Emergence d'un locus producteur de piRNAs chez la drosophile : mise en place de l'épigénome

DNA transposable elements are almost ubiquitous in the living world and their mobility can be deleterious for the genome. Their regulation in germaria is mediated by the piRNAsilencing pathway (PIWI-interacting RNAs). piRNAs are produced by loci formed by clusters of fragments of transposable elements. We are studying the emergence of these piRNA-producing clusters in Drosophila.We have de novo activated a cluster of transgenes via maternal inheritance of homologous piRNAs. This is a case of paramutation i.e. a stable and recurrent epigenetic conversion process.We have shown that this paramutated cluster produces de novo piRNAs and, surprisingly, also siRNAs. I have characterized this paramutation functionally and molecularly, by a mutant approach. I have shown that its silencing properties, as well as piRNA and siRNA production are abolished in mutant contexts for all the genes from the primary and secondary piRNA pathways I have tested. At the same time, I have studied the case of a partially homologous paramutation, in which piRNAs maternally inherited by the cluster are homologous to only a part of its sequence. I have shown that piRNA are produced all along the cluster as early as the 3rdgeneration.Finally, I have shown that a cluster activated de novo by an environmental stress shows the same functional and molecular properties as a cluster paramutated via maternal piRNA inheritance.These studies provide key elements for understanding the emergence of the epigenome from a mechanistic and an evolutionary perspective.Les éléments transposables d’ADN sont presque ubiquitaires dans le monde vivant et leur mobilité peut être délétère pour le génome. Leur régulation dans les tissus germinaux animaux passe par la voie de silencing des piRNAs (PIWI-interacting RNAs). Les piRNAs sont produits à partir de loci contenant des fragments d’éléments transposables insérés en clusters. Nous étudions l’émergence de ces clusters de piRNAs chez la drosophile.Nous avons activé de novo un cluster de transgènes par héritage maternel de piRNAs homologues. Il s’agit d’un cas de paramutation, ou conversion épigénétique stable et récurrente.Nous avons montré que ce cluster paramuté produit de novo des piRNAs, et étonnement dessiRNAs.J’ai caractérisé de façon fonctionnelle et moléculaire ce phénomène de paramutation par l’utilisation de mutants. J’ai montré que les propriétés de silencing, ainsi que la production depiRNAs et de siRNAs, sont abolies en contexte mutant pour tous les gènes testés de la voie despiRNAs (voies primaire et secondaire). Parallèlement, j’ai étudié un cas de paramutation« partiellement homologue » dans laquelle le cluster reçoit des piRNAs homologues seulement àune partie de sa séquence. J’ai montré qu’il y a production de piRNAs par la totalité du cluster dès la 3e génération.J’ai montré, enfin, que des clusters activés de novo par la chaleur, présentent des propriétés fonctionnelles et moléculaires semblables aux clusters activés par les piRNAsmaternels.Ces travaux apportent des éléments clés pour la compréhension de la mise en place de l’épigénome, tant d’un point de vue mécanistique qu’évolutif

Dedukti: a Logical Framework based on the λ\lambdaΠ\Pi-Calculus Modulo Theory

Dedukti is a Logical Framework based on the $\lambda$$\Pi$-Calculus Modulo Theory. We show that many theories can be expressed in Dedukti: constructive and classical predicate logic, Simple type theory, programming languages, Pure type systems, the Calculus of inductive constructions with universes, etc. and that permits to used it to check large libraries of proofs developed in other proof systems: Zenon, iProver, FoCaLiZe, HOL Light, and Matita

Emergence of a piRNA-producing locus in drosophila

Les éléments transposables d’ADN sont presque ubiquitaires dans le monde vivant et leur mobilité peut être délétère pour le génome. Leur régulation dans les tissus germinaux animaux passe par la voie de silencing des piRNAs (PIWI-interacting RNAs). Les piRNAs sont produits à partir de loci contenant des fragments d’éléments transposables insérés en clusters. Nous étudions l’émergence de ces clusters de piRNAs chez la drosophile.Nous avons activé de novo un cluster de transgènes par héritage maternel de piRNAs homologues. Il s’agit d’un cas de paramutation, ou conversion épigénétique stable et récurrente.Nous avons montré que ce cluster paramuté produit de novo des piRNAs, et étonnement dessiRNAs.J’ai caractérisé de façon fonctionnelle et moléculaire ce phénomène de paramutation par l’utilisation de mutants. J’ai montré que les propriétés de silencing, ainsi que la production depiRNAs et de siRNAs, sont abolies en contexte mutant pour tous les gènes testés de la voie despiRNAs (voies primaire et secondaire). Parallèlement, j’ai étudié un cas de paramutation« partiellement homologue » dans laquelle le cluster reçoit des piRNAs homologues seulement àune partie de sa séquence. J’ai montré qu’il y a production de piRNAs par la totalité du cluster dès la 3e génération.J’ai montré, enfin, que des clusters activés de novo par la chaleur, présentent des propriétés fonctionnelles et moléculaires semblables aux clusters activés par les piRNAsmaternels.Ces travaux apportent des éléments clés pour la compréhension de la mise en place de l’épigénome, tant d’un point de vue mécanistique qu’évolutif.DNA transposable elements are almost ubiquitous in the living world and their mobility can be deleterious for the genome. Their regulation in germaria is mediated by the piRNAsilencing pathway (PIWI-interacting RNAs). piRNAs are produced by loci formed by clusters of fragments of transposable elements. We are studying the emergence of these piRNA-producing clusters in Drosophila.We have de novo activated a cluster of transgenes via maternal inheritance of homologous piRNAs. This is a case of paramutation i.e. a stable and recurrent epigenetic conversion process.We have shown that this paramutated cluster produces de novo piRNAs and, surprisingly, also siRNAs. I have characterized this paramutation functionally and molecularly, by a mutant approach. I have shown that its silencing properties, as well as piRNA and siRNA production are abolished in mutant contexts for all the genes from the primary and secondary piRNA pathways I have tested. At the same time, I have studied the case of a partially homologous paramutation, in which piRNAs maternally inherited by the cluster are homologous to only a part of its sequence. I have shown that piRNA are produced all along the cluster as early as the 3rdgeneration.Finally, I have shown that a cluster activated de novo by an environmental stress shows the same functional and molecular properties as a cluster paramutated via maternal piRNA inheritance.These studies provide key elements for understanding the emergence of the epigenome from a mechanistic and an evolutionary perspective

Optical pancreatic beta cell based biosensor, applications and glucose monitoring

An optical pancreatic beta-cell based biosensor for measuring cytosolic [Ca2+] is presented in this work. An in vitro bio-electronic system is built for testing various applications and characterizing features of the biosensor. A panoply of applications such as effect of temperature and response of the cells in culture media with 11 mM glucose vs. KRBH with 0 mM glucose are explored. The effect of the excitation light intensity and integration time on the fluorescence performance of the biosensor FRET probe are elucidated experimentally

Paramutation in Drosophila Requires Both Nuclear and Cytoplasmic Actors of the piRNA Pathway and Induces Cis-spreading of piRNA Production

International audienceTransposable element activity is repressed in the germline in animals by PIWI-interacting RNAs (piRNAs), a class of small RNAs produced by genomic loci mostly composed of TE sequences. The mechanism of induction of piRNA production by these loci is still enigmatic. We have shown that, in Drosophila melanogaster, a cluster of tandemly repeated P-lacZ-white transgenes can be activated for piRNA production by maternal inheritance of a cytoplasm containing homologous piRNAs. This activated state is stably transmitted over generations and allows trans-silencing of a homologous transgenic target in the female germline. Such an epigenetic conversion displays the functional characteristics of a paramutation, i.e., a heritable epigenetic modification of one allele by the other. We report here that piRNA production and trans-silencing capacities of the paramutated cluster depend on the function of the rhino, cutoff, and zucchini genes involved in primary piRNA biogenesis in the germline, as well as on that of the aubergine gene implicated in the ping-pong piRNA amplification step. The 21-nt RNAs, which are produced by the paramutated cluster, in addition to 23- to 28-nt piRNAs are not necessary for paramutation to occur. Production of these 21-nt RNAs requires Dicer-2 but also all the piRNA genes tested. Moreover, cytoplasmic transmission of piRNAs homologous to only a subregion of the transgenic locus can generate a strong paramutated locus that produces piRNAs along the whole length of the transgenes. Finally, we observed that maternally inherited transgenic small RNAs can also impact transgene expression in the soma. In conclusion, paramutation involves both nuclear (Rhino, Cutoff) and cytoplasmic (Aubergine, Zucchini) actors of the piRNA pathway. In addition, since it is observed between nonfully homologous loci located on different chromosomes, paramutation may play a crucial role in epigenome shaping in Drosophila natural populations

piRNAs and epigenetic conversion in Drosophila

International audienceTransposable element (TE) activity is repressed in the Drosophila germline by Piwi-Interacting RNAs (piRNAs), a class of small non-coding RNAs. These piRNAs are produced by discrete genomic loci containing TE fragments. In a recent publication, we tested for the existence of a strict epigenetic induction of piRNA production capacity by a locus in the D. melanogaster genome. We used 2 lines carrying a transgenic 7-copy tandem cluster (P-lacZ-white) at the same genomic site. This cluster generates in both lines a local heterochromatic sector. One line (T-1) produces high levels of ovarian piRNAs homologous to the P-lacZ-white transgenes and shows a strong capacity to repress homologous sequences in trans, whereas the other line (BX2) is devoid of both of these capacities. The properties of these 2 lines are perfectly stable over generations. We have shown that the maternal transmission of a cytoplasm carrying piRNAs from the first line can confer to the inert transgenic locus of the second, a totally de novo capacity to produce high levels of piRNAs as well as the ability to induce homology-dependent silencing in trans. These new properties are stably inherited over generations (n > 50). Furthermore, the converted locus has itself become able to convert an inert transgenic locus via cytoplasmic maternal inheritance. This results in a stable epigenetic conversion process, which can be performed recurrently-a phenomenon termed paramutation and discovered in Maize 60 y ago. Paramutation in Drosophila corresponds to the first stable paramutation in animals and provides a model system to investigate the epigenetically induced emergence of a piRNA-producing locus, a crucial step in epigenome shaping. In this Extra View, we discuss some additional functional aspects and the possible molecular mechanism of this piRNA-linked paramutation

Inheritance of paramutation in plants and Drosophila

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