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See detailTowards the identification of cis-elements responsible for the high expression of HMA4, an essential gene for Zn hyperaccumulation in Arabidopsis halleri.
Spielmann, Julien ULg; Nouet, Cécile ULg; Scheepers, Maxime ULg et al

Conference (2015, June 26)

A. halleri is a Zn and Cd hypertolerant and hyperaccumulator plant which stores metals in shoot vacuoles. One key step for hyperaccumulation is a highly efficient root-to-shoot translocation of metals ... [more ▼]

A. halleri is a Zn and Cd hypertolerant and hyperaccumulator plant which stores metals in shoot vacuoles. One key step for hyperaccumulation is a highly efficient root-to-shoot translocation of metals. The Zn/Cd plasmamembrane pump HMA4 (heavy metal ATPase 4) plays a central role in this process and is involved in xylem loading/unloading. Moreover, HMA4 is overexpressed in A. halleri compare to its non-tolerant and non-hyperaccumulator sister, Arabidopsis thaliana. The HMA4 overexpression is determined by a combination of gene triplication and cis-regulatory changes increasing transcription levels of each three HMA4 copies. In this study, we are focusing on the two most active HMA4 promoters and we aim to identify the cis-elements underlying high expression of HMA4 in A. halleri using promoter-reporter constructs. Through deletion analysis, two important regions for a high expression have been identified. Ongoing analysis of more detailed deletions will be presented. [less ▲]

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See detailTowards the identification of mechanisms underlying the high expression of HMA4, an essential gene for zinc hyperaccumulation in Arabidopsis halleri.
Spielmann, Julien ULg; Nouet, Cécile ULg; Scheepers, Maxime ULg et al

Poster (2015, June 08)

To maintain metal homeostasis within physiological limits independently of metal concentrations present in soil, plants developed a complex network including metal uptake, chelation, trafficking, and ... [more ▼]

To maintain metal homeostasis within physiological limits independently of metal concentrations present in soil, plants developed a complex network including metal uptake, chelation, trafficking, and storage processes. In this network, transporters have a central role. To study the molecular basis of zinc homeostasis in plants, we are using A. halleri, a zinc- and cadmium-tolerant and zinc-hyperaccumulating species representing the extreme end of natural variation in terms of metal homeostasis. Indeed, A. halleri stores high amount of metals in vacuoles of above-ground tissues (>1% Zn or 0.01% Cd in shoot dry weight). Cross-species transcriptomic studies identified about thirty genes which are constitutively over-expressed in A. halleri compared to its non-tolerant and non-hyperaccumulator sister species, Arabidopsis thaliana (1-3). HMA4 is one of these genes. It encodes a Zn/Cd plasmamembrane pump involved in xylem loading/unloading which is a key step in the hyperaccumulation process (4-5). The HMA4 overexpression is determined by a combination of gene triplication and cis-regulatory changes increasing transcription levels of each three HMA4 copies (5). We aim to identify the cis-elements underlying high expression of HMA4 in A. halleri using promoter-reporter constructs. [less ▲]

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See detailExpression study of FRD3 in Arabidopsis relatives
Scheepers, Maxime ULg; Charlier, Jean-Benoit ULg; Spielmann, Julien ULg et al

Poster (2015, June 08)

Transcriptomic studies identified genes which are constitutively over-expressed in A. halleri compared to A. thaliana and which may have a role in metal tolerance or accumulation (1-3). A candidate gene ... [more ▼]

Transcriptomic studies identified genes which are constitutively over-expressed in A. halleri compared to A. thaliana and which may have a role in metal tolerance or accumulation (1-3). A candidate gene encodes FRD3, a member of the MATE family of membrane transporters (56 members in A. thaliana). It is a citrate transporter involved in iron homeostasis (4-6) and playing a role in zinc tolerance in A. thaliana (7). We are aiming to analyse the FRD3 high expression in A. halleri and the FRD3 function in zinc and iron homeostasis in A. thaliana. [less ▲]

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See detailFunctional analysis of FRD3 in Arabidopsis
Scheepers, Maxime ULg; Charlier, Jean-Benoit ULg; Spielmann, Julien ULg et al

Poster (2015, June)

Zinc and iron are two essential micronutrients for plants. The homeostasis networks of the two metals are intertwined. The FRD3 (FERRIC REDUCTASE DEFECTIVE 3) protein, a member of the MATE family of ... [more ▼]

Zinc and iron are two essential micronutrients for plants. The homeostasis networks of the two metals are intertwined. The FRD3 (FERRIC REDUCTASE DEFECTIVE 3) protein, a member of the MATE family of membrane transporters, is a citrate transporter involved in iron homeostasis and playing a role in zinc tolerance in Arabidopsis. The FRD3 gene displays a complex regulation. Alternative transcript initiation for FRD3 determines two transcripts, which differ in their 5'UTRs and have differential translation efficiency. The two transcripts are selectively regulated under stress conditions: iron and zinc depletion, zinc excess or cadmium presence. We are aiming to determine the FRD3 function in zinc and iron homeostasis in Arabidopsis. We will present data (i) on the functional characterization of the alternative transcripts and their role in metal homeostasis in Arabidopsis and (ii) on the zinc phenotypes of the frd3 mutant. [less ▲]

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See detailTowards the identification of cis-elements responsible for the high expression of HMA4, an essential gene for zinc hyperaccumulation in Arabidopsis halleri.
Spielmann, Julien ULg; Nouet, Cécile ULg; Scheepers, Maxime ULg et al

Poster (2015, May 13)

A. halleri is a Zn and Cd hypertolerant and hyperaccumulator plant which stores metals in shoot vacuoles. One key step for hyperaccumulation is a highly efficient root-to-shoot translocation of metals ... [more ▼]

A. halleri is a Zn and Cd hypertolerant and hyperaccumulator plant which stores metals in shoot vacuoles. One key step for hyperaccumulation is a highly efficient root-to-shoot translocation of metals. The Zn/Cd plasmamembrane pump HMA4 (heavy metal ATPase 4) plays a central role in this process and is involved in xylem loading/unloading. Moreover, HMA4 is overexpressed in A. halleri compare to its non-tolerant and non-hyperaccumulator sister, Arabidopsis thaliana. The HMA4 overexpression is determined by a combination of gene triplication and cis-regulatory changes increasing transcription levels of each three HMA4 copies. In this study, we are focusing on the two most active HMA4 promoters and we aim to identify the cis-elements underlying high expression of HMA4 in A. halleri using promoter-reporter constructs. Through deletion analysis, two important regions for a high expression have been identified. Ongoing analysis of more detailed deletions will be presented. [less ▲]

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See detailZinc triggers a complex transcriptional and post-transcriptional regulation of the metal homeostasis gene FRD3 in Arabidopsis relatives
Charlier, Jean_Benoit; Polese, Catherine; Nouet, Cécile ULg et al

in Journal of Experimental Botany (2015)

In Arabidopsis thaliana, FRD3 (Ferric Chelate Reductase Defective 3) plays a central role in metal homeostasis. FRD3 is among a set of metal homeostasis genes that are constitutively highly expressed in ... [more ▼]

In Arabidopsis thaliana, FRD3 (Ferric Chelate Reductase Defective 3) plays a central role in metal homeostasis. FRD3 is among a set of metal homeostasis genes that are constitutively highly expressed in roots and shoots of Arabidopsis halleri, a zinc hyperaccumulating and hypertolerant species. Here, we examined the regulation of FRD3 by zinc in both species to shed light on the evolutionary processes underlying the evolution of hyperaccumulation in A. halleri. We combined gene expression studies with the use of GUS and GFP reporter constructs to compare the expression profile, transcriptional and post-transcriptional regulation of FRD3 in both species. The AtFRD3 and AhFRD3 genes display a conserved expression profile. In A. thaliana, alternative transcription initiation sites from two promoters determine transcript variants which are differentially regulated by zinc supply in roots and shoots to favour the most highly translated variant under zinc excess conditions. In A. halleri, a single transcript variant with higher transcript stability and enhanced translation has been maintained. The FRD3 gene thus undergoes complex transcriptional and post-transcriptional regulation in Arabidopsis relatives. Our study reveals that a diverse set of mechanisms underlie increased gene dosage in the A. halleri lineage and illustrates how an environmental challenge can alter gene regulation. [less ▲]

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See detailFunctional analysis of the three HMA4 copies of the metal hyperaccumulator Arabidopsis halleri
Nouet, Cécile ULg; Charlier, Jean-Benoit; Carnol, Monique ULg et al

in Journal of Experimental Botany (2015), in press

In Arabidopsis halleri, the AhHMA4 gene has an essential function in Zn/Cd hypertolerance and hyperaccumulation by mediating root to shoot translocation of metals. Constitutive high expression of AhHMA4 ... [more ▼]

In Arabidopsis halleri, the AhHMA4 gene has an essential function in Zn/Cd hypertolerance and hyperaccumulation by mediating root to shoot translocation of metals. Constitutive high expression of AhHMA4 results from a tandem triplication and cis-activation of the promoter of all three copies. The three AhHMA4 copies possess divergent promoter sequences, but highly conserved coding sequences, and display identical expression profiles in the root and shoot vascular system. Here, we expressed an AhHMA4::GFP fusion under the control of each three A. halleri HMA4 promoters in a hma2hma4 double mutant of Arabidopsis thaliana to individually examine the function of each A. halleri AhHMA4 copy. The protein localized non-polarly at the plasma membrane of the root pericycle cells of both A. thaliana and A. halleri. The expression of each AhHMA4::GFP copy complemented the severe Zn deficiency phenotype of the hma2hma4 mutant by restoring root-to-shoot translocation of zinc. However, each copy had different impact on metal homeostasis in the A. thaliana genetic background: AhHMA4 copies 2 and 3 were more highly expressed and provided higher Zn tolerance in roots and accumulation in shoots than copy 1, whereas AhHMA4 copy 3 also increased Cd tolerance in roots. Our data suggest a certain extent of functional differentiation among the three A. halleri HMA4 copies, stemming from differences in expression levels rather than in expression profile. HMA4 is a key node of the Zn homeostasis network and small changes in expression level can have major impact on Zn allocation to root or shoot tissues. [less ▲]

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See detailFunctional analysis of FRD3 in Arabidopsis relatives
Scheepers, Maxime ULg; Charlier, Jean-Benoit ULg; Motte, Patrick ULg et al

Poster (2014, November 28)

Transcriptomic studies identified genes which are constitutively over-expressed in A. halleri compared to A. thaliana and which may have a role in metal tolerance or accumulation (1-3). A candidate gene ... [more ▼]

Transcriptomic studies identified genes which are constitutively over-expressed in A. halleri compared to A. thaliana and which may have a role in metal tolerance or accumulation (1-3). A candidate gene encodes FRD3, a member of the MATE family of membrane transporters (56 members in A. thaliana). It is a citrate transporter involved in iron homeostasis (4-6) and playing a role in zinc tolerance in A. thaliana (7). We are aiming to analyse the FRD3 high expression in A. halleri and the FRD3 function in zinc and iron homeostasis in A. thaliana. [less ▲]

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See detailFunctional study of the Ser/Arg-rich splicing factor SRSF5a during zebrafish embryonic development
Joris, Marine ULg; Muller, Marc ULg; Motte, Patrick ULg

Poster (2014, June 05)

Nuclear pre-mRNA splicing is a key process regulating gene expression in eukaryotes. Splicing consists in the removal of introns and the joining of exons within a dynamic macromolecular complex called the ... [more ▼]

Nuclear pre-mRNA splicing is a key process regulating gene expression in eukaryotes. Splicing consists in the removal of introns and the joining of exons within a dynamic macromolecular complex called the spliceosome, which consist of five small nuclear ribonucleoproteins (snRNPs) and numerous non snRNPs proteins (1). Amongst these non snRNPs proteins, the SR proteins family constituted an important group of splicing factors that are involved in constitutive and alternative splicing (2,3). SR proteins are structurally related as they are characterized by one or two RNA-recognition motifs (RRMs) in N-ter and a C-terminal domain enriched in dipeptide Ser/Arg. Phylogenetic inference using the RRM domain allowed us to identify 13 encoding genes for SR proteins in the vertebrate model organism, Danio rerio. The Zebrafish is increasingly recognized as a powerful model for the study of vertebrate embryonic development in a physiological context. The roles of SR splicing factors during animal cell differentiation and development are largely unknown. The aim of the present research is to investigate SR proteins functions during zebrafish development by using molecular and genetic approaches. In this study, we investigated the role of the SR splicing factor SRSF5a. The expression profile was determined by in situ hybridization at 24, 48 and 72 hours post-fertilization and showed SRSF5a expression mainly in brain, retina and pharyngeal arches at these stages. Furthermore, SRSF5a knock-down by morpholinos microinjection strongly suggests an important role of this specific splicing factor during eyes and brain development. In order to gain insight into the molecular function of SRSF5a, we analysed control and morphant transcriptomes using high throughput RNA sequencing. Finally, we use a complementary approach to morpholinos and generate SRSF5a mutant fishes using TALENs (Tal effector nucleases)(4,5). 1. Wahl MC, Will CL, & Luhrmann R (2009) The spliceosome: design principles of a dynamic RNP machine. Cell 136(4):701-718. 2. Long JC & Caceres JF (2009) The SR protein family of splicing factors: master regulators of gene expression. Biochem J 417(1):15-27. 3. Graveley BR (2000) Sorting out the complexity of SR protein functions. RNA 6(9):1197-1211. 4. Moore et al. (2012) Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs). Plos One 120(1):1-12. 5. Cade et al (2012) Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs. Nucleic acid research Vol 40, No 16:8001-8010. [less ▲]

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See detailComplex regulation of the FRD3 gene in Arabidopsis relatives
Charlier, Jean Benoit; Polese, Catherine ULg; Nouet, Cécile ULg et al

Poster (2014, March 31)

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See detailFunctional study of the Ser/Arg-rich splicing factor SRSF5a during zebrafish embryonic development.
Joris, Marine ULg; Larbuisson, Arnaud ULg; Muller, Marc ULg et al

Poster (2013, April 18)

To investigate the role of the splicing factor SRSF5a during zebrafish embryonic development, we performed SRSF5a knockdown by morpholino microinjection and we analysed control and morphant transcriptomes ... [more ▼]

To investigate the role of the splicing factor SRSF5a during zebrafish embryonic development, we performed SRSF5a knockdown by morpholino microinjection and we analysed control and morphant transcriptomes using RNA sequencing. [less ▲]

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See detailComplex regulation of the FRD3 gene in Arabidopsis relatives
Charlier, Jean-Benoit ULg; Polese, Catherine ULg; Nouet, Cécile ULg et al

Poster (2013, February 26)

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See detailHard Selective Sweep and Ectopic Gene Conversion in a Gene Cluster Affording Environmental Adaptation
Hanikenne, Marc ULg; Kroymann, Juergen; Trampczynska, Aleksandra et al

in PLoS Genetics (2013), 9

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See detailUse of red autofluorescence for monitoring prodiginine biosynthesis
Tenconi, Elodie ULg; Guichard, Paul; Motte, Patrick ULg et al

in Journal of Microbiological Methods (2013), 93

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See detailInhibition of Tumor Angiogenesis and Growth by a Small-Molecule Multi-FGF Receptor Blocker with Allosteric Properties.
Bono, Francoise; De Smet, Frederik; Herbert, Corentin et al

in Cancer Cell (2013), 23(4), 477-88

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the ... [more ▼]

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment. [less ▲]

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