CICEFEDER- P12-CTS-2256, Plan Nacional de I+D+I 2008-2011 and 2013-2016 (FISFEDER- PI14/02152), PCIN-2014-115-ERA-NET NEURON II, the European Research Council (ERC-Consolidator ERC-STG-2012-233764), by an International Early Career Scientist grant from the Howard Hughes Medical Institute (IECS-55007420), by The Wellcome Trust-University of Edinburgh Institutional Strategic Support Fund (ISFF2) and by a private donation from Ms Francisca Serrano (Trading y Bolsa para Torpes, Granada, Spain)

Alu retrotransposons promote differentiation of human carcinoma cells through the aryl hydrocarbon receptor

Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions.Trabajo financiado por: Ministerio de Economía y Competitividad. Proyectos BFU2011-22678, SAF2014-51813-R (I+D+i) para Pedro María Fernández Salguero Ministerio de Economía y Competitividad, Instituto Carlos III, Red Temática de Investigación Cooperativa en Cáncer Junta de Extremadura. Ayudas GR10008, GR15008 CICE-FEDER-P09-CTS-4980, CICE-FEDERP12-CTS-2256, Plan Nacional de I+D+I 2008–2011 y 2013–2016 (FIS-FEDER-PI11/01489, FIS-FEDERPI14/02152), PCIN-2014-115-ERA-NET NEURON II, para José Luis García Pérez European Research Council ERC-Consolidator ERC-STG-2012-233764 International Early Career Scientist Beca de la Howard Hughes Medical Institute IECS-55007420 Programa Unión Europea de Fondos FEDERpeerReviewe

LINE-1 Evasion of Epigenetic Repression in Humans

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\ua0vitro. 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

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types