Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Aryl hydrocarbon receptor-dependent genomic effects of environmental polycyclic aromatic hydrocarbons (PAHs) have been shown to be modulated by non-genomic protein kinase C (PKC)-related pathways. The present study was designed to determine whether PKC activation may also impair estrogenic genomic response triggered by PAHs. Treatment by the PKC activator phorbol 12-myristate 13-acetate (PMA) was found to markedly and differentially impair the up-regulation of estrogenic markers triggered by the estrogenic PAH benzanthracene (BZA) in cultured human mammary cells; BZA-mediated mRNA up-regulation of pS2 and amphiregulin was thus increased, whereas that of progesterone receptor and CXCL12 was repressed. BZA/PMA cotreatment however failed to alter BZA-mediated increase of activity of a luciferase gene reporter construct driven by an estrogen response element, thus discarding any global effect of PMA toward BZA-triggered estrogen receptor activation. Various chemicals inhibiting PKCs or extracellular signal-regulated kinase (ERK) as well as the knock-down of PKCδ expression counteracted the PMA-mediated increase of pS2 mRNA up-regulation triggered by BZA, demonstrating that it was dependent on PKCs, including PKCδ isoform, and ERKs. This non-genomic modulation of estrogenic effects of PAHs by PKC activation may have to be considered when considering the deleterious effects of these environmental contaminants towards the endocrine system.
ISSN: 0887-2333

inserm-00872862
http://www.hal.inserm.fr/inserm-00872862
DOI : 10.1016/j.tiv.2012.05.006
PUBMED : 22643241

Éditeur(s) : HAL CCSD Springer Verlag
Résumé : International audience
Chromatin modification enzymes are important regulators of gene expression and some are evolutionarily conserved from yeast to human. Saccharomyces cerevisiae is a major model organism for genome-wide studies that aim at the identification of target genes under the control of conserved epigenetic regulators. Ume6 interacts with the upstream repressor site 1 (URS1) and represses transcription by recruiting both the conserved histone deacetylase Rpd3 (through the co-repressor Sin3) and the chromatin-remodeling factor Isw2. Cells lacking Ume6 are defective in growth, stress response, and meiotic development. RNA profiling studies and in vivo protein-DNA binding assays identified mRNAs or transcript isoforms that are directly repressed by Ume6 in mitosis. However, a comprehensive understanding of the transcriptional alterations, which underlie the complex ume6Δ mutant phenotype during fermentation, respiration, or sporulation, is lacking. We report the protein-coding transcriptome of a diploid MAT a/α wild-type and ume6/ume6 mutant strains cultured in rich media with glucose or acetate as a carbon source, or sporulation-inducing medium. We distinguished direct from indirect effects on mRNA levels by combining GeneChip data with URS1 motif predictions and published high-throughput in vivo Ume6-DNA binding data. To gain insight into the molecular interactions between successive waves of Ume6-dependent meiotic genes, we integrated expression data with information on protein networks. Our work identifies novel Ume6 repressed genes during growth and development and reveals a strong effect of the carbon source on the derepression pattern of transcripts in growing and developmentally arrested ume6/ume6 mutant cells. Since yeast is a useful model organism for chromatin-mediated effects on gene expression, our results provide a rich source for further genetic and molecular biological work on the regulation of cell growth and cell differentiation in eukaryotes.
ISSN: 1617-4615

hal-01151619
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01151619
DOI : 10.1007/s00438-015-1051-5
PUBMED : 25957495

Éditeur(s) : HAL CCSD
Résumé : contient des versions PDF d'articles scientifiques (versions éditeur)
Spectrins are actin binding proteins that constitute the main components of membrane skeletons, the protein network located under the plasma membrane. Erythroid or non erythroid (in this case, they are called fodrins), spectrins play an important structural function in the membrane as demonstrated in the red blood cell. Spectrins are composed of two long α and β chains, associated in tetramers (αβ)2, forming the long flexible filaments of the network. Both chains are composed of repeating units which are folded in a triple helical arrangement (A, B and C helices), except for segment α10 which corresponds to a Src Homology 3 domain (SH3). The head-to-head interaction between dimers implies the N-terminal end of α chain and the C-terminal end of β chain. A model was proposed for the tetramerization site of spectrin : the two A and B helices of the last repeat of α chain interact with the C helix of the β chain in order to constitute a triple-helical structure similar to the repeats along the remainder of spectrin molecule. This model was first inferred from the relationship between the severity of the spectrin self-association defect and the location of the underlying mutations found in hereditary elliptocytosis (HE). Using recombinant peptides we defined the region, on both chains, which was sufficient and necessary to generate a fully functional tetramerization site. Producing peptides carrying a βHE mutation, we reproduced the tetramerization defect as observed in red blood cells of patients.
Finally, the presence of an SH3 domain probably gives to spectrins new functions which are different from the stability of the membrane. After contesting the existence of interaction between the fodrin SH3 domain and the α subunit of epithelial sodium channel sensitive to amiloride, we searched for partners of this domain, by screening a rat kidney library with the yeast two-hybrid system. We identified severals proteins implicated in the actin polymerization asoociated with motility or cell differenciation, suggesting that SH3 domain could play a part in this process.
Les spectrines, protéines liant l'actine, sont des constituants majeurs du squelette membranaire, réseau multiprotéique localisé sous la membrane plasmique. Érythroïdes ou non érythroïdes (dans ce cas, on les appelle fodrines), les spectrines ont un rôle structural important dans la membrane comme cela a déjà été démontré, pour la spectrine, dans le globule rouge. Elles sont constituées de deux longues chaînes a et ß associées côte à côte en tétramères (aß)2 qui forment les longs filaments du réseau. Chacune des chaînes est composée par la répétition de segments homologues, 22 pour a et 17 pour ß. Ces segments sont constitués de trois hélices a (hélices A, B et C) repliées sur elles-mêmes. Cette succession de structures trihélicoïdales est parfois interrompue par un domaine particulier comme le domaine SH3 (Src Homology 3) présent au milieu de la chaîne a. Les tétramères (aß)2 de spectrine constituent les filaments du squelette membranaire. L'interaction tête-à-tête de deux dimères aß implique les extrémités NH2 de la chaîne a et COOH de la chaîne ß. D'une part la sévérité du défaut d'auto-association et la localisation des mutations associées à celui-ci et d'autre part, les séquences en acides aminés des extrémités impliquées dans le site d'auto-association ont permis de proposer un modèle : la première hélice C isolée de la chaîne a pourrait s'associer aux deux dernières hélices A et B de la chaîne ß pour reconstituer une unité conformationnelle trihélicoïdale semblable à celles observées le long de la molécule. Un défaut dans la formation du tétramère est le support moléculaire le plus fréquemment observé dans les elliptocytoses héréditaires (HE). À l'aide de peptides recombinants, nous avons défini, sur les deux chaînes, les régions nécessaires et suffisantes possédant les caractéristiques pleinement fonctionnelles du site de tétramérisation. Nous avons ensuite, par mutagenèse dirigée, reproduit le lien entre la présence de mutations HE localisées dans les hélices A ou B et le défaut d'auto-association observé dans les globules rouges de patients HE. La présence d'un domaine SH3 localisé au milieu de la chaîne a confère probablement aux spectrine des fonctions autres que le maintien et la stabilité de la membrane. Les SH3, petits domaines protéiques, participent aux interactions protéine/protéine. Le seul partenaire connu du domaine SH3 de la fodrine était la sous-unité a du canal sodium sensible à l'amiloride (ENaC) mais la fonction de ce complexe n'était pas encore caractérisée. À l'aide de différentes méthodes, nous avons remis en cause l'interaction directe entre ENaC et le SH3 de la fodrine. La fonction de ce domaine SH3 étant liée à la nature de son ligand, nous avons donc recherché les partenaires putatifs du domaine SH3 de la fodrine par la technique du double-hybride. 29 partenaires ont été identifiés, regroupés en 19 familles et 10 clones isolés. La spécificité des interactions a été étudiée à la fois par double-hybride, à l'aide de mutants du domaine SH3 de la fodrine produits par mutagenèse dirigée. Les interactions vis-à-vis d'autres domaines SH3 (spectrine ou une protéine de levure non relatée Scd2) ont été également analysées. Enfin, la spécificité de certains partenaires a été confirmée par des études d'interactions in vitro. Deux des protéines les plus spécifiques du domaine SH3 de la fodrine sont des protéines tyrosine-phosphatase PTP. La première est l'isoforme A de PTP de faible poids moléculaire (LMPTPA) mais l'interaction n'a pas été confirmée in vitro. La deuxième, TD14, est une nouvelle PTP dont la seule fonction connue est d'inhiber la formation des foyers tumoraux des cellules surexprimant l'oncogène Ha-ras. Ces PTP pourraient soit déphosphoryler la fodrine, soit être recrutées sous la membrane pour déphosphoryler une autre cible. Nous avons également identifié trois partenaires (N-WASP, Evl et une protéine de la famille des formines) suggérant que les domaines SH3 des spectrines pourraient être impliqués dans les processus de polymérisation de l'actine liés à la mobilité ou la différenciation cellulaire. Mot clés : spectrine, fodrine, tétramérisation, SH3, double-hybride, polymérisation de l'actine
https://tel.archives-ouvertes.fr/tel-00284819

Droits : info:eu-repo/semantics/OpenAccess
tel-00284819
https://tel.archives-ouvertes.fr/tel-00284819
https://tel.archives-ouvertes.fr/tel-00284819/document
https://tel.archives-ouvertes.fr/tel-00284819/file/Nicolas_1999_-_These.pdf

Éditeur(s) : HAL CCSD Endocrine Society
Résumé : International audience
Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.
ISSN: 0888-8809

hal-01146846
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01146846
DOI : 10.1210/me.2014-1356
PUBMED : 25710594

Éditeur(s) : HAL CCSD BioScientifica
Résumé : International audience
Spermatogenesis is a complex and tightly regulated process leading to the continuous production of male gametes, the spermatozoa. This developmental process requires the sequential and coordinated expression of thousands of genes, including many that are testis-specific. The molecular networks underlying normal and pathological spermatogenesis have been widely investigated in recent decades, and many high-throughput expression studies have studied genes and proteins involved in male fertility. In this review, we focus on studies that have attempted to correlate transcription and translation during spermatogenesis by comparing the testicular transcriptome and proteome. We also discuss the recent development and use of new transcriptomic approaches that provide a better proxy for the proteome, from both qualitative and quantitative perspectives. Finally, we provide illustrations of how testis-derived transcriptomic and proteomic data can be integrated to address new questions and how the 'proteomics informed by transcriptomics' technique, by combining RNA-seq and MS-based proteomics, can contribute significantly to the discovery of new protein-coding genes or new protein isoforms expressed during spermatogenesis.
ISSN: 1470-1626

hal-01214005
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01214005
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01214005/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01214005/file/Linking%20transcriptomics%20and%20proteomics%20in%20spermatogenesis.pdf
DOI : 10.1530/REP-15-0073

Éditeur(s) : HAL CCSD Oxford University Press (OUP)
Résumé : International audience
Hepatocellular carcinoma treatment by arterial infusion of cis-diamminedichloroplatinum-II (cisplatin) exhibits certain therapeutic efficacy. However, optimizations are required and the mechanisms underlying cisplatin proapoptotic effect remain unclear. The mitogen-activated protein kinase (MAPK) pathway plays a key role in cell response to cisplatin and the functional specificity of the isoform MAPK/ERK kinase 1 and 2 (MEK1/2) and ERK1/2 could influence this response. The individual contribution of each kinase on cisplatin-induced death was thus analyzed after a transient or stable specific inhibition by RNA interference in the human hepatocellular carcinoma cells Huh-7 or in knockout mice. We demonstrated here that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas MEK1 and MEK2 acted in a redundant manner. Indeed, at clinically relevant concentrations of cisplatin, ERK1 silencing alone was sufficient to protect cells from cisplatin-induced death both in vitro, in Huh-7 cells and ERK1(-/-) hepatocytes, and in vivo, in ERK1-deficient mice. Moreover, we showed that ERK1 activity correlated with the induction level of the proapoptotic BH3-only protein Noxa, a critical mediator of cisplatin toxicity. On the contrary, ERK2 inhibition upregulated ERK1 activity, favored Noxa induction and sensitized hepatocarcinoma cells to cisplatin. Our results point to a crucial role of ERK1 in cisplatin-induced proapoptotic signal and lead us to propose that ERK2-specific targeting could improve the efficacy of cisplatin therapy by increasing ERK1 prodeath functions.
ISSN: 0143-3334

inserm-00814096
http://www.hal.inserm.fr/inserm-00814096
DOI : 10.1093/carcin/bgs317
PUBMED : 23042098

Éditeur(s) : HAL CCSD Springer New York
Résumé : International audience
Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1. Several ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene
Estrogen Receptors

ISBN : 978-1-4939-3126-2
hal-01237073
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01237073
DOI : 10.1007/978-1-4939-3127-9_22

Éditeur(s) : HAL CCSD Springer Verlag (Germany) Springer Verlag
Résumé : Dihydroceramide ?4-desaturase 1 (DES1) catalyzes the last step of the de novo ceramide biosynthesis, which consists of the introduction of a trans ?4-double bond in the carbon chain of the dihydroceramide. It was previously observed that myristic acid binds DES1 through N-myristoylation. This N-terminal modification significantly increased the activity of the recombinant DES1 in COS-7 cells and targeted part of the enzyme initially present in the endoplasmic reticulum to the mitochondrial outer membrane, leading to an increase in ceramide levels. Since these results were obtained in a recombinant COS-7 cell model with high expression of rat DES1, the purpose of the present study was to investigate if the native DES1 enzyme was really upregulated by its N-myristoylation in cultured rat hepatocytes. We first showed that DES1 was the main dihydroceramide desaturase isoform expressed in rat hepatocytes. In this model, the wild-type myristoylable recombinant form of rat DES1 was found in both the endoplasmic reticulum and the mitochondria whereas the mutated non-myristoylable recombinant form (N-terminal glycine replaced by an alanine) was almost exclusively localized in the endoplasmic reticulum, which evidenced the importance of the myristoylation. Then, we showed that compared to other fatty acids, myristic acid was the only one to increase native DES1 activity, in both total cell lysates and mitochondrial fractions. The myristic acid-associated increase in DES1 activity was not linked to elevated mRNA or protein expression but more likely to its N-terminal myristoylation. Finally, the myristic acid-associated increase in DES1 activity slightly enhanced the number of apoptotic cells.
ISSN: 0024-4201

hal-00808026
https://hal-agrocampus-ouest.archives-ouvertes.fr/hal-00808026
DOI : 10.1007/s11745-011-3638-x