Hoxd gene regulation in the developing caecum

[...] In this project, we investigated the qualitative and quantitative profile of expression of the HoxD complex in the developing caecum of wild-type animals. [... click on "Download fulltext" for full abstract

Multiple Enhancers Regulate Hoxd Genes and the Hotdog LncRNA during Cecum Budding

Hox genes are required for the development of the intestinal cecum, a major organ of plant-eating species. We have analyzed the transcriptional regulation of Hoxd genes in cecal buds and show that they are controlled by a series of enhancers located in a gene desert flanking the HoxD cluster. The start site of two opposite long noncoding RNAs (lncRNAs), Hotdog and Twin of Hotdog, selectively contacts the expressed Hoxd genes in the framework of a topological domain, coinciding with robust transcription of these genes during cecum budding. Both lncRNAs are specifically transcribed in the cecum, albeit bearing no detectable function in trans. Hedgehogs have kept this regulatory potential despite the absence of the cecum, suggesting that these mechanisms are used in other developmental situations. In this context, we discuss the implementation of a common "budding toolkit" between the cecum and the limbs

Gene activation by metazoan enhancers: diverse mechanisms stimulate distinct steps of transcription

Enhancers can stimulate transcription by a number of different mechanisms which control different stages of the transcription cycle of their target genes, from recruitment of the transcription machinery to elongation by RNA polymerase. These mechanisms may not be mutually exclusive, as a single enhancer may act through different pathways by binding multiple transcription factors. Multiple enhancers may also work together to regulate transcription of a shared target gene. Most of the evidence supporting different enhancer mechanisms comes from the study of single genes, but new high-throughput experimental frameworks offer the opportunity to integrate and generalize disparate mechanisms identified at single genes. This effort is especially important if we are to fully understand how sequence variation within enhancers contributes to human disease

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences

siRNA biogenesis in Arabidopsis thaliana : functional study of a new double-stranded RNA binding protein, DRB7.2 and developement of molecular tools for DCL4 study

Les ARN double brin (ARNdb) sont les molécules clés initiatrices du RNA silencing, à partir desquelles les différentes classes de petits ARN (sRNAs), conférant la séquence spécificité de ce mécanisme, vont être produit. Chez la plante modèle Arabidopsis thaliana, le clivage des divers ARNdb en sRNAs est opéré par quatre enzymes de type RNase III, nommées DCL1 à DCL4, dont l’activité peut être assistée par des protéines fixant l’ARNdb (DRBs). Au cours de cette thèse, j’ai pu caractériser la fonction d’une nouvelle DRB, DRB7.2. Les résultats obtenus m’ont permis de démontrer que cette protéine régule la production d’une classe particulière de sRNAs endogènes, les endoIR-siRNAs, en séquestrant spécifiquement leurs précurseurs ARNdb, inhibant ainsi leur clivage par les différents DCLs. En parallèle, j’ai également développé des outils moléculaires afin d’étudier le mode d’action du DCL le plus polyvalent chez les plantes, DCL4. La caractérisation détaillée de ces outils a permis de révéler le rôle clé de déterminant structuraux distinct (protéiques ou nucléiques) impliqués dans la spécificité de reconnaissance et de clivage des divers substrats ARNdb par cette enzyme.RNA silencing is initiated by double-stranded RNA (dsRNA) molecules that will be processed into various classes of small RNAs (sRNAs), which confer the sequence-specificity of this mechanism. In the model plant Arabidopsis thaliana, dsRNA processing is mediated by four distinct RNaseIII-like enzymes, named DCL1 to DCL4, which can be assisted by dsRNA-binding proteins (DRBs). During my PhD, I was able to characterize in details the function of a new DRB protein, DRB7.2. Our results revealed that this protein regulates the accumulation of a specific class of endogenous sRNAs, the endoIR-siRNAs, by selectively sequestering their dsRNA precursors and inhibiting their cleavage by the DCLs. In parallel, I also developed molecular tools to study the mode of action of the most versatile DCL in plants, DCL4. Detailed characterization of these tools revealed key roles of distinct structural determinants (at the protein or RNA level), implicated in the specificity and cleavage efficiency of the various dsRNA susbtrates by DCL4

Mechanisms of "Hoxd" genes collinearity in developing digits and external genitalia

During the development of mammalian digits, clustered "Hoxd" genes are expressed following a collinear strategy, such that their expression levels correlate with gene order in the complex. By analyzing the distribution of transcript levels and the spatial organization of the "HoxD" locus in a variety of genetic configurations, we found that the locus adopts an active conformation in digits, bringing two distant enhancers in physical proximity with the 5' extremity of the complex. These interactions result in the polar activation of the locus, gene rank relative to the 5' extremity of the cluster controlling the respective expression levels. This remote regulation is accompanied by a widespread intergenic transcription in the posterior "HoxD" complex, leading to the synthesis of various non-coding RNAs of unknown function. Furthermore, as similar analysis of transcript levels distribution in the genital bud suggests a common regulatory mechanism for "HoxD" genes in digits and external genitalia

Biogénèse des siRNAs endogènes chez Arabidopsis thaliana : étude fonctionnelle de DRB7.2, une nouvelle protéine de fixation à l'ARN double brin et développement d'outils moléculaires pour la caractérisation du mode d'action de DCL4

RNA silencing is initiated by double-stranded RNA (dsRNA) molecules that will be processed into various classes of small RNAs (sRNAs), which confer the sequence-specificity of this mechanism. In the model plant Arabidopsis thaliana, dsRNA processing is mediated by four distinct RNaseIII-like enzymes, named DCL1 to DCL4, which can be assisted by dsRNA-binding proteins (DRBs). During my PhD, I was able to characterize in details the function of a new DRB protein, DRB7.2. Our results revealed that this protein regulates the accumulation of a specific class of endogenous sRNAs, the endoIR-siRNAs, by selectively sequestering their dsRNA precursors and inhibiting their cleavage by the DCLs. In parallel, I also developed molecular tools to study the mode of action of the most versatile DCL in plants, DCL4. Detailed characterization of these tools revealed key roles of distinct structural determinants (at the protein or RNA level), implicated in the specificity and cleavage efficiency of the various dsRNA susbtrates by DCL4.Les ARN double brin (ARNdb) sont les molécules clés initiatrices du RNA silencing, à partir desquelles les différentes classes de petits ARN (sRNAs), conférant la séquence spécificité de ce mécanisme, vont être produit. Chez la plante modèle Arabidopsis thaliana, le clivage des divers ARNdb en sRNAs est opéré par quatre enzymes de type RNase III, nommées DCL1 à DCL4, dont l’activité peut être assistée par des protéines fixant l’ARNdb (DRBs). Au cours de cette thèse, j’ai pu caractériser la fonction d’une nouvelle DRB, DRB7.2. Les résultats obtenus m’ont permis de démontrer que cette protéine régule la production d’une classe particulière de sRNAs endogènes, les endoIR-siRNAs, en séquestrant spécifiquement leurs précurseurs ARNdb, inhibant ainsi leur clivage par les différents DCLs. En parallèle, j’ai également développé des outils moléculaires afin d’étudier le mode d’action du DCL le plus polyvalent chez les plantes, DCL4. La caractérisation détaillée de ces outils a permis de révéler le rôle clé de déterminant structuraux distinct (protéiques ou nucléiques) impliqués dans la spécificité de reconnaissance et de clivage des divers substrats ARNdb par cette enzyme

siRNA biogenesis in Arabidopsis thaliana : functional study of a new double-stranded RNA binding protein, DRB7.2 and developement of molecular tools for DCL4 study

Les ARN double brin (ARNdb) sont les molécules clés initiatrices du RNA silencing, à partir desquelles les différentes classes de petits ARN (sRNAs), conférant la séquence spécificité de ce mécanisme, vont être produit. Chez la plante modèle Arabidopsis thaliana, le clivage des divers ARNdb en sRNAs est opéré par quatre enzymes de type RNase III, nommées DCL1 à DCL4, dont l’activité peut être assistée par des protéines fixant l’ARNdb (DRBs). Au cours de cette thèse, j’ai pu caractériser la fonction d’une nouvelle DRB, DRB7.2. Les résultats obtenus m’ont permis de démontrer que cette protéine régule la production d’une classe particulière de sRNAs endogènes, les endoIR-siRNAs, en séquestrant spécifiquement leurs précurseurs ARNdb, inhibant ainsi leur clivage par les différents DCLs. En parallèle, j’ai également développé des outils moléculaires afin d’étudier le mode d’action du DCL le plus polyvalent chez les plantes, DCL4. La caractérisation détaillée de ces outils a permis de révéler le rôle clé de déterminant structuraux distinct (protéiques ou nucléiques) impliqués dans la spécificité de reconnaissance et de clivage des divers substrats ARNdb par cette enzyme.RNA silencing is initiated by double-stranded RNA (dsRNA) molecules that will be processed into various classes of small RNAs (sRNAs), which confer the sequence-specificity of this mechanism. In the model plant Arabidopsis thaliana, dsRNA processing is mediated by four distinct RNaseIII-like enzymes, named DCL1 to DCL4, which can be assisted by dsRNA-binding proteins (DRBs). During my PhD, I was able to characterize in details the function of a new DRB protein, DRB7.2. Our results revealed that this protein regulates the accumulation of a specific class of endogenous sRNAs, the endoIR-siRNAs, by selectively sequestering their dsRNA precursors and inhibiting their cleavage by the DCLs. In parallel, I also developed molecular tools to study the mode of action of the most versatile DCL in plants, DCL4. Detailed characterization of these tools revealed key roles of distinct structural determinants (at the protein or RNA level), implicated in the specificity and cleavage efficiency of the various dsRNA susbtrates by DCL4