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International audience; Glioblastomas (GBM) are heterogeneous tumors with high metabolic plasticity. Their poor prognosis is linked to the presence of glioblastoma stem cells (GSC), which support resistance to therapy, notably to temozolomide (TMZ). Mesenchymal stem cells (MSC) recruitment to GBM contributes to GSC chemoresistance, by mechanisms still poorly understood. Here, we provide evidence that MSCs transfer mitochondria to GSCs through tunneling nanotubes, which enhances GSCs resistance to TMZ. More precisely, our metabolomics analyses reveal that MSC mitochondria induce GSCs metabolic reprograming, with a nutrient shift from glucose to glutamine, a rewiring of the tricarboxylic acid cycle from glutaminolysis to reductive carboxylation and increase in orotate turnover as well as in pyrimidine and purine synthesis. Metabolomics analysis of GBM patient tissues at relapse after TMZ treatment documents increased concentrations of AMP, CMP, GMP, and UMP nucleotides and thus corroborate our in vitro analyses. Finally, we provide a mechanism whereby mitochondrial transfer from MSCs to GSCs contributes to GBM resistance to TMZ therapy, by demonstrating that inhibition of orotate production by Brequinar (BRQ) restores TMZ sensitivity in GSCs with acquired mitochondria. Altogether, these results identify a mechanism for GBM resistance to TMZ and reveal a metabolic dependency of chemoresistant GBM following the acquisition of exogenous mitochondria, which opens therapeutic perspectives based on synthetic lethality between TMZ and BRQ.Significance: Mitochondria acquired from MSCs enhance the chemoresistance of GBMs. The discovery that they also generate metabolic vulnerability in GSCs paves the way for novel therapeutic approaches.

Metabolites produced by GSCs following MSC mitochondria acquisition and TMZ treatment

TMZ dose-response assay Caspase-GloⓇ 3/7 assay Cytotox assay Extracellular Flux Assays Cell Proliferation Western Blot Flow Cytometry Metabolite usage Mitoplates Mass Spectrometry Quantification 13C stable isotope tracing experiments Preparation and quantification of MSC mtDNA in GSCs Imaging

Cocultures of red MitoTracker-labeled MSCs and green CellTracker-labeled GSCs

Glioblastomas (GBM) are heterogeneous tumors with high metabolic plasticity. Their poor prognosis is linked to the presence of glioblastoma stem cells (GSCs), which support resistance to therapy, notably to temozolomide (TMZ). Mesenchymal stem cells (MSCs) recruitment to GBM contributes to GSC chemoresistance, by mechanisms still poorly understood. Here, we provide evidence that MSCs transfer mitochondria to GSCs through tunneling nanotubes, which enhances GSCs resistance to TMZ. More precisely, our metabolomics analyses reveal that MSC mitochondria induce GSCs metabolic reprograming, with a nutrient shift from glucose to glutamine, a rewiring of the TCA cycle from glutaminolysis to reductive carboxylation and increase in orotate turnover as well as in pyrimidine and purine synthesis. Metabolomics analysis of GBM patient tissues at relapse after TMZ treatment documents increased concentrations of AMP, CMP, GMP and UMP nucleotides and thus corroborate our in vitro analyses. Finally, we provide a mechanism whereby mitochondrial transfer from MSCs to GSCs contributes to GBM resistance to TMZ therapy, by demonstrating that inhibition of orotate production by Brequinar (BRQ) restores TMZ sensitivity in GSCs with acquired mitochondria. Altogether, these results identify a mechanism for GBM resistance to TMZ and reveal a metabolic dependency of chemoresistant GBM following the acquisition of exogenous mitochondria, which opens therapeutic perspectives based on synthetic lethality between TMZ and BRQ.

Thyroid hormones (TH) and glucocorticoids (GC) are involved in numerous developmental and physiological processes. The effects of individual hormones are well documented, but little is known about the joint actions of the two hormones. To decipher the crosstalk between these two hormonal pathways, we conducted a transcriptional analysis of genes regulated by TH, GC, or both hormones together in liver of

Amphibians display very diverse life cycles and development can be direct, where it occurs in ovo and a juvenile hatches directly, or biphasic, where an aquatic larva hatches and later undergoes metamorphosis followed by sexual maturation. In both cases, metamorphosis, corresponds to the Post Embryonic Transition (PETr). A third strategy, only found in Urodeles, is more complex as larvae reach sexual maturity before metamorphosis, which can become accessory. The resulting paedomorphs retain their larval characters and keep their aquatic habitat. Does it mean that paedomorphs do not undergo PETr ? Recent work using high throughput technologies coupled to system biology and developmental endocrinology revisited this question and provided novel datasets indicating that a paedomorph's 'larval' tissue undergoes a proper developmental transition. Together with historical data, we propose that this transition is a marker of the PETr, which would be distinct from metamorphosis. This implies that 1) complex life cycles would result from the uncoupling of PETr and metamorphosis, and 2), biphasic life cycles would be a special cases where they occur simultaneously. This article is protected by copyright. All rights reserved.

Frogs are an ecologically diverse and phylogenetically ancient group of living amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., centric) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding, surrounded by pericentromeric LINE/L1 elements. We explored chromosome structure across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (HiC) data for all species. Abundant satellite repeats occupy the unusually long (∼20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible association of centromeric chromatin, and of telomeres, reflecting a Rabl configuration similar to the “bouquet” structure of meiotic cells. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

Background The 7SK small nuclear RNA (snRNA) found in most metazoans is a key regulator of P-TEFb which in turn regulates RNA polymerase II elongation. Although its primary sequence varies in protostomes, its secondary structure and function are conserved across evolutionary distant taxa. Results Here, we describe a novel ncRNA sharing many features characteristic of 7SK RNAs, in D. melanogaster. We examined the structure of the corresponding gene and determined the expression profiles of the encoded RNA, called snRNA:7SK:94F, during development. It is probably produced from the transcription of a lncRNA which is processed into a mature snRNA. We also addressed its biological function and we show that, like dm7SK, this alternative 7SK interacts in vivo with the different partners of the P-TEFb complex, i.e. HEXIM, LARP7 and Cyclin T. This novel RNA is widely expressed across tissues. Conclusion We propose that two distinct 7SK genes might contribute to the formation of the 7SK snRNP complex in D. melanogaster.

Thyroid hormones (THs) are important regulators of growth, development, and homeostasis of all vertebrates. There are many environmental contaminants that are known to disrupt TH action, yet their mechanisms are only partially understood. While the effects of Endocrine Disrupting Chemicals (EDCs) are mostly studied as "hormone system silos", the present critical review highlights the complexity of EDCs interfering with TH function through their interactions with other hormonal axes involved in reproduction, stress, and energy metabolism. The impact of EDCs on components that are shared between hormone signaling pathways or intersect between pathways can thus extend beyond the molecular ramifications to cellular, physiological, behavioral, and whole-body consequences for exposed organisms. The comparatively more extensive studies conducted in mammalian models provides encouraging support for expanded investigation and highlight the paucity of data generated in other non-mammalian vertebrate classes. As greater genomics-based resources become available across vertebrate classes, better identification and delineation of EDC effects, modes of action, and identification of effective biomarkers suitable for HPT disruption is possible. EDC-derived effects are likely to cascade into a plurality of physiological effects far more complex than the few variables tested within any research studies. The field should move towards understanding a system of hormonal systems' interactions rather than maintaining hormone system silos.

International audience; Setmar is a gene specific to simian genomes. The function(s) of its isoforms are poorly understood and their existence in healthy tissues remains to be validated. Here we profiled SETMAR expression and its genome-wide binding landscape in colon tissue. We found isoforms V3 and V6 in healthy and tumour colon tissues as well as incell lines. In two colorectal cell lines SETMAR binds to several thousand Hsmar1 and MADE1 terminal ends, transposons mostly located in non-genic regions of active chromatin including in enhancers. It also binds to a 12-bp motifs similar to an inner motif in Hsmar1 and MADE1 terminal ends. This motif is interspersed throughout the genome and is enriched in GC-rich regions as well as in CpG islands that contain constitutive replication origins. It is also found in enhancers other than those associated with Hsmar1 and MADE1. The role of SETMAR in the expression of genes, DNA replication and in DNA repair are discussed.

Thyroid hormones (TH) act mainly on the expression of the genome. It is well accepted that differential gene expression following treatment with TH recapitulates the expression profiles taking place during natural processes induced by these hormones. Although both processes seem to correlate at the scale of a few target genes, this hasn’t been addressed in a systematic manner. Our objectives were first to compare transcriptome variations after TH treatment with transcriptome variations during a TH controlled natural process and second to evaluate the proportion of the direct TH-response. The measurement of gene expression at genome scale (transcriptome) is obtained by sequencing all the messenger RNAs (RNA-seq). Direct response was sorted out by linking the transcription start sites of target genes using RNA-PET analysis with TR binding sites mapping using chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). Indeed, ChIA-PET not only allow to map TR binding sites but also the physical interactions between them and transcription start sites of regulated genes. Our model is one of the striking developmental processes orchestrated by TH: amphibian metamorphosis. Tadpole transformation is marked by dramatic changes including de novo morphogenesis (limb), tissue remodelling (brain, intestine...) and organ resorption through apoptosis (tail). These changes involve cascades of gene regulation initiated by TH and their receptors. Because metamorphosis has close and interesting parallels with the perinatal period in mammals (including human), metamorphosis is thus an attractive model to analyze in a physiological context, the functions and mechanisms of action of TH. Here, we have focused on one Xenopus tropicalis organs, the tail fin skin which will disappear through cell death. We have compared natural metamorphosis with 24h of 10nM triiodothyronine (T3) treatment. We were able to observe several differences between T3 treatment and natural development. First, the genes regulated by T3 only correspond to a proportion of genes differentially expressed during metamorphosis. Second, T3-response genes start to be regulated well before tail regression (several days). Finally, T3-direct target genes represent a few percent of all the genes differentially expressed during tail regression. In conclusion, the comparison of transcriptomes of natural and induced metamorphosis allow us to reach a more precise understanding of TH action

International audience; Methylation of cytosine residues in DNA influences chromatin structure and gene transcription, and its regulation is crucial for brain development. There is mounting evidence that DNA methylation can be modulated by hormone signaling. We analyzed genome-wide changes in DNA methylation and their relationship to gene regulation in the brain of Xenopus tadpoles during metamorphosis, a thyroid hormone-dependent developmental process. We studied the region of the tadpole brain containing neurosecretory neurons that control pituitary hormone secretion, a region that is highly responsive to thyroid hormone action. Using Methylated DNA Capture sequencing (MethylCap-seq) we discovered a diverse landscape of DNA methylation across the tadpole neural cell genome, and pairwise stage comparisons identified several thousand differentially methylated regions (DMRs). During the pre-to pro-metamorphic period, the number of DMRs was lowest (1,163), with demethylation predominating. From pre-metamorphosis to metamorphic climax DMRs nearly doubled (2,204), with methylation predominating. The largest changes in DNA methylation were seen from metamorphic climax to the completion of metamorphosis (2960 DMRs), with 80% of the DMRs representing demethylation. Using RNA sequencing, we found negative correlations between differentially expressed genes and DMRs localized to gene bodies and regions upstream of transcription start sites. DNA demethylation at metamorphosis revealed by MethylCap-seq was corroborated by increased immunoreactivity for the DNA demethylation intermediates 5-hydroxymethylcytosine and 5-carboxymethylcytosine, and the methylcytosine dioxygenase ten eleven translocation 3 that catalyzes DNA demethylation. Our findings show that the genome of tadpole neural cells undergoes significant changes in DNA methylation during metamorphosis, and these changes likely influence chromatin architecture, and gene regulation programs occurring during this developmental period.

International audience; Previous work identified a transcribed locus, Str. 34945, induced by the frog stress hormone corticos-terone (CORT) in Xenopus tropicalis tails. Because thyroid hormone had no influence on its expression, Str. 34945 was dubbed the first ''CORT-only" gene known from tadpoles. Here, we examine the genomic annotation for this transcript, hormone specificity, time course of induction, tissue distribution, and developmental expression profile. The location of Str. 34945 on the X. tropicalis genome lies between the genes ush1g (Usher syndrome 1G) and fads6 (fatty acid desaturase 6). A blast search showed that it maps to the same region on the X. laevis genome, but no hits were found in the human genome. Using RNA-seq data and conventional reverse transcriptase PCR and sequencing, we show that Str. 34945 is part of the 3 0 untranslated region of ush1g. We find that CORT but not aldosterone or thyroid hormone treatment induces Str. 34945 in tadpole tails and that expression of Str. 34945 achieves maximal expression within 12-24 h of CORT treatment. Among tissues, Str. 34945 is induced to the highest degree in tail, with lesser induction in lungs, liver, and heart, and no induction in the brain or kidney. During natural metamorphosis , Str. 34945 expression in tails peaks at metamorphic climax. The role of ush1g in metamorphosis is not understood, but the specificity of its hormone response and its expression in tail make ush1g valuable as a marker of CORT-response gene induction independent of thyroid hormone.

Background: Most work in endocrinology focus on the action of a single hormone, and very little on the cross-talks between two hormones. Here we characterize the nature of interactions between thyroid hormone and glucocorticoid signaling during Xenopus tropicalis metamorphosis. Methods: We used functional genomics to derive genome wide profiles of methylated DNA and measured changes of gene expression after hormonal treatments of a highly responsive tissue, tailfin. Clustering classified the data into four types of biological responses, and biological networks were modeled by system biology. Results: We found that gene expression is mostly regulated by either T3 or CORT, or their additive effect when they both regulate the same genes. A small but non-negligible fraction of genes (12%) displayed non-trivial regulations indicative of complex interactions between the signaling pathways. Strikingly, DNA methylation changes display the opposite and are dominated by cross-talks. Conclusion: Cross-talks between thyroid hormones and glucocorticoids are more complex than initially envisioned and are not limited to the simple addition of their individual effects, a statement that can be summarized with the pseudo-equation: TH ∙ GC &gt, TH + GC. DNA methylation changes are highly dynamic and buffered from genome expression.

International audience; Amphibian post-embryonic development and Thyroid Hormones (TH) signaling are deeply and intimately connected. In anuran amphibians, TH induce the spectacular and complex process known as metamorphosis. In paedomorphic salamanders, at similar development time, raising levels of TH fail to induce proper metamorphosis, as many "larval" tissues (e.g., gills, tailfin) are maintained. Why does the same evolutionary conserved signaling pathway leads to alternative phenotypes? We used a combination of developmental endocrinology, functional genomics and network biology to compare the transcriptional response of tailfin to TH, in the post-hatching paedormorphic Axolotl salamander and Xenopus tadpoles. We also provide a technological framework that efficiently reduces large lists of regulated genes down to a few genes of interest, which is well-suited to dissect endocrine regulations. We first show that Axolotl tailfin undergoes a strong and robust TH-dependent transcriptional response at post embryonic transition, despite the lack of visible anatomical changes. We next show that Fos and Actg1, which structure a single and dense subnetwork of cellular sensors and regulators, display opposite regulation between the two species. We finally show that TH treatments and natural variations of TH levels follow similar transcriptional dynamics. We suggest that, at the molecular level, tailfin fate correlates with the alternative transcriptional states of an fos-actg1 sub-network, which also includes transcription factors and regulators of cell fate. We propose that this subnetwork is one of the molecular switches governing the initiation of distinct TH responses, with transcriptional programs conducting alternative tailfin fate (maintenance vs. resorption) 2 weeks post-hatching.

Thyroid hormones (TH) have been shown to govern post-embryonic development in vertebrates inducing multiples effects. Recently, TH were shown to regulate the expression of genes coding for DNA methylation modifying enzymes. Acting on DNA methylation is an important means of regulating the expression of gene clusters and a central mechanism for coordinating developmental transitions. However, DNA methylation is dynamic and also integrate signals from the constantly changing environment. Certain variation of DNA methylation at these developmental transitions can confer on the genome a risk situation that can have lifelong consequences (cardiovascular and metabolism diseases, cancer or nervous system associated illness). Our objective is to locate TH induced variation of DNA methylation profiles to characterize potential determinants for certain pathologies and / or adaptation to a variation of the environment. We have thus initiated the mapping of DNA methylation following treatment with T3, using a method for capturing methylated DNA (MethylCap) combined with high-throughput sequencing (MethylCap-Seq). Magnetic beads allow for capture and fractionation of methylated DNA by CpG density. Here, we will focus on the highly methylated fraction of DNA. Our model of developmental transition is the amphibian metamorphosis, a post-embryonic process regulated by TH. Additionally, stress during metamorphosis might also induce potential phenotype propagation to adult forms, highlighting its importance in driving adaptation to fluctuating environments. We have compared TH induced methylation profiles in the hind limb and caudal epidermal, two organs with contrasted fate during metamorphosis (respectively morphogenesis and cell death). The results shown that TH affect the methylome with a strong tendency for DNA methylation marks to be removed. The majority of differential methylated regions (DMR) are tissue specific. However, few DMRs are found in both tissues and show either identical or opposite variations. The DMRs are generally not localize near genes regulated or not by the hormones. In conclusion, at post-embryonic transition in amphibians, TH induce mainly tissues-specific DNA demethylation not directly related to gene transcription.

International audience; PiggyBac(PB)-like elements (pble) are members of a eukaryotic DNA transposon family. This family is of interest to evolutionary genomics because pble transposases have been domesticated at least 9 times in vertebrates. The amino acid sequence of pble transposases can be split into three regions: an acidic N-terminal domain (similar to 100 aa), a central domain (similar to 400 aa) containing a DD[D/E] catalytic triad, and a cysteine-rich domain (CRD; similar to 90 aa). Two recent reports suggested that a functional CRD is required for pble transposase activity. Here we found that two CRD-deficient pble transposases, a PB variant and an isoform encoded by the domesticated PB-derived vertebrate transposase gene 5 (pgbd5) trigger transposition of the Ifp2 pble. When overexpressed in HeLa cells, these CRD-deficient transposases can insert Ifp2 elements with proper and improper transposon ends, associated with deleterious effects on cells. Finally, we found that mouse CRD-deficient transposase Pgbd5, as well as PB, do not insert pbles at random into chromosomes. Transposition events occurred more often in genic regions, in the neighbourhood of the transcription start sites and were often found in genes predominantly expressed in the human central nervous system.

International audience; Proper gene expression involves communication between the regulatory elements and promoters of genes. Because regulatory elements can be located over a large range of genomic distances (from as close as a few hundred bp to as much as several Mb away), contact and communication between regulators and the core transcriptional machinery at promoters are mediated through DNA looping. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity between en-hancer(s) and their target promoter(s). One such method, chromatin interaction analysis using paired-end-tag (ChIA-PET) sequencing, is a leading high-throughput method for detection of genome wide chromatin interactions. Briefly, the method involves cross-linkage of chromatin (-DNA) fibers in cells in situ, fragmentation of the fixed chromatin-DNA complexes by sonication, followed by enrichment of the chromatin complexes with a dedicated antibody through the process of immunoprecipitation (IP). Next, application of the ChIA-PET protocol followed by deep sequencing and mapping of reads to the reference genome reveals both binding sites and remote chromatin interactions mediated by the protein factors of interest. The method detailed here focuses on ChIP sample preparation and can be completed in 5 d. The ChIA-PET method is detailed in an associated protocol. Because not all chromatin immunoprecipitation protocols are suitable for ChIA-PET, it is important to strictly follow this procedure before performing the ChIA-PET protocol. MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at

Thyroid hormones are pleiotropic hormones involved in chordates physiology. Understanding their functions and mechanisms is also instrumental to diagnose dys-regulations and get a predictive power that can applied to medicine, ecology, etc. Today, high-throughput sequencing technologies offer the opportunity to address this issue not only in model organisms but also in non-model organisms. Here, we describe a method that makes use of RNA-seq to address differential expression analysis in non-model organism.

International audience; Thyroid hormones are pleiotropic hormones involved in chordates physiology. Understanding their functions and mechanisms is also instrumental to diagnose dys-regulations and get a predictive power that can applied to medicine, ecology, etc. Today, high-throughput sequencing technologies offer the opportunity to address this issue not only in model organisms but also in non-model organisms. Here, we describe a method that makes use of RNA-seq to address differential expression analysis in non-model organism.

International audience

Cooperative, synergistic gene regulation by nuclear hormone receptors can increase sensitivity and amplify cellular responses to hormones. We investigated thyroid hormone (TH) and glucocorticoid (GC) synergy on the Krüppel-like factor 9 (Klf9) gene, which codes for a zinc finger transcription factor involved in development and homeostasis of diverse tissues. We identified regions of the Xenopus and mouse Klf9 genes 5–6 kb upstream of the transcription start sites that supported synergistic transactivation by TH plus GC. Within these regions, we found an orthologous sequence of approximately 180 bp that is highly conserved among tetrapods, but absent in other chordates, and possesses chromatin marks characteristic of an enhancer element. The Xenopus and mouse approximately 180-bp DNA element conferred synergistic transactivation by hormones in transient transfection assays, so we designate this the Klf9 synergy module (KSM). We identified binding sites within the mouse KSM for TH receptor, GC receptor, and nuclear factor κB. TH strongly increased recruitment of liganded GC receptor and serine 5 phosphorylated (initiating) RNA polymerase II to chromatin at the KSM, suggesting a mechanism for transcriptional synergy. The KSM is transcribed to generate long noncoding RNAs, which are also synergistically induced by combined hormone treatment, and the KSM interacts with the Klf9 promoter and a far upstream region through chromosomal looping. Our findings support that the KSM plays a central role in hormone regulation of vertebrate Klf9 genes, it evolved in the tetrapod lineage, and has been maintained by strong stabilizing selection.

Setmar is a 3-exons gene coding a SET domain fused to a Hsmar1 transposase. Its different transcripts theoretically encode 8 isoforms with SET moieties differently spliced. In vitro, the largest isoform binds specifically to Hsmar1 DNA ends and with no specificity to DNA when it is associated with hPso4. In colon cell lines, we found they bind specifically to two chromosomal targets depending probably on the isoform, Hsmar1 ends and sites with no conserved motifs. We also discovered that the isoforms profile was different between cell lines and patient tissues, suggesting the isoforms encoded by this gene in healthy cells and their functions are currently not investigated.

This article is distributed under the terms of a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license)., There is increasing evidence that the thyroid hormone (TH) receptors (THRs) can play a role in aging, cancer and degenerative diseases. In this paper, we demonstrate that binding of TH T3 (triiodothyronine) to THRB induces senescence and deoxyribonucleic acid (DNA) damage in cultured cells and in tissues of young hyperthyroid mice. T3 induces a rapid activation of ATM (ataxia telangiectasia mutated)/PRKAA (adenosine monophosphate- activated protein kinase) signal transduction and recruitment of the NRF1 (nuclear respiratory factor 1) and THRB to the promoters of genes with a key role on mitochondrial respiration. Increased respiration leads to production of mitochondrial reactive oxygen species, which in turn causes oxidative stress and DNA double- strand breaks and triggers a DNA damage response that ultimately leads to premature senescence of susceptible cells. Our findings provide a mechanism for integrating metabolic effects of THs with the tumor suppressor activity of THRB, the effect of thyroidal status on longevity, and the occurrence of tissue damage in hyperthyroidism. © 2014 Zambrano et al., This work was supported by grants from Ministerio de Economía y Competitividad (BFU2011-28958 to A. Aranda and SAF2009-11150 to A. Pascual), from the Instituto de Salud Carlos III (RD012/0036/0030 to A. Aranda; and PI 07/0167 and PI 10/0703 to R. Garesse), from the Comunidad de Madrid (S2011/BMD-2328 TIRONET to A. Aranda), and European Union grant project CRESCENDO (FP6-018652 to A. Aranda and L.M. Sachs).

Background Thyroid hormone (TH) receptor (TR) plays critical roles in vertebrate development. However, the in vivo mechanism of TR action remains poorly explored. Scope of review Frog metamorphosis is controlled by TH and mimics the postembryonic period in mammals when high levels of TH are also required. We review here some of the findings on the developmental functions of TH and TR and the associated mechanisms obtained from this model system. Major conclusion A dual function model for TR in Anuran development was proposed over a decade ago. That is, unliganded TR recruits corepressors to TH response genes in premetamorphic tadpoles to repress these genes and prevent premature metamorphic changes. Subsequently, when TH becomes available, liganded TR recruits coactivators to activate these same genes, leading to metamorphic changes. Over the years, molecular and genetic approaches have provided strong support for this model. Specifically, it has been shown that unliganded TR recruits histone deacetylase containing corepressor complexes during larval stages to control metamorphic timing, while liganded TR recruits multiple histone modifying and chromatin remodeling coactivator complexes during metamorphosis. These complexes can alter chromatin structure via nucleosome position alterations or eviction and histone modifications to contribute to the recruitment of transcriptional machinery and gene activation. General significance The molecular mechanisms of TR action in vivo as revealed from studies on amphibian metamorphosis are very likely applicable to mammalian development as well. These findings provide a new perspective for understanding the diverse effects of TH in normal physiology and diseases caused by TH dysfunction. This article is part of a Special Issue entitled Thyroid hormone signalling.

The emergence of vertebrates is closely associated to the evolution of mineralized bone tissue. However, the molecular basis underlying the origin and subsequent diversification of the skeletal mineralized matrixisstillpoorlyunderstood. Oneefficientway totackle thisissueis tocomparethe expression, between vertebrate species, of osteoblastic genes coding for bone matrix proteins. In this work, wehave focusedon theevolution ofthenetwork formingcollagen familywhichcontains the Col8a1, Col8a2, and Col10a1 genes. Both phylogeny and synteny reveal that these three paralogues are vertebrate-specific and derive from two independent duplications in the vertebrate lineage. To shed light on the evolution of this family, we have analyzed the osteoblastic expression of the network forming collagens in endochondral and intramembraneous skeletal elements of the amphibian Xenopus tropicalis. Remarkably, we find that amphibian osteoblasts express Col10a1 ,a gene strongly expressed in osteoblasts in actinopterygians but not in amniotes. In addition, while Col8a1 is known to be robustly expressed in mammalian osteoblasts, the expression levels of its amphibian orthologue are dramatically reduced. Our work reveals that while a skeletal expression of network forming collagen members is widespread throughout vertebrates, osteoblasts from divergent vertebrate lineages express different combinations of network forming collagen paralogues. J. Exp. Zool. (Mol. Dev. Evol.) 9999B: 1-11, 2013. © 2013 Wiley Periodicals, Inc. How to cite this article: Aldea D, Hanna P, Munoz D, Espinoza J, Torrejon M, Sachs L, Buisine N, Oulion S, Escriva H, Marcellini S. 2013. Evolution of the vertebrate bone matrix: An expression analysisofthenetworkformingcollagenparaloguesinamphibianosteoblasts.J.Exp.Zool.(Mol. Dev. Evol.) 9999:1-11.

International audience; Studying the dynamic of gene regulatory networks is essential in order to understand the specific signals and factors that govern cell proliferation and differentiation during development. This also has direct implication in human health and cancer biology. The general transcrip-tional elongation regulator P-TEFb regulates the transcriptional status of many developmental genes. Its biological activity is controlled by an inhibitory complex composed of HEXIM and the 7SK snRNA. Here, we examine the function of HEXIM during Drosophila development. Our key finding is that HEXIM affects the Hedgehog signaling pathway. HEXIM knockdown flies display strong phenotypes and organ failures. In the wing imaginal disc, HEXIM knock-down initially induces ectopic expression of Hedgehog (Hh) and its transcriptional effector Cubitus interuptus (Ci). In turn, deregulated Hedgehog signaling provokes apoptosis, which is continuously compensated by apoptosis-induced cell proliferation. Thus, the HEXIM knock-down mutant phenotype does not result from the apoptotic ablation of imaginal disc; but rather from the failure of dividing cells to commit to a proper developmental program due to Hedgehog signaling defects. Furthermore, we show that ci is a genetic suppressor of hexim. Thus, HEXIM ensures the integrity of Hedgehog signaling in wing imaginal disc, by a yet unknown mechanism. To our knowledge, this is the first time that the physiological function of HEXIM has been addressed in such details in vivo.

The origin of bone and cartilage, and their subsequent diversification in specific vertebrate lineages, is intimately linked to the precise transcriptional control of genes involved in matrix mineralization. It is not yet clear, however, to which extent the osteoblasts, osteocytes, and chondrocytes of each of the major vertebrate groups express similar sets of genes. In this study we have focused on the evolution of two independent families of genes that code for extracellular matrix components of the skeleton and that include secreted protein, acidic, cysteine-rich (SPARC), bone sialoprotein (BSP) and dentin matrix protein 1 (DMP1) paralogues, and the osteocalcin (OC) and matrix gla protein (MGP) paralogues. Analyzing developing Xenopus tropicalis skeletal elements, we show that the expression patterns of these genes are well conserved with mammals. The fact that only a few osteoblasts express DMP1, while only some osteocytes express SPARC and BSP, reveals a significant degree of molecular heterogeneity for these two populations of X. tropicalis cells, similarly to what has been described in mouse. Although the cis-regulatory modules (CRM) of the mammalian OC, DMP1, and BSP orthologs have been functionally characterized, we found no evidence of sequence similarity between these regions and the X. tropicalis genome. Furthermore, these regulatory elements evolve rapidly, as they are only poorly conserved between human and rodents. Therefore, the SPARC/DMP1/BSP and the OC/MGP families provide a good paradigm to study how transcriptional output can be maintained in skeletal cells despite extensive sequence divergence of CRM.

Proper gene expression involves communication between the regulatory elements and promoters of genes. Today, chromosome conformation capture (3C)-based methods efficiently probe chromosome folding in the nucleus and thus provide a molecular description of physical proximity through DNA looping between enhancer(s) and their target promoter(s). One such method, chromatin interaction analysis using paired-end-tag (ChIA-PET) sequencing is a powerful high-throughput method for detection of genome-wide chromatin interactions. Following enrichment of the chromatin complexes with a dedicated antibody, through a process of immunoprecipitation (IP), DNA fragments are end-joined with specifically designed DNA-linkers through proximity ligation. The DNA-linkers contain the binding site for the type II restriction enzyme MmeI, which cleaves 20 bp from each end of the ligated fragments, thus releasing a “paired end tag” (PET): [20 bp tag]-[linker]-[20 bp tag]. The PETs are then deep-sequenced and reads are mapped to the reference genome, revealing both binding sites, as well as remote chromatin interactions mediated by the protein factors of interest. The method detailed here focuses on ChIA-PET library construction and can be completed in 2 wk.

Bioinformatique et génomique (Resp. Hadi Quesneville); International audience; Transposable elements (TEs) are ubiquitous components of eukaryotic genomes that impact many aspects of genome function. TE detection in genomic sequences is typically performed using similarity searches against a set of reference sequences built from previously identified TEs. Here, we demonstrate that this process can be improved by designing reference sets that incorporate key aspects of the structure and evolution of TEs and by combining these sets with Repbase Update (RU), which is composed mainly of consensus sequences. Using the Arabidopsis genome as a test case, our approach leads to the detection of an extra 12.4% of TE sequences. These correspond to novel TE fragments as well as to the extension of TE fragments already detected by RU. Significantly, we find that TE detection could be readily optimized using only two reference sets, one containing true consensus sequences and the other mosaic sequences that capture the structural diversity of TE copies within a family.

Transposons have contributed protein coding sequences to a unexpectedly large number of human genes. Except for the V(D)J recombinase and telomerase, all remain of unknown function. Here we investigate the activity of the human SETMAR protein, a highly expressed fusion between a histone H3 methylase and a mariner family transposase. Although SETMAR has demonstrated methylase activity and a DNA repair phenotype, its mode of action and the role of the transposase domain remain obscure. As a starting point to address this problem, we have dissected the activity of the transposase domain in the context of the full-length protein and the isolated transposase domain. Complete transposition of an engineered Hsmar1 transposon by the transposase domain was detected, although the extent of the reaction was limited by a severe defect for cleavage at the 3 ends of the element. Despite this problem, SETMAR retains robust activity for the other stages of the Hsmar1 transposition reaction, namely, site-specific DNA binding to the transposon ends, assembly of a paired-ends complex, cleavage of the 5 end of the element in Mn 2 , and integration at a TA dinucleotide target site. SETMAR is unlikely to catalyze transposition in the human genome, although the nicking activity may have a role in the DNA repair phenotype. The key activity for the mariner domain is therefore the robust DNA-binding and looping activity which has a high potential for targeting the histone methylase domain to the many thousands of specific binding sites in the human genome provided by copies of the Hsmar1 transposon. DNA transposons are genomic parasites that exist purely at the molecular level. Although ubiquitous, they are short-lived in any given eukaryotic genome and rely on frequent horizontal transfer to new hosts (19, 24). At present, DNA transposons are accepted as extinct in humans, and the youngest family identified thus far appeared some 50 million years ago, after the divergence of the

Vitellogenins (Vtg) are usually encoded by small multigene families containing up to six genes. With 20 tandemly arranged genes, the rainbow trout ( Oncorhynchus mykiss) is an exception to this rule. PCR amplification, cloning and sequence analysis of Vtg genes in other salmonid species revealed the existence of two paralogous gene clusters, designated Vtg-A and Vtg-B. Southern hybridization showed that the number of genes varies from 2 to 30 copies from one species to another, as well as between the two gene clusters. All Coregonus, Thymallus, Salmo and Salvelinus species studied have both gene clusters, while Oncorhynchus species possess only the Vtg-A locus. Phylogenetic trees constructed from Vtg sequences revealed conflicting nodes with the consensus tree based on morphological and anatomical data. Vtg sequences support the grouping ( Salmo, ( Salvelinus, Oncorhynchus)) instead of the accepted consensus ( Salvelinus, ( Salmo, Oncorhynchus)). Structural data on gene organization also support the contention that Salvelinus and Oncorhynchus are sister taxa. Evolutionary implications for the Vtg gene clusters in salmonids are discussed.

International audience

Amphibian metamorphosis is marked by dramatic thyroid hormone (T(3))-induced changes including de novo morphogenesis, tissue remodeling, and organ resorption through programmed cell death. These changes involve cascades of gene regulation initiated by thyroid hormone (TH). TH functions by regulating gene expression through TH receptors (TR). TR are DNA-binding transcription factors that belong to the steroid hormone receptor superfamily. In the absence of ligand, TR can repress gene expression by recruiting a corepressor complex, whereas liganded TR recruits a coactivator complex for gene activation. Earlier studies have led us to propose a dual function model for TR during development. In premetamorphic tadpoles, unliganded TR represses transcription involving corepressors. During metamorphosis, endogenous T(3) allows TR to activate gene expression. To fully understand the diversity of T(3) effects during metamorphosis, whole genome analysis of transcriptome and mechanism of TR action should be carried out. To this end, the new sequencing technologies have dramatically changed how fundamental questions in biology are being addressed and is now making the transition from technology development to being a standard for genomic and functional genomic analysis. This review focuses on the applications of high-throughput technologies to the field of amphibian metamorphosis.