Sophie Lanciano

Les éléments transposables (ET) sont des éléments génétiques ubiquitaires et potentiellement mobiles dans les génomes eucaryotes. Les génomes hôtes ont développé des mécanismes épigénétiques pour contrôler et prévenir la prolifération des ET. Néanmoins, certains ET semblent capables de s’activer en réponses à des stress ou à des facteurs développementaux. Les méthodes disponibles pour détecter l’activité transpositionnelle d’un ET sont souvent limitées au stade transcriptionnel ou sont adaptées à des génomes de petite taille. Relativement peu d’ET sont actuellement connus pour être actifs et les mécanismes spécifiques qui les contrôlent ne sont pas clairement identifiés.Durant mes travaux de thèse, nous avons développé une stratégie de séquençage à haut débit qui permet la détection d’ADN extrachromosomique circulaire (ADNecc) témoignant notamment de l’activité des ET et de la stabilité d’un génome. Ainsi nous avons pu caractériser chez plusieurs espèces le mobilome, défini comme l’ensemble des ADNecc présents dans un tissu.La technique du mobilome-seq s’est avérée être un outil puissant pour la détection des ET actifs notamment chez le riz asiatique Oryza sativa. Notre analyse du mobilome a permis l’identification d’un rétrotransposon PopRice actif dans l’albumen (tissu nourricier du grain) chez différentes variétés de riz. Pour la première fois chez les plantes, nous avons également détecté des insertions somatiques d’ET par re-séquençage de génome entier. À partir de nos résultats, nous avons combiné nos données mobilomiques avec une analyse GWAS pour proposer des pistes afin d’identifier de nouveaux mécanismes de régulation de cet élément.En parallèle, nous avons appliqué la technique du mobilome-seq à différents organismes animaux et végétaux révélant ainsi des spécificités de mobilome propre à chaque espèce. Nos travaux en collaboration avec d’autres équipes ont notamment contribué à préciser le rôle de l’ARN polymérase II dans le contrôle des ET chez O. sativa et à mettre en évidence le lien entre la présence d’ADNecc viral et la réponse immunitaire chez Drosophila melanogaster.Mes travaux de thèse ouvrent des perspectives pour l’étude du mobilome, ce répertoire génomique encore largement inexploré et qui se révèle être à la fois une source d’information au niveau des mouvements des ET mais aussi de la stabilité des génomes. L’étude future des mobilomes promet d’apporter des réponses sur notre compréhension de la dynamique des génomes.

Immunotherapy is one of the most promising advances in the fight against cancer and is beginning to join more conventional treatment approaches such as surgery, radiation therapy, and chemotherapy. A set of strategies consists of ensuring that the immune cells recognize tumor-specific antigens (TSAs) exposed at the surface of tumor cells and eliminate the tumor. TSAs can be processed within tumor cells and presented by human leukocyte antigen (HLA) molecules at the cell membrane, where they can be recognized by cytotoxic T lymphocytes. Additionally, the external region of membrane proteins can act as TSAs independently of HLA presentation. They can be recognized by antibodies, including therapeutic monoclonal antibodies, and thus mark tumor cells for elimination by another set of immune cells. Membrane proteins are also potential targets for chimeric antigen receptor T cells, which are patient-derived T cells engineered ex vivo to recognize a specific antigen in an HLA-independent manner, then expanded and reinjected into the patient. Discovering TSAs with the strongest immunogenicity and specificity is essential for the development of efficient and safe immunotherapies. Moreover, the identification of TSAs that are shared by many tumor types and cases could enable the development of universal and off-the-shelf cancer vaccines or therapies, as opposed to the current state of more personalized but expensive and time-consuming therapies.

SUMMARY: Long interspersed element-1 (L1) retrotransposons play important roles in human disease and evolution. Their global activity is repressed by DNA methylation, but studying the regulation of individual copies has been difficult. Here, we combine short- and long-read sequencing to resolve the DNA methylation profiles of these repeated sequences in a panel of normal and cancer cells genome-wide at single-locus resolution. We unveil key principles underpinning L1 methylation heterogeneity among cell-types, families and integration sites. First, intronic L1 methylation is intimately associated with gene transcription. Conversely, L1s can influence the methylation status of the upstream region over short distances (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 generally insufficient to trigger L1 expression due to redundant silencing pathways. Our results illuminate the epigenetic and transcriptional interplay between retrotransposons and their host genome. GRAPHICAL HIGHLIGHTS: Bs-ATLAS-seq profiles L1 position and methylation genome-wide L1 has a frequent but short-range (300 bp) influence on the DNA methylation status of the upstream sequence Hypomethylated L1s are bound by tissue-specific transcription factors which drive L1 and chimeric transcripts synthesis L1 hypomethylation alone is insufficient to enable its transcription at most loci

Summary: Retrotransposition of LINE-1 (L1) elements represent 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.

The Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin recruits the histone methyl-transferase SETDB1 to spread H3K9me3 repressive marks across genes and transgenes in an integration site-dependent manner. The deposition of these repressive marks leads to heterochromatin formation and inhibits gene expression, but the underlying mechanism is not fully understood. Here, we show that TASOR silencing or HIV-2 Vpx expression, which induces TASOR degradation, increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. Furthermore, using a yeast 2-hybrid screen, we identify new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 synergistically repress HIV expression from its LTR. Similar to the RNA-induced transcriptional silencing complex found in fission yeast, we show that TASOR interacts with the RNA exosome and RNA Polymerase II, predominantly under its elongating state. Finally, we show that TASOR facilitates the association of RNA degradation proteins with RNA polymerase II and is detected at transcriptional centers. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral expression.

Summary: Trees are long‐lived organisms that continuously adapt to their environments, a process in which epigenetic mechanisms are likely to play a key role. Via downregulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula × Populus alba) RNAi lines, we examined how DNA methylation coordinates genomic and physiological responses to moderate water deficit. We compared the growth and drought response of two RNAi‐ddm1 lines to wild‐type (WT) trees under well‐watered and water deficit/rewatering conditions, and analyzed their methylomes, transcriptomes, mobilomes and phytohormone contents in the shoot apical meristem. The RNAi‐ddm1 lines were more tolerant to drought‐induced cavitation but did not differ in height or stem diameter growth. About 5000 differentially methylated regions were consistently detected in both RNAi‐ddm1 lines, colocalizing with 910 genes and 89 active transposable elements. Under water deficit conditions, 136 differentially expressed genes were found, including many involved in phytohormone pathways; changes in phytohormone concentrations were also detected. Finally, the combination of hypomethylation and drought led to the mobility of two transposable elements. Our findings suggest major roles for DNA methylation in regulation of genes involved in hormone‐related stress responses, and the maintenance of genome integrity through repression of transposable elements.

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.

Like many other crops, the cultivated peanut (Arachis hypogaea L.) is of hybrid origin and has a polyploid genome that contains essentially complete sets of chromosomes from two ancestral species. Here we report the genome sequence of peanut and show that after its polyploid origin, the genome has evolved through mobile-element activity, deletions and by the flow of genetic information between corresponding ancestral chromosomes (that is, homeologous recombination). Uniformity of patterns of homeologous recombination at the ends of chromosomes favors a single origin for cultivated peanut and its wild counterpart A. monticola. However, through much of the genome, homeologous recombination has created diversity. Using new polyploid hybrids made from the ancestral species, we show how this can generate phenotypic changes such as spontaneous changes in the color of the flowers. We suggest that diversity generated by these genetic mechanisms helped to favor the domestication of the polyploid A. hypogaea over other diploid Arachis species cultivated by humans.