Real-Time Study of Interactions between a Composite DNA Regulatory Region (HIV-1 LTR NRE) and Several Transcription Factors of Nuclear Extracts

International audienceHere we describe the first real-time study of nuclear protein interaction with a composite DNA regulatory region. We studied the interplay between the three target sites of the negative regulatory element (NRE) of HIV-1 LTR, comprising a noncanonical GATA site overlapping two negative regulatory regions, USF and NFIL-6, and their corresponding transcription factors in nuclear extracts. By bandshift analysis, no GATA binding activity could be detected between LTR NRE and different nuclear extracts, although evidenced by in vitro footprinting. Additionally, the LTR NRE and a USF oligonucleotide showed identical retarded complexes. BIAcore study of these interactions revealed the binding of huGATA-3, as well as USF, to the immobilized LTR NRE oligonucleotide. Competition analyses , performed with GATA, USF, and NFIL-6 oligonu-cleotides, clearly showed that this regulatory region could bind both huGATA-3 and USF factors. Finally, the presence of USF and huGATA-3 proteins in the complexes formed with LTR NRE was ascertained using specific anti-huGATA-3 and anti-USF2 polyclonal antibodies

Clonage de microARN dans la glande mammaire ovine

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La monotraite modifie l'expression d'environ 500 gènes

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Clonage de microARN dans la glande mammaire ovine

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Specific nuclear positioning of the casein gene cluster in luminal mammary epithelial cells

International audienceThe nuclear organization of mammary epithelial cells has been shown to be sensitive to the three-dimensional microenvironment in several models of cultured cells. However, the relationships between the expression and position of genes have not often been explored in animal tissues. We therefore studied the localization of milk protein genes in the nuclei of luminal mammary epithelial cells during lactation as well as in two non- expressing cells, i.e. hepatocytes and the less differentiated embryonic fibroblasts. We compared the position of a cluster of co-regulated genes, encoding caseins (CSN), with that of the whey acidic protein (WAP) gene which is surrounded by genes displaying different expression profiles. We show that the position of the CSN cluster relative to various nuclear compartments is correlated with its activity. In luminal cells, the CSN cluster loops out from its chromosome territory and is positioned in the most euchromatic regions, and frequently associated with elongating RNA polymerase II rich zones. In hepatocytes and embryonic fibroblasts, the cluster is found preferentially closer to the nuclear periphery. Interestingly, we had previously observed a very peripheral position of the CSN locus in the nuclei of HC11 mammary epithelial cells weakly expressing milk protein genes. We thus show that cultured cell lines are not fully representative of the nuclear organization of genes in a complex and highly organized tissue such as the mammary gland and propose that the spatial positioning of the locus is important to ensuring the optimum control of CSN gene activity observed in the mammary tissue

Specific nuclear positioning of the casein gene cluster in luminal mammary epithelial cells

International audienceThe nuclear organization of mammary epithelial cells has been shown to be sensitive to the three-dimensional microenvironment in several models of cultured cells. However, the relationships between the expression and position of genes have not often been explored in animal tissues. We therefore studied the localization of milk protein genes in the nuclei of luminal mammary epithelial cells during lactation as well as in two non- expressing cells, i.e. hepatocytes and the less differentiated embryonic fibroblasts. We compared the position of a cluster of co-regulated genes, encoding caseins (CSN), with that of the whey acidic protein (WAP) gene which is surrounded by genes displaying different expression profiles. We show that the position of the CSN cluster relative to various nuclear compartments is correlated with its activity. In luminal cells, the CSN cluster loops out from its chromosome territory and is positioned in the most euchromatic regions, and frequently associated with elongating RNA polymerase II rich zones. In hepatocytes and embryonic fibroblasts, the cluster is found preferentially closer to the nuclear periphery. Interestingly, we had previously observed a very peripheral position of the CSN locus in the nuclei of HC11 mammary epithelial cells weakly expressing milk protein genes. We thus show that cultured cell lines are not fully representative of the nuclear organization of genes in a complex and highly organized tissue such as the mammary gland and propose that the spatial positioning of the locus is important to ensuring the optimum control of CSN gene activity observed in the mammary tissue

Specific positioning of the casein gene cluster in active nuclear domains in luminal mammary epithelial cells

International audienceThe nuclear organization of mammary epithelial cells has been shown to be sensitive to the three-dimensional microenvironment in several models of cultured cells. However, the relationships between the expression and position of genes have not often been explored in animal tissues. We therefore studied the localization of milk protein genes in the nuclei of luminal mammary epithelial cells during lactation as well as in two non- expressing cells, i.e., hepatocytes and the less differentiated embryonic fibroblasts. We compared the position of a cluster of co-regulated genes, encoding caseins (CSN), with that of the whey acidic protein (WAP) gene which is surrounded by genes displaying different expression profiles. We show that the position of the CSN cluster relative to various nuclear compartments is correlated with its activity. In luminal cells, the CSN cluster loops out from its chromosome territory and is positioned in the most euchromatic regions, and frequently associated with elongating RNA polymerase II-rich zones. In hepatocytes and embryonic fibroblasts, the cluster is found preferentially closer to the nuclear periphery. Interestingly, we had previously observed a very peripheral position of the CSN locus in the nuclei of HC11 mammary epithelial cells weakly expressing milk protein genes. We thus show that cultured cell lines are not fully representative of the nuclear organization of genes in a complex and highly organized tissue such as the mammary gland and propose that the spatial positioning of the locus is important to ensuring the optimum control of CSN gene activity observed in the mammary tissue

Expression des microARN pendant le développement de la glande mammaire ovine adulte

National audienceIntroduction : La glande mammaire subit de nombreux remodelages durant la gestation et la lactation mettant en œuvre de nombreux processus cellulaires (i.e. prolifération et différenciation cellulaire, apoptose...). Ces processus sont sous le contrôle de nombreux types de régulateurs, notamment les microARN (miRNA). Résultats: Après construction d’une banque de miRNA dans la glande mammaire ovine en début de gestation, nous avons cloné 54 nouveaux miRNA jamais décrits chez le mouton. Les séquences de ces miRNA sont très conservées par rapport à leur équivalent chez la vache, la souris ou l’homme. Ces résultats ont été complétés par une étude suivant l’expression des miRNA pendant le développement de la glande mammaire ovine adulte, en utilisant la technique de microarray. Nous avons montré qu’une centaine de miRNA est régulée suivant trois principaux profils d’expression: une baisse d’expression pendant la gestation et la lactation, un pic d’expression pendant la gestation ou une augmentation progressive d’expression à partir de la fin de gestation et au cours de la lactation. L’accumulation d’un miRNA représentatif de chacun de ces profils (respectivement miR-21, miR-205 et miR-200b) a été suivie par hybridation in situ à plusieurs stades du développement de la glande mammaire ovine. MiR-21 et miR-200b ont été détectés dans les cellules épithéliales luminales, alors que miR-205, quant à lui, l’a été dans des cellules basales pendant la première moitié de la gestation puis dans les cellules luminales pendant la deuxième moitié. Conclusion: Nos résultats décrivent une forte expression de miR-21 dans la glande mammaire pendant la gestation précoce, période correspondant à une prolifération cellulaire intense. Par ailleurs, nous montrons que miR-205 commence à être exprimé dans les cellules luminales pendant la seconde moitié de la gestation, alors que miR-200b est déjà présent dans ces cellules. Ces deux miRNA pourraient ainsi coopérer, comme cela a déjà été décrit dans une lignée cellulaire épithéliale (MDCK), notamment afin de maintenir le statut épithélial de ces cellules en réprimant un programme comparable à l’EMT (transition epithélio-mésenchymateuse) de façon à atteindre puis préserver le phénotype sécrétoire des cellules épithéliales mammaires. Perspectives : Afin de déterminer l’implication de ces miRNA dans ces régulations, nous envisageons de bloquer l’action de ces miRNA dans un modèle de dédifférenciation cellulaire (acini mammaires de souris en culture in vitro), puis de suivre la morphologie de ces cellules ainsi que l’expression de marqueurs épithéliaux et mésenchymaux

Etude des protéines communes aux mécanismes d'exocytose et de bourgeonnement lors de la sécrétion des produits du lait

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Characterization of bovine oviductal exosomes from in vivo and in vitro origin

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