Stéphanie Rialle

A Duplicated NUCLEOLIN Gene with Antagonistic Activity Is Required for Chromatin Organization of Silent 45S rDNA in Arabidopsis

The nucleolus, the most prominent nuclear structure, is involved in ribosome biogenesis and nuclear and nucleolar chromatin organization. Nucleolins are among the most abundant nucleolar proteins. In contrast with mammals, plants encode two nucleolins. Characterization of Arabidopsis thaliana nuc2 mutants shows the importance of these proteins in chromatin silencing and ribosomal DNA organization. In plants as well as in animals, hundreds to thousands of 45S rRNA gene copies localize in Nucleolus Organizer Regions (NORs), and the activation or repression of specific sets of rDNA depends on epigenetic mechanisms. Previously, we reported that the Arabidopsis thaliana nucleolin protein NUC1, an abundant and evolutionarily conserved nucleolar protein in eukaryotic organisms, is required for maintaining DNA methylation levels and for controlling the expression of specific rDNA variants in Arabidopsis. Interestingly, in contrast with animal or yeast cells, plants contain a second nucleolin gene. Here, we report that Arabidopsis NUC1 and NUC2 nucleolin genes are both required for plant growth and survival and that NUC2 disruption represses flowering. However, these genes seem to be functionally antagonistic. In contrast with NUC1, disruption of NUC2 induces CG hypermethylation of rDNA and NOR association with the nucleolus. Moreover, NUC2 loss of function triggers major changes in rDNA spatial organization, expression, and transgenerational stability. Our analyses indicate that silencing of specific rRNA genes is mostly determined by the active or repressed state of the NORs and that nucleolin proteins play a key role in the developmental control of this process.

Neural stem cell-encoded temporal patterning delineates an early window of malignant susceptibility in Drosophila

Pediatric neural tumors are often initiated during early development and can undergo very rapid transformation. However, the molecular basis of this early malignant susceptibility remains unknown. During Drosophila development, neural stem cells (NSCs) divide asymmetrically and generate intermediate progenitors that rapidly differentiate in neurons. Upon gene inactivation, these progeny can dedifferentiate and generate malignant tumors. Here, we find that intermediate progenitors are prone to malignancy only when born during an early window of development while expressing the transcription factor Chinmo, and the mRNA-binding proteins Imp/IGF2BP and Lin-28. These genes compose an oncogenic module that is coopted upon dedifferentiation of early-born intermediate progenitors to drive unlimited tumor growth. In late larvae, temporal transcription factor progression in NSCs silences the module, thereby limiting mitotic potential and terminating the window of malignant susceptibility. Thus, this study identifies the gene regulatory network that confers malignant potential to neural tumors with early developmental origins. DOI: http://dx.doi.org/10.7554/eLife.13463.001

Transcriptional Profiling of Cutaneous MRGPRD Free Nerve Endings and C-LTMRs.

Summary Cutaneous C-unmyelinated MRGPRD+ free nerve endings and C-LTMRs innervating hair follicles convey two opposite aspects of touch sensation: a sensation of pain and a sensation of pleasant touch. The molecular mechanisms underlying these diametrically opposite functions are unknown. Here, we used a mouse model that genetically marks C-LTMRs and MRGPRD+ neurons in combination with fluorescent cell surface labeling, flow cytometry, and RNA deep-sequencing technology (RNA-seq). Cluster analysis of RNA-seq profiles of the purified neuronal subsets revealed 486 and 549 genes differentially expressed in MRGPRD-expressing neurons and C-LTMRs, respectively. We validated 48 MRGPD- and 68 C-LTMRs-enriched genes using a triple-staining approach, and the Cav3.3 channel, found to be exclusively expressed in C-LTMRs, was validated using electrophysiology. Our study greatly expands the molecular characterization of C-LTMRs and suggests that this particular population of neurons shares some molecular features with Aβ and Aδ low-threshold mechanoreceptors.

RNA-seq de novo Assembly Reveals Differential Gene Expression in Glossina palpalis gambiensis Infected with Trypanosoma brucei gambiense vs. Non-Infected and Self-Cured Flies

Trypanosoma brucei gambiense (Tbg), causing the sleeping sickness chronic form, completes its developmental cycle within the tsetse fly vector Glossina palpalis gambiensis (Gpg) before its transmission to humans. Within the framework of an anti-vector disease control strategy, a global gene expression profiling of trypanosome infected (susceptible), non-infected, and self-cured (refractory) tsetse flies was performed, on their midguts, to determine differential genes expression resulting from in vivo trypanosomes, tsetse flies (and their microbiome) interactions. An RNAseq de novo assembly was achieved. The assembled transcripts were mapped to reference sequences for functional annotation. Twenty-four percent of the 16,936 contigs could not be annotated, possibly representing untranslated mRNA regions, or Gpg- or Tbg-specific ORFs. The remaining contigs were classified into 65 functional groups. Only a few transposable elements were present in the Gpg midgut transcriptome, which may represent active transpositions and play regulatory roles. One thousand three hundred and seventy three genes differentially expressed (DEGs) between stimulated and non-stimulated flies were identified at day-3 post-feeding; 52 and 1025 between infected and self-cured flies at 10 and 20 days post-feeding, respectively. The possible roles of several DEGs regarding fly susceptibility and refractoriness are discussed. The results provide new means to decipher fly infection mechanisms, crucial to develop anti-vector control strategies.

FliZ Is a Global Regulatory Protein Affecting the Expression of Flagellar and Virulence Genes in Individual Xenorhabdus nematophila Bacterial Cells

Heterogeneity in the expression of various bacterial genes has been shown to result in the presence of individuals with different phenotypes within clonal bacterial populations. The genes specifying motility and flagellar functions are coordinately regulated and form a complex regulon, the flagellar regulon. Complex interplay has recently been demonstrated in the regulation of flagellar and virulence gene expression in many bacterial pathogens. We show here that FliZ, a DNA-binding protein, plays a key role in the insect pathogen, Xenorhabdus nematophila, affecting not only hemolysin production and virulence in insects, but efficient swimming motility. RNA-Seq analysis identified FliZ as a global regulatory protein controlling the expression of 278 Xenorhabdus genes either directly or indirectly. FliZ is required for the efficient expression of all flagellar genes, probably through its positive feedback loop, which controls expression of the flhDC operon, the master regulator of the flagellar circuit. FliZ also up- or downregulates the expression of numerous genes encoding non-flagellar proteins potentially involved in key steps of the Xenorhabdus lifecycle. Single-cell analysis revealed the bimodal expression of six identified markers of the FliZ regulon during exponential growth of the bacterial population. In addition, a combination of fluorescence-activated cell sorting and RT-qPCR quantification showed that this bimodality generated a mixed population of cells either expressing (“ON state”) or not expressing (“OFF state”) FliZ-dependent genes. Moreover, studies of a bacterial population exposed to a graded series of FliZ concentrations showed that FliZ functioned as a rheostat, controlling the rate of transition between the “OFF” and “ON” states in individuals. FliZ thus plays a key role in cell fate decisions, by transiently creating individuals with different potentials for motility and host interactions.

Identification of Plagl1/Zac1 binding sites and target genes establishes its role in the regulation of extracellular matrix genes and the imprinted gene network

Abstract PLAGL1/ZAC1 undergoes parental genomic imprinting, is paternally expressed, and is a member of the imprinted gene network (IGN). It encodes a zinc finger transcription factor with anti-proliferative activity and is a candidate tumor suppressor gene on 6q24 whose expression is frequently lost in various neoplasms. Conversely, gain of PLAGL1 function is responsible for transient neonatal diabetes mellitus, a rare genetic disease that results from defective pancreas development. In the present work, we showed that Plagl1 up-regulation was not associated with DNA damage-induced cell cycle arrest. It was rather associated with physiological cell cycle exit that occurred with contact inhibition, growth factor withdrawal, or cell differentiation. To gain insights into Plagl1 mechanism of action, we identified Plagl1 target genes by combining chromatin immunoprecipitation and genome-wide transcriptomics in transfected cell lines. Plagl1-elicited gene regulation correlated with multiple binding to the proximal promoter region through a GC-rich motif. Plagl1 target genes included numerous genes involved in signaling, cell adhesion, and extracellular matrix composition, including collagens. Plagl1 targets also included 22% of the 409 genes that make up the IGN. Altogether, this work identified Plagl1 as a transcription factor that coordinated the regulation of a subset of IGN genes and controlled extracellular matrix composition.

Comparative Genomics of Glossina palpalis gambiensis and G. morsitans morsitans to Reveal Gene Orthologs Involved in Infection by Trypanosoma brucei gambiense

Blood-feeding Glossina palpalis gambiense (Gpg) fly transmits the single-celled eukaryotic parasite Trypanosoma brucei gambiense (Tbg), the second Glossina fly African trypanosome pair being Glossina morsitans/T.brucei rhodesiense. Whatever the T. brucei subspecies, whereas the onset of their developmental program in the zoo-anthropophilic blood feeding flies does unfold in the fly midgut, its completion is taking place in the fly salivary gland where does emerge a low size metacyclic trypomastigote population displaying features that account for its establishment in mammals-human individuals included. Considering that the two Glossina—T. brucei pairs introduced above share similarity with respect to the developmental program of this African parasite, we were curious to map on the Glossina morsitans morsitans (Gmm), the Differentially Expressed Genes (DEGs) we listed in a previous study. Briefly, using the gut samples collected at days 3, 10, and 20 from Gpg that were fed or not at day 0 on Tbg—hosting mice, these DGE lists were obtained from RNA seq—based approaches. Here, post the mapping on the quality controlled DEGs on the Gmm genome, the identified ortholog genes were further annotated, the resulting datasets being compared. Around 50% of the Gpg DEGs were shown to have orthologs in the Gmm genome. Under one of the three Glossina midgut sampling conditions, the number of DEGs was even higher when mapping on the Gmm genome than initially recorded. Many Gmm genes annotated as “Hypothetical” were mapped and annotated on many distinct databases allowing some of them to be properly identified. We identify Glossina fly candidate genes encoding (a) a broad panel of proteases as well as (b) chitin—binding proteins, (c) antimicrobial peptide production—Pro3 protein, transferrin, mucin, atttacin, cecropin, etc—to further select in functional studies, the objectives being to probe and validated fly genome manipulation that prevents the onset of the developmental program of one or the other T. brucei spp. stumpy form sampled by one of the other bloodfeeding Glossina subspecies.

In Vitro Corticogenesis from Embryonic Stem Cells Recapitulates the In Vivo Epigenetic Control of Imprinted Gene Expression

Abstract In vitro corticogenesis from embryonic stem cells (ESCs) is an attractive model of cortical development and a promising tool for cortical therapy. It is unknown to which extent epigenetic mechanisms crucial for cortex development and function, such as parental genomic imprinting, are recapitulated by in vitro corticogenesis. Here, using genome‐wide transcriptomic and methylation analyses on hybrid mouse tissues and cells, we find a high concordance of imprinting status between in vivo and ESC‐derived cortices. Notably, in vitro corticogenesis strictly reproduced the in vivo parent‐of‐origin‐dependent expression of 41 imprinted genes (IGs), including Mest and Cdkn1c known to control corticogenesis. Parent‐of‐origin‐dependent DNA methylation was also conserved at 14 of 18 imprinted differentially methylated regions. The least concordant imprinted locus was Gpr1‐Zdbf2, where the aberrant bi‐allelic expression of Zdbf2 and Adam23 was concomitant with a gain of methylation on the maternal allele in vitro. Combined, our data argue for a broad conservation of the epigenetic mechanisms at imprinted loci in cortical cells derived from ESCs. We propose that in vitro corticogenesis helps to define the still poorly understood mechanisms that regulate imprinting in the brain and the roles of IGs in cortical development.

Larval transcriptomic response to host plants in two related phytophagous lepidopteran species : implications for host specialization and species divergence

BackgroundMost phytophagous insects have morphological, behavioral and physiological adaptations allowing them to specialize on one or a few plant species. Identifying the mechanisms involved in host plant specialization is crucial to understand the role of divergent selection between different environments in species diversification, and to identify sustainable targets for the management of insect pest species. In the present study, we measured larval phenotypic and transcriptomic responses to host plants in two related phytophagous lepidopteran species: the European corn borer (ECB), a worldwide pest of maize, and the adzuki bean borer (ABB), which feeds of various dicotyledons. Our aim was to identify the genes and functions underlying host specialization and/or divergence between ECB and ABB.ResultsAt the phenotypic level, we observed contrasted patterns of survival, weight gain and developmental time between ECB and ABB, and within ECB and ABB reared on two different host plants. At the transcriptomic level, around 8% of the genes were differentially expressed (DE) between species and/or host plant. 70% of these DE genes displayed a divergent pattern of expression between ECB and ABB, regardless of the host, while the remaining 30% were involved in the plastic response between hosts. We further categorized plastic DE genes according to their parallel or opposite pattern between ECB and ABB to specifically identify candidate genes involved in the species divergence by host specialization. These candidates highlighted a comprehensive response, involving functions related to plant recognition, digestion, detoxification, immunity and development. Last, we detected viral, bacterial, and yeast genes whose incidence contrasted ECB and ABB samples, and maize and mugwort conditions. We suggest that these microorganism communities might influence the survival, metabolism and defense patterns observed in ECB and ABB larvae.ConclusionsThe comprehensive approach developed in the present study allowed to identify phenotypic specialization patterns and underlying candidate molecular mechanisms, and highlighted the putative role of microorganisms in the insect-host plant interaction. These findings offer the opportunity to pinpoint specific and sustainable molecular or physiological targets for the regulation of ECB pest populations.

Cell Type-Specific mRNA Dysregulation in Hippocampal CA1 Pyramidal Neurons of the Fragile X Syndrome Mouse Model

Fragile X syndrome (FXS) is a genetic disorder due to the silencing of the Fmr1 gene, causing intellectual disability, seizures, hyperactivity, and social anxiety. All these symptoms result from the loss of expression of the RNA binding protein fragile X mental retardation protein (FMRP), which alters the neurodevelopmental program to abnormal wiring of specific circuits. Aberrant mRNAs translation associated with the loss of Fmr1 product is widely suspected to be in part the cause of FXS. However, precise gene expression changes involved in this disorder have yet to be defined. The objective of this study was to identify the set of mistranslated mRNAs that could contribute to neurological deficits in FXS. We used the RiboTag approach and RNA sequencing to provide an exhaustive listing of genes whose mRNAs are differentially translated in hippocampal CA1 pyramidal neurons as the integrative result of FMRP loss and subsequent neurodevelopmental adaptations. Among genes differentially regulated between adult WT and Fmr1−/y mice, we found enrichment in FMRP-binders but also a majority of non-FMRP-binders. Interestingly, both up- and down-regulation of specific gene expression is relevant to fully understand the molecular deficiencies triggering FXS. More importantly, functional genomic analysis highlighted the importance of genes involved in neuronal connectivity. Among them, we show that Klk8 altered expression participates in the abnormal hippocampal dendritic spine maturation observed in a mouse model of FXS.