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Jesper Q. Svejstrup - One of the best experts on this subject based on the ideXlab platform.
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role of elongator subunit ELP3 in drosophila melanogaster larval development and immunity
Genetics, 2011Co-Authors: Jane Walker, So Yeon Kwon, Paul Badenhorst, Phil East, Helen Mcneill, Jesper Q. SvejstrupAbstract:The Elongator complex has been implicated in several cellular processes, including gene expression and tRNA modification. We investigated the biological importance of the ELP3 gene in Drosophila melanogaster. Deletion of ELP3 results in larval lethality at the pupal stage. During early development, larval growth is dramatically impaired, with progression to the third instar delayed for ∼24 hr, and pupariation occurring only at day 14 after egg laying. Melanotic nodules appear after 4 days. Microarray analysis shows that stress response genes are induced and ecdysone-induced transcription factors are severely repressed in the mutant. Interestingly, the phenotypes of ELP3 flies are similar to those of flies lacking the domino gene, encoding a SWI/SNF-like ATP-dependent chromatin-remodeling enzyme. Indeed, the gene expression profiles of these mutants are also remarkably similar. Together, these data demonstrate that Drosophila ELP3 is essential for viability, normal development, and hematopoiesis and suggest a functional overlap with the chromatin remodeler Domino.
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An iron-sulfur cluster domain in ELP3 important for the structural integrity of elongator
The Journal of biological chemistry, 2008Co-Authors: Catherine Greenwood, Luke A. Selth, A. Barbara Dirac-svejstrup, Jesper Q. SvejstrupAbstract:The Elongator complex functions in diverse cellular processes, such as RNA polymerase II transcription and tRNA modification. The ELP3 subunit possesses a C-terminal histone acetyltransferase (HAT) domain and an N-terminal sequence that resembles an iron-sulfur (FeS) cluster motif. The HAT domain is well characterized, but the role of the FeS cluster is unknown, although one report proposed that it might be involved in catalyzing histone demethylation. We investigated the importance and function of the yeast ELP3 FeS cluster by a combination of genetic and biochemical means. To minimize oxidation of the ELP3 FeS cluster during purification, we also developed a novel tandem affinity tag and an accompanying isolation procedure that enables purification of tagged proteins to virtual homogeneity within a few hours of cell disruption. Our results failed to support a role for Elongator in histone demethylation. Moreover FeS cluster integrity was not required for the HAT or RNA binding activities of Elongator. However, a fully functional FeS cluster was required for Elongator integrity and for the association of the complex with its accessory factors Kti11 and Kti12. In contrast, the association of Elongator with RNA polymerase II in chromatin was unaffected by FeS cluster mutations. Together our data support the idea that the ELP3 FeS cluster is essential for normal Elongator function in vivo primarily as a structural, rather than catalytic, domain.
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molecular architecture structure function relationship and importance of the ELP3 subunit for the rna binding of holo elongator
Journal of Biological Chemistry, 2004Co-Authors: Thodoris G Petrakis, Birgitte O Wittschieben, Jesper Q. SvejstrupAbstract:The molecular architecture of six-subunit yeast holo-Elongator complex was investigated by the use of immunoprecipitation, two-hybrid interaction mapping, and in vitro studies of binary interactions between individual subunits. Surprisingly, Elp2 is dispensable for the integrity of the holo-Elongator complex, and a purified five-subunit elp2Δ Elongator complex retains histone acetyltransferase activity in vitro. These results indicate that the WD40 repeats in Elp2 are required neither for subunit-subunit interactions within Elongator nor for Elongator interaction with histones during catalysis. Elp2 and Elp4 were largely dispensable for the association of Elongator with nascent RNA transcript in vivo.In contrast, Elongator-RNA interaction requires the ELP3 protein. Together, these data shed light on the structure-function relationship of the Elongator complex.
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elongator is a histone h3 and h4 acetyltransferase important for normal histone acetylation levels in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Sebastiaan G Winkler, Paul Tempst, Hediye Erdjumentbromage, Arnold Kristjuhan, Jesper Q. SvejstrupAbstract:The elongating, hyperphosphorylated form of RNA polymerase II is associated with the Elongator complex, which has the histone acetyltransferase (HAT) ELP3 as a subunit. Here we show that, in contrast to the isolated ELP3 subunit, the activity of intact Elongator complex is directed specifically toward the amino-terminal tails of histone H3 and H4, and that Elongator can acetylate both core histones and nucleosomal substrates. The predominant acetylation sites are lysine-14 of histone H3 and lysine-8 of histone H4. The three smallest Elongator subunits—Elp4, Elp5, and Elp6—are required for HAT activity, and Elongator binds to both naked and nucleosomal DNA. By using chromatin immunoprecipitation, we show that the levels of multiply acetylated histone H3 and H4 in chromatin are decreased in vivo in yeast cells lacking ELP3.
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rna polymerase ii elongator holoenzyme is composed of two discrete subcomplexes
Journal of Biological Chemistry, 2001Co-Authors: Sebastiaan G Winkler, Hediye Erdjumentbromage, Paul Tempst, Thodoris G Petrakis, Steen Ethelberg, Masao Tokunaga, Jesper Q. SvejstrupAbstract:Elongator is a histone acetyltransferase complex that associates with the elongating form of RNA polymerase II. We purified Elongator to virtual homogeneity via a rapid three-step procedure based largely on affinity chromatography. The purified factor, holo-Elongator, is a labile six-subunit factor composed of two discrete subcomplexes: one comprised of the previously identified Elp1, Elp2, and ELP3 proteins and another comprised of three novel polypeptides, termed Elp4, Elp5, and Elp6. Disruption of the yeast genes encoding the new Elongator proteins confers phenotypes indistinguishable from those previously described for the otherelp mutants, and concomitant disruption of genes encoding proteins in either subcomplex does not confer new phenotypes. Taken together, our results indicate that holo-Elongator is a functional entity in vitro as well as in vivo. Metazoan homologues of Elp1 and ELP3 have previously been reported. We cloned the human homologue of yeast ELP4 and show that this gene is ubiquitously expressed in human tissues.
Raffael Schaffrath - One of the best experts on this subject based on the ideXlab platform.
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Unfolded Protein Response Suppression in Yeast by Loss of tRNA Modifications
'MDPI AG', 2018Co-Authors: Alexander Bruch, Roland Klassen, Raffael SchaffrathAbstract:Modifications in the anticodon loop of transfer RNAs (tRNAs) have been shown to ensure optimal codon translation rates and prevent protein homeostasis defects that arise in response to translational pausing. Consequently, several yeast mutants lacking important anticodon loop modifications were shown to accumulate protein aggregates. Here we analyze whether this includes the activation of the unfolded protein response (UPR), which is commonly triggered by protein aggregation within the endoplasmic reticulum (ER). We demonstrate that two different aggregation prone tRNA modification mutants (elp6 ncs2; ELP3 deg1) lacking combinations of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U: ELP3; elp6; ncs2) and pseudouridine (Ψ: deg1) reduce, rather than increase, splicing of HAC1 mRNA, an event normally occurring as a precondition of UPR induction. In addition, tunicamycin (TM) induced HAC1 splicing is strongly impaired in the ELP3 deg1 mutant. Strikingly, this mutant displays UPR independent resistance against TM, a phenotype we found to be rescued by overexpression of tRNAGln(UUG), the tRNA species usually carrying the mcm5s2U34 and Ψ38 modifications. Our data indicate that proper tRNA anticodon loop modifications promote rather than impair UPR activation and reveal that protein synthesis and homeostasis defects in their absence do not routinely result in UPR induction but may relieve endogenous ER stress
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loss of anticodon wobble uridine modifications affects trnalys function and protein levels in saccharomyces cerevisiae
PLOS ONE, 2015Co-Authors: Roland Klassen, Pia Grunewald, Kathrin Thuring, Christian Eichler, Mark Helm, Raffael SchaffrathAbstract:In eukaryotes, wobble uridines in the anticodons of tRNALysUUU, tRNAGluUUC and tRNAGlnUUG are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm5s2U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm5U and s2U) without a lethal effect. However, an ELP3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm5s2U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an ELP3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNALysUUU. These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNALysUUU hypomodification and malfunction.
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elongator function in trna wobble uridine modification is conserved between yeast and plants
Molecular Microbiology, 2010Co-Authors: Constance Mehlgarten, Raffael Schaffrath, Anders S Bystrom, Daniel Jablonowski, Uta Wrackmeyer, Susan Tschitschmann, David Sondermann, Gunilla Jager, Zhizhong Gong, Karin D. BreunigAbstract:Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast ELP3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator-dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an ELP3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 ELP3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast–plant ELP3 hybrids suggesting that AtElp1 has less stringent sequence requirements for ELP3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life.
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molecular analysis of kti12 tot4 a saccharomyces cerevisiae gene required for kluyveromyces lactis zymocin action
Molecular Microbiology, 2002Co-Authors: Lars Fichtner, Karin D. Breunig, Frank Frohloff, Konstanze Burkner, Michael Larsen, Raffael SchaffrathAbstract:Summary TOT, the putative Kluyveromyces lactis zymocin target complex from Saccharomyces cerevisiae, is encoded by TOT1–7, six loci of which are isoallelic to RNA polymerase II (RNAPII) Elongator genes (ELP1–6). Unlike TOT1–3 (ELP1–3) and TOT5–7 (ELP5, ELP6 and ELP4 respectively), which display zymocin resistance when deleted, TOT4 (KTI12) also renders cells refractory to zymocin when maintained in multicopy or overexpressed from the GAL10 promoter. Elevated TOT4 copy number results in an intermediate tot phenotype, which includes mild sensitivities towards caffeine, Calcofluor white and elevated growth temperature, suggesting that TOT4 influences TOT/Elongator function. Tot4p interacts with Elongator, as shown by co-immunoprecipitation, and cell fractionation studies demonstrate partial co-migration with RNAPII and Elongator. As Elongator subunit interaction is not affected by either deletion of TOT4 or multicopy TOT4, Tot4p may not be a structural Elongator subunit but, rather, may regulate TOT/Elongator in a fashion that requires transient physical contact with TOT/Elongator. Consistent with a regulatory role, the presence of a potential P-loop motif conserved between yeast and human TOT4 homologues suggests capability of ATP or GTP binding and P-loop deletion renders Tot4p biologically inactive.
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kluyveromyces lactis zymocin mode of action is linked to rna polymerase ii function via elongator
Molecular Microbiology, 2001Co-Authors: Daniel Jablonowski, Lars Fichtner, Frank Frohloff, Michael J R Stark, Raffael SchaffrathAbstract:The putative Kluyveromyces lactis zymocin target complex, TOT, from Saccharomyces cerevisiae comprises five Tot proteins, four of which are RNA polymerase II (RNAP II) Elongator subunits. Recently, two more Elongator subunit genes, ELP6 (TOT6) and ELP4 (TOT7), have been identified. Deletions of both TOT6 and TOT7 result in the complex tot phenotype, including resistance to zymocin, thermosensitivity, slow growth and hypersensitivity towards drugs, thus reinforcing the notion that TOT/Elongator may be crucial in signalling zymocicity. Mutagenesis of ELP3/TOT3, the Elongator histone acetyltransferase (HAT) gene, revealed that zymocin sensitivity could be uncoupled from Elongator wild-type function, indicating that TOT interacts genetically with zymocin. To test the possibility that zymocin functions by affecting RNAP II activity in a TOT/Elongator-dependent manner, global poly(A)+ mRNA levels were found to decline drastically on zymocin treatment. Moreover, cells overexpressing Fcp1p, the RNAP II carboxy-terminal domain phosphatase, acquired partial zymocin resistance, whereas cells underproducing RNAP II became zymocin hypersensitive. This suggests that zymocin may convert TOT/Elongator into a cellular poison toxic for RNAP II function and eventually leading to the observed G1 cell cycle arrest.
Hediye Erdjumentbromage - One of the best experts on this subject based on the ideXlab platform.
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elongator is a histone h3 and h4 acetyltransferase important for normal histone acetylation levels in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Sebastiaan G Winkler, Paul Tempst, Hediye Erdjumentbromage, Arnold Kristjuhan, Jesper Q. SvejstrupAbstract:The elongating, hyperphosphorylated form of RNA polymerase II is associated with the Elongator complex, which has the histone acetyltransferase (HAT) ELP3 as a subunit. Here we show that, in contrast to the isolated ELP3 subunit, the activity of intact Elongator complex is directed specifically toward the amino-terminal tails of histone H3 and H4, and that Elongator can acetylate both core histones and nucleosomal substrates. The predominant acetylation sites are lysine-14 of histone H3 and lysine-8 of histone H4. The three smallest Elongator subunits—Elp4, Elp5, and Elp6—are required for HAT activity, and Elongator binds to both naked and nucleosomal DNA. By using chromatin immunoprecipitation, we show that the levels of multiply acetylated histone H3 and H4 in chromatin are decreased in vivo in yeast cells lacking ELP3.
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rna polymerase ii elongator holoenzyme is composed of two discrete subcomplexes
Journal of Biological Chemistry, 2001Co-Authors: Sebastiaan G Winkler, Hediye Erdjumentbromage, Paul Tempst, Thodoris G Petrakis, Steen Ethelberg, Masao Tokunaga, Jesper Q. SvejstrupAbstract:Elongator is a histone acetyltransferase complex that associates with the elongating form of RNA polymerase II. We purified Elongator to virtual homogeneity via a rapid three-step procedure based largely on affinity chromatography. The purified factor, holo-Elongator, is a labile six-subunit factor composed of two discrete subcomplexes: one comprised of the previously identified Elp1, Elp2, and ELP3 proteins and another comprised of three novel polypeptides, termed Elp4, Elp5, and Elp6. Disruption of the yeast genes encoding the new Elongator proteins confers phenotypes indistinguishable from those previously described for the otherelp mutants, and concomitant disruption of genes encoding proteins in either subcomplex does not confer new phenotypes. Taken together, our results indicate that holo-Elongator is a functional entity in vitro as well as in vivo. Metazoan homologues of Elp1 and ELP3 have previously been reported. We cloned the human homologue of yeast ELP4 and show that this gene is ubiquitously expressed in human tissues.
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the elp2 subunit of elongator and elongating rna polymerase ii holoenzyme is a wd40 repeat protein
Journal of Biological Chemistry, 2000Co-Authors: Jane Fellows, Hediye Erdjumentbromage, Paul Tempst, Jesper Q. SvejstrupAbstract:A novel yeast gene, ELP2, is shown to encode the 90-kDa subunit of the Elongator complex and elongating RNA polymerase II holoenzyme. ELP2 encodes a protein with eight WD40 repeats, and cells lacking the gene display typicalelp phenotypes, such as temperature and salt sensitivity. Generally, different combinations of double and triple ELPgene deletions cause the same phenotypes as single ELP1,ELP2, or ELP3 deletion, providing genetic evidence that the ELP gene products work together in a complex.
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a novel histone acetyltransferase is an integral subunit of elongating rna polymerase ii holoenzyme
Molecular Cell, 1999Co-Authors: Birgitte O Wittschieben, Gabriel Otero, Therese De Bizemont, Jane Fellows, Hediye Erdjumentbromage, Reiko Ohba, David C Allis, Paul Tempst, Jesper Q. SvejstrupAbstract:The elongator complex is a major component of the RNA polymerase II (RNAPII) holoenzyme responsible for transcriptional elongation in yeast. Here we identify ELP3, the 60-kilodalton subunit of elongator/RNAPII holoenzyme, as a highly conserved histone acetyltransferase (HAT) capable of acetylating core histones in vitro. In vivo, ELP3 gene deletion confers typical elp phenotypes such as slow growth adaptation, slow gene activation, and temperature sensitivity. These results suggest a role for a novel, tightly RNAPII-associated HAT in transcription of DNA packaged in chromatin.
Paul Tempst - One of the best experts on this subject based on the ideXlab platform.
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elongator is a histone h3 and h4 acetyltransferase important for normal histone acetylation levels in vivo
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Sebastiaan G Winkler, Paul Tempst, Hediye Erdjumentbromage, Arnold Kristjuhan, Jesper Q. SvejstrupAbstract:The elongating, hyperphosphorylated form of RNA polymerase II is associated with the Elongator complex, which has the histone acetyltransferase (HAT) ELP3 as a subunit. Here we show that, in contrast to the isolated ELP3 subunit, the activity of intact Elongator complex is directed specifically toward the amino-terminal tails of histone H3 and H4, and that Elongator can acetylate both core histones and nucleosomal substrates. The predominant acetylation sites are lysine-14 of histone H3 and lysine-8 of histone H4. The three smallest Elongator subunits—Elp4, Elp5, and Elp6—are required for HAT activity, and Elongator binds to both naked and nucleosomal DNA. By using chromatin immunoprecipitation, we show that the levels of multiply acetylated histone H3 and H4 in chromatin are decreased in vivo in yeast cells lacking ELP3.
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rna polymerase ii elongator holoenzyme is composed of two discrete subcomplexes
Journal of Biological Chemistry, 2001Co-Authors: Sebastiaan G Winkler, Hediye Erdjumentbromage, Paul Tempst, Thodoris G Petrakis, Steen Ethelberg, Masao Tokunaga, Jesper Q. SvejstrupAbstract:Elongator is a histone acetyltransferase complex that associates with the elongating form of RNA polymerase II. We purified Elongator to virtual homogeneity via a rapid three-step procedure based largely on affinity chromatography. The purified factor, holo-Elongator, is a labile six-subunit factor composed of two discrete subcomplexes: one comprised of the previously identified Elp1, Elp2, and ELP3 proteins and another comprised of three novel polypeptides, termed Elp4, Elp5, and Elp6. Disruption of the yeast genes encoding the new Elongator proteins confers phenotypes indistinguishable from those previously described for the otherelp mutants, and concomitant disruption of genes encoding proteins in either subcomplex does not confer new phenotypes. Taken together, our results indicate that holo-Elongator is a functional entity in vitro as well as in vivo. Metazoan homologues of Elp1 and ELP3 have previously been reported. We cloned the human homologue of yeast ELP4 and show that this gene is ubiquitously expressed in human tissues.
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the elp2 subunit of elongator and elongating rna polymerase ii holoenzyme is a wd40 repeat protein
Journal of Biological Chemistry, 2000Co-Authors: Jane Fellows, Hediye Erdjumentbromage, Paul Tempst, Jesper Q. SvejstrupAbstract:A novel yeast gene, ELP2, is shown to encode the 90-kDa subunit of the Elongator complex and elongating RNA polymerase II holoenzyme. ELP2 encodes a protein with eight WD40 repeats, and cells lacking the gene display typicalelp phenotypes, such as temperature and salt sensitivity. Generally, different combinations of double and triple ELPgene deletions cause the same phenotypes as single ELP1,ELP2, or ELP3 deletion, providing genetic evidence that the ELP gene products work together in a complex.
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a novel histone acetyltransferase is an integral subunit of elongating rna polymerase ii holoenzyme
Molecular Cell, 1999Co-Authors: Birgitte O Wittschieben, Gabriel Otero, Therese De Bizemont, Jane Fellows, Hediye Erdjumentbromage, Reiko Ohba, David C Allis, Paul Tempst, Jesper Q. SvejstrupAbstract:The elongator complex is a major component of the RNA polymerase II (RNAPII) holoenzyme responsible for transcriptional elongation in yeast. Here we identify ELP3, the 60-kilodalton subunit of elongator/RNAPII holoenzyme, as a highly conserved histone acetyltransferase (HAT) capable of acetylating core histones in vitro. In vivo, ELP3 gene deletion confers typical elp phenotypes such as slow growth adaptation, slow gene activation, and temperature sensitivity. These results suggest a role for a novel, tightly RNAPII-associated HAT in transcription of DNA packaged in chromatin.
Malgrange Brigitte - One of the best experts on this subject based on the ideXlab platform.
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ELP3 Lysine Acetyl-Transferase Controls Neuronal Survival in the Developing Inner Ear and is Crucial to Balance and Hearing
2016Co-Authors: Delacroix Laurence, Mateo Sanchez Susana, Nguyen Laurent, Freeman Stephen, Malgrange BrigitteAbstract:ELP3 lysine acetyl-transferase, the catalytic subunit of the Elongator complex, has been assigned multiple roles in gene transcription, DNA methylation and protein translation efficiency. Given the importance of acetylation homeostasis in controlling developmental processes together with recent reports implicating ELP3 in cortical neurogenesis, we investigated its role during inner ear formation. In the inner ear, we detected ELP3 transcript in the sensory epithelia of the entire otic vesicle at embryonic day E11.5. At later stages, ELP3 mRNA is strongly expressed in the vestibular and spiral ganglion neurons. To investigate the role of ELP3 in vivo, we used a conditional knock-out mice (Foxg1Cre) in which the expression of the acetyl-transferase is lost in early otocyst. These mice show obvious vestibular defects as indicated by a stereotyped circling ambulation, head bobbing, retropulsion and the absence of a reaching response in the tail-hanging test. Furthermore, we identified a severe hearing loss in ELP3cKO mice through Auditory Brainstem Responses. We show that ELP3 enzyme is crucial for neuronal survival in the spiral ganglion and in the vestibule and that it ensures a correct innervation pattern in the developing inner ear. In the absence of ELP3, a drastic increase in the number of apoptotic neurons was detected by active Caspase-3 and pH2AX immunostainings, particularly during the early stages of development (between E12.5 and E14.5). Postnatally, the neurons remaining in ELP3cKO cochleae seem to establish synaptic contacts with the sensory cells but show obvious signs of cell damage as evidenced by Transmission Electron Microscopy. Taken together, these data support a role for ELP3 in hearing and balance and point out an important role for acetylation homeostasis during inner ear formation. We are currently investigating the molecular mechanisms underlying ELP3 effect on neuronal survival and pathfinding.Peer reviewe
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ELP3 Lysine Acetyl-Transferase Controls Neuronal Survival in the Developing Inner Ear and is Crucial to Balance and Hearing
2016Co-Authors: Delacroix Laurence, Mateo Sanchez Susana, Nguyen Laurent, Freeman Stephen, Malgrange BrigitteAbstract:peer reviewedaudience: researcherELP3 lysine acetyl-transferase, the catalytic subunit of the Elongator complex, has been assigned multiple roles in gene transcription, DNA methylation and protein translation efficiency. Given the importance of acetylation homeostasis in controlling developmental processes together with recent reports implicating ELP3 in cortical neurogenesis, we investigated its role during inner ear formation. In the inner ear, we detected ELP3 transcript in the sensory epithelia of the entire otic vesicle at embryonic day E11.5. At later stages, ELP3 mRNA is strongly expressed in the vestibular and spiral ganglion neurons. To investigate the role of ELP3 in vivo, we used a conditional knock-out mice (Foxg1Cre) in which the expression of the acetyl-transferase is lost in early otocyst. These mice show obvious vestibular defects as indicated by a stereotyped circling ambulation, head bobbing, retropulsion and the absence of a reaching response in the tail-hanging test. Furthermore, we identified a severe hearing loss in ELP3cKO mice through Auditory Brainstem Responses. We show that ELP3 enzyme is crucial for neuronal survival in the spiral ganglion and in the vestibule and that it ensures a correct innervation pattern in the developing inner ear. In the absence of ELP3, a drastic increase in the number of apoptotic neurons was detected by active Caspase-3 and pH2AX immunostainings, particularly during the early stages of development (between E12.5 and E14.5). Postnatally, the neurons remaining in ELP3cKO cochleae seem to establish synaptic contacts with the sensory cells but show obvious signs of cell damage as evidenced by Transmission Electron Microscopy. Taken together, these data support a role for ELP3 in hearing and balance and point out an important role for acetylation homeostasis during inner ear formation. We are currently investigating the molecular mechanisms underlying ELP3 effect on neuronal survival and pathfinding.Unravelling the roles of Lysine acetylation during inner ear developmen
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Unravelling the roles of lysine acetyl-transferase activity of Elongator complex during inner ear development
2014Co-Authors: Mateo Sanchez Susana, Delacroix Laurence, Laguesse Sophie, Huysseune Sandra, Chariot Alain, Nguyen Laurent, Malgrange BrigitteAbstract:Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on ELP3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. To determine the role of ELP3 in the inner ear, we first analysed the spatio-temporal pattern of ELP3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of ELP3 in the inner ear, we used conditional knock-out mice in which ELP3 gene is deleted from early otocyst (ELP3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, the auditory brain stem response of ELP3 cKO indicated that these mice are severely deaf. At the cellular level, we did not find any structural abnormalities nor cell patterning defects that could explain deafness or balance dysfunction in ELP3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. In addition, the length of the kinocilium was significantly reduced both in vestibular and cochlear hair cells from ELP3 cKO mice compared with wild type littermates. We were also able to demonstrate an increased level of apoptosis in the ELP3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons at P15. To find new potential targets for ELP3, transcriptomes from wild-type, heterozygous and ELP3 cKO mice were analysed by RNA-Seq at E14.5 and E18.5. Surprisingly, we observed that hair cell markers were upregulated in the ELP3 cKO at E14.5, suggesting a premature differentiation in these mice that was confirmed by in situ hybridisation. In conclusion, our results clearly show a role for ELP3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by ELP3
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Unravelling the roles of lysine acetylation by ELP3 during inner ear development
2014Co-Authors: Mateo Sanchez Susana, Delacroix Laurence, Laguesse Sophie, Huysseune Sandra, Chariot Alain, Nguyen Laurent, Malgrange BrigitteAbstract:Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on ELP3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. To determine the role of ELP3 in the inner ear, we first analysed the spatio-temporal pattern of ELP3 mRNA expression and showed that it was expressed in the entire early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea (the organ of Corti), in the spiral ganglion, in the stria vascularis and in the vestibule. To unravel in vivo functions of ELP3 in the inner ear, we used conditional knock-out mice in which ELP3 gene is deleted from early otocyst (ELP3 cKO). We submitted these mice to a battery of vestibular testing (i.e. stereotyped circling ambulation, head bobbing, retropulsion, and absence of reaching response in the tail-hanging test) and found significant abnormalities. Besides, the auditory brain stem response of ELP3 cKO indicated that these mice are severely deaf. At the cellular level, we did not find any structural abnormalities nor cell patterning defects that could explain deafness or balance dysfunction in ELP3 cKO mice. However, we detected some defaults in the planar orientation of their auditory hair cell bundle. In addition, the length of the kinocilium was significantly reduced both in vestibular and cochlear hair cells from ELP3 cKO mice compared with wild type littermates. We were also able to demonstrate an increased level of apoptosis in the ELP3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons P15. In conclusion, our results clearly show a role for ELP3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by ELP3
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Rôles du complexe Elongator dans le développement embryonnaire de l'oreille interne
2013Co-Authors: Mateo Sanchez Susana, Delacroix Laurence, Laguesse Sophie, Huysseune Sandra, Chariot Alain, Nguyen Laurent, Malgrange BrigitteAbstract:Given the importance of acetylation homeostasis in controlling developmental processes, we planned to investigate its role in inner ear formation and focused our attention on ELP3 acetyl-transferase, a member of the Elongator complex recently implicated in neurogenesis. We first analysed the spatio-temporal pattern of ELP3 mRNA expression and showed that it was expressed in the early otocyst at E11.5 and persisted later in the sensory epithelium of the cochlea, the spiral ganglion, the stria vascularis and the vestibule. To unravel functions of ELP3 in the inner ear, we used conditional knock-out mice in which ELP3 gene is deleted from early otocyst (ELP3 cKO). We submitted these mice to a battery of vestibular testing and found significant abnormalities. Besides, the auditory brain stem response of ELP3 cKO indicated that these mice are severely deaf. At the cellular level, we detected some defaults in the planar orientation of the auditory hair cell bundle. In addition, the length of the kinocilium was significantly reduced both in vestibular and cochlear hair cells from ELP3 cKO mice. We were also able to demonstrate an increased level of apoptosis in the ELP3 cKO spiral ganglion at E14.5 leading to a reduced number of fibers innervating the cochlear hair cells as well as a reduced number of their synaptic ribbons. In conclusion, our results clearly show a role for ELP3 both in hearing and balance. We plan to go deeper in the mechanisms involved through the identification of the proteins that are targeted for acetylation by ELP3.Unravelling the roles of lysine acetylation by ELP3 during inner ear developmen