Clonage de microARN dans la glande mammaire ovine

National audienc

Clonage de microARN dans la glande mammaire ovine

National audienc

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

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

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

MicroRNA in the ovine mammary gland during early pregnancy: spatial and temporal expression of miR-21, miR-205, and miR-200

The mammary gland undergoes extensive remodeling between the beginning of pregnancy and lactation; this involves cellular processes including cell proliferation, differentiation, and apoptosis, all of which are under the control of numerous regulators. To unravel the role played by miRNA, we describe here 47 new ovine miRNA cloned from mammary gland in early pregnancy displaying strong similarities with those already identified in the cow, human, or mouse. A microarray study of miRNA variations in the adult ovine mammary gland during pregnancy and lactation showed that 100 miRNA are regulated according to three principal patterns of expression: a decrease in early pregnancy, a peak at midpregnancy, or an increase throughout late pregnancy and lactation. One miRNA displaying each pattern (miR-21, miR-205, and miR-200b) was analyzed by qRT-PCR. Variations in expression were confirmed for all three miRNA. Using in situ hybridization, we detected both miR-21 and miR-200 in luminal mammary epithelial cells when expressed, whereas miR-205 was expressed in basal cells during the first half of pregnancy and then in luminal cells during the second half. We therefore conclude that miR-21 is strongly expressed in the luminal cells of the normal mammary gland during early pregnancy when extensive cell proliferation occurs. In addition, we show that miR-205 and miR-200 are coexpressed in luminal cells, but only during the second half of pregnancy. These two miRNA may cooperate to maintain epithelial status by repressing an EMT-like program, to achieve and preserve the secretory phenotype of mammary epithelial cells

Action différentielle des acides gras saturés et insaturés sur la formation des gouttelettes lipidiques et la synthèse de la caséine-beta par la cellule épithéliale mammaire murine HC11

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Oleate and linoleate stimulate degradation of β-casein in prolactin-treated HC11 mouse mammary epithelial cells

International audienceAlthough virtually all cells store neutral lipids as cytoplasmic lipid droplets, mammary epithelial cells have developed a specialized function to secrete them as milk fat globules. We have used the mammary epithelial cell line HC11 to evaluate the potential connections between the lipid and protein synthetic pathways. We show that unsaturated fatty acids induce a pronounced proliferation of cytoplasmic lipid droplets and stimulate the synthesis of adipose differentiation-related protein. Unexpectedly, the cellular level of β-casein, accumulated under lactogenic hormone treatment, decreases following treatment of the cells with unsaturated fatty acids. In contrast, saturated fatty acids have no significant effect on either cytoplasmic lipid droplet proliferation or cellular β-casein levels. We demonstrate that the action of unsaturated fatty acids on the level of β-casein is post- translational and requires protein synthesis. We have also observed that proteasome inhibitors potentiate β-casein degradation, indicating that proteasomal activity can destroy some cytosolic protein(s) involved in the process that negatively controls β-casein levels. Finally, lysosome inhibitors block the effect of unsaturated fatty acids on the cellular level of β-casein. Our data thus suggest that the degradation of β-casein occurs via the microautophagic pathway

Localisation de microARN et organisation nucléaire dans le tissu mammaire de vache et notamment les cellules progénitrices

National audienceLa glande mammaire se développe de manière cyclique à partir de cellules souches et progénitrices, en réponse à une régulation hormonale complexe. Plusieurs articles ont souligné le rôle que pouvaient jouer les microARN à la fois dans le maintien et la maturation des cellules souches. L’organisation spatiale du noyau, qui participe à la régulation l’expression des gènes et change au cours de la différenciation, n’a pas encore été étudiée dans ces cellules. Chez la génisse, les cellules progénitrices de la glande mammaire peuvent être identifiées sur les coupes de tissu par coloration à la fuchsine basique. Nous avons mis au point la détection de microARN par hybridation in situ sur cryo-coupes à l’aide de sondes oligonucléotidiques modifiées. L’expression de plusieurs microARN, notamment miR-21 et miR-205, a été étudiée au cours du développement de la glande mammaire de la vache et de la brebis. Mir-21 est un microARN oncogénique (oncomir) fortement exprimé dans de nombreux cancers et notamment les cancers du sein. MiR-205 a été montré par plusieurs équipes comme étant fortement exprimé dans les progénitrices mammaires chez la souris. Parallèlement, nous avons utilisé l’immunofluorescence sur cryo-coupes pour localiser les compartiments nucléaires, notamment les domaines d’hétérochromatine potentiellement impliqués dans l’épigénétique des cellules progénitrices. Nous avons montré qu’un simple marquage de l’ADN au DAPI permettait de distinguer les cellules progénitrices des cellules épithéliales en cours de différenciation, sur la base de la forme et de l’organisation interne des noyaux. L’identification des types cellulaires est optimale lorsque le tissu est inclus en paraffine. Les conditions d’hybridation in situ et d’immunofluorescence sont en cours d’optimisation afin d’examiner l’expression des microARN et l’organisation des noyaux à partir des coupes de tissu mammaire de génisse préparées avec cette méthode d’inclusion