DNA methylation and transcription in a distal region upstream from the bovine AlphaS1 casein gene after once or twice daily milking.

Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1 ± 0.2 kg/day instead of 16.2 ± 0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.

Unilateral once daily milking locally induces differential gene expression in both mammary tissue and milk epithelial cells revealing mammary remodeling.

Once daily milking reduces milk yield, but the underlying mechanisms are not yet fully understood. Local regulation due to milk stasis in the tissue may contribute to this effect, but such mechanisms have not yet been fully described. To challenge this hypothesis, one udder half of six Holstein dairy cows was milked once a day (ODM), and the other twice a day (TDM). On the 8th day of unilateral ODM, mammary epithelial cells (MEC) were purified from the milk using immunomagnetic separation. Mammary biopsies were harvested from both udder halves. The differences in transcript profiles between biopsies from ODM and TDM udder halves were analyzed by a 22k bovine oligonucleotide array, revealing 490 transcripts that were differentially expressed. The principal category of upregulated transcripts concerned mechanisms involved in cell proliferation and death. We further confirmed remodeling of the mammary tissue by immunohistochemistry, which showed less cell proliferation and more apoptosis in ODM udder halves. Gene expression analyzed by RT-qPCR in MEC purified from milk and mammary biopsies showed a common downregulation of six transcripts (ABCG2, FABP3, NUCB2, RNASE1 and 5, and SLC34A2) but also some discrepancies. First, none of the upregulated transcripts in biopsies varied in milk-purified MEC. Second, only milk-purified MEC showed significant LALBA downregulation, which suggests therefore that they correspond to a mammary epithelial cell subpopulation. Our results, obtained after unilateral milking, suggest that cell remodeling during ODM is due to a local effect, which may be triggered by milk accumulation.

Leptin gene in rabbit: cloning and expression in mammary epithelial cells during pregnancy and lactation.

Leptin is known as a cytokine mostly produced by fat cells and implicated in regulation of energy metabolism and food intake but has also been shown to be involved in many physiological mechanisms such as tissue metabolism and cell differentiation and proliferation. In particular, leptin influences the development of mammary gland. Although leptin expression in mammary gland has been studied in several species, no data are available in the rabbit. Leptin transcripts in this species have been described as being encoded by only two exons rather than three as in other species. Our focus was to clone and sequence the rabbit leptin cDNA and to prepare the recombinant biologically active protein for validation of the proper sequence and then to describe leptin expression in rabbit mammary gland during different stages of pregnancy and lactation. The leptin sequence obtained was compared with those of other species, and genome alignment demonstrated that the rabbit leptin gene is also encoded by three exons. Additionally, we analyzed the expression of leptin during pregnancy and lactation. Leptin mRNA was weakly expressed throughout pregnancy, whereas mRNA levels were higher during lactation, with a significant increase between days 3 and 16. Leptin transcripts and protein were localized in luminal epithelial cells, thus indicating that leptin synthesis occurs in this compartment. Therefore, mammary synthesized leptin may constitute a major regulator of mammary gland development by acting locally as an autocrine and/or paracrine factor. Furthermore, our results support the possible physiological role of leptin in newborns through consumption of milk.

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.

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

The 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.

An obesogenic diet started before puberty leads to abnormal mammary gland development during pregnancy in the rabbit.

Alterations to the metabolic environment during puberty can impact future lactation efficiency and mammary tumorigenesis. During this study, we used a model of rabbits receiving an obesogenic diet (OD), starting before puberty and extending until mid-pregnancy. Three months later, the body weight of OD animals was significantly higher than that of controls and their mammary glands displayed a precocious and abnormal development at mid-pregnancy. OD mammary ducts were filled with dense products, while alveolar structures invaded most of the fat pad. The proportion of secretory epithelium was significantly higher in OD mammary tissue, which contained an abundant accumulation of milk proteins and lipids. In conclusion, an obesogenic diet started before puberty induced an accelerated development of the rabbit mammary gland, leading to an accumulation of secretory products at mid-pregnancy. These results support the critical influence of nutrition on mammary growth and differentiation, which may be deleterious to mammary development and subsequent lactation.

[Organization of the nucleus during cell differentiation in the mammary tissue]. / L'organisation du noyau au cours de la différenciation cellulaire dans le tissu mammaire.

In many tissues, the features of cell nuclei are specific to their differentiated state, notably in terms of the nature and distribution of nuclear compartments and the position of chromosomes and genes. This spatial organization of the nucleus reveals domains that are differentially permissive for gene expression and may constitute an epigenetic mechanism that is involved in maintaining tissue-specific expression profiles. The mammary gland is a complex tissue in which mammary epithelial cells (MECs), which synthesize and secrete milk components, interact with other cell types (myoepithelial cells, adipocytes) and the extracellular matrix. MECs cultures can to some extent recreate cell differentiation in vitro and have been used to follow the development and functional importance of nuclear organization. They have made it possible to show how hormonal stimulation can lead to a remodeling of nuclear domains and the repositioning of genes specific to the mammary gland, such as milk protein genes. By modulating the growth conditions of culture in order to replace cells in a microenvironment similar to that of mammary gland tissue, it should be possible to study the role of this cellular microenvironment in nuclear organization.

Statistical analysis of 3D images detects regular spatial distributions of centromeres and chromocenters in animal and plant nuclei.

In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear "compartments". Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types.

Epigenetic modifications in 3D: nuclear organization of the differentiating mammary epithelial cell.

During the development of tissues, complex programs take place to reach terminally differentiated states with specific gene expression profiles. Epigenetic regulations such as histone modifications and chromatin condensation have been implicated in the short and long-term control of transcription. It has recently been shown that the 3D spatial organization of chromosomes in the nucleus also plays a role in genome function. Indeed, the eukaryotic interphase nucleus contains sub-domains that are characterized by their enrichment in specific factors such as RNA Polymerase II, splicing machineries or heterochromatin proteins which render portions of the genome differentially permissive to gene expression. The positioning of individual genes relative to these sub-domains is thought to participate in the control of gene expression as an epigenetic mechanism acting in the nuclear space. Here, we review what is known about the sub-nuclear organization of mammary epithelial cells in connection with gene expression and epigenetics. Throughout differentiation, global changes in nuclear architecture occur, notably with respect to heterochromatin distribution. The positions of mammary-specific genes relative to nuclear sub-compartments varies in response to hormonal stimulation. The contribution of tissue architecture to cell differentiation in the mammary gland is also seen at the level of nuclear organization, which is sensitive to microenvironmental stimuli such as extracellular matrix signaling. In addition, alterations in nuclear organization are concomitant with immortalization and carcinogenesis. Thus, the fate of cells appears to be controlled by complex pathways connecting external signal integration, gene expression, epigenetic modifications and chromatin organization in the nucleus.

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

Although 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 beta-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 beta-casein levels. We demonstrate that the action of unsaturated fatty acids on the level of beta-casein is post-translational and requires protein synthesis. We have also observed that proteasome inhibitors potentiate beta-casein degradation, indicating that proteasomal activity can destroy some cytosolic protein(s) involved in the process that negatively controls beta-casein levels. Finally, lysosome inhibitors block the effect of unsaturated fatty acids on the cellular level of beta-casein. Our data thus suggest that the degradation of beta-casein occurs via the microautophagic pathway.

The epigenetic landscape of mammary gland development and functional differentiation.

Most of the development and functional differentiation in the mammary gland occur after birth. Epigenetics is defined as the stable alterations in gene expression potential that arise during development and proliferation. Epigenetic changes are mediated at the biochemical level by the chromatin conformation initiated by DNA methylation, histone variants, post-translational modifications of histones, non-histone chromatin proteins, and non-coding RNAs. Epigenetics plays a key role in development. However, very little is known about its role in the developing mammary gland or how it might integrate the many signalling pathways involved in mammary gland development and function that have been discovered during the past few decades. An inverse relationship between marks of closed (DNA methylation) or open chromatin (DnaseI hypersensitivity, certain histone modifications) and milk protein gene expression has been documented. Recent studies have shown that during development and functional differentiation, both global and local chromatin changes occur. Locally, chromatin at distal regulatory elements and promoters of milk protein genes gains a more open conformation. Furthermore, changes occur both in looping between regulatory elements and attachment to nuclear matrix. These changes are induced by developmental signals and environmental conditions. Additionally, distinct epigenetic patterns have been identified in mammary gland stem and progenitor cell sub-populations. Together, these findings suggest that epigenetics plays a role in mammary development and function. With the new tools for epigenomics developed in recent years, we now can begin to establish a framework for the role of epigenetics in mammary gland development and disease.

Modeling the 3D functional architecture of the nucleus in animal and plant kingdoms.

Compartmentalization is one of the fundamental principles which underly nuclear function. Numerous studies describe complex and sometimes conflicting relationships between nuclear gene positioning and transcription regulation. Therefore the question is whether topological landmarks and/or organization principles exist to describe the nuclear architecture and, if existing, whether these principles are identical in the animal and plant kingdoms. In the frame of an agroBI-INRA program on nuclear architecture, we set up a multidisciplinary approach combining biological studies, spatial statistics and 3D modeling to investigate spatial organization of a nuclear compartment in both plant and animal cells in their physiological contexts. In this article, we review the questions addressed in this program and the methodology of our work.

The nuclear localization of WAP and CSN genes is modified by lactogenic hormones in HC11 cells.

Whey acidic protein (WAP) and casein (CSN) genes are among the most highly expressed milk protein genes in the mammary gland of the lactating mouse. Their tissue-specific regulation depends on the activation and recruitment of transcription factors, and chromatin modifications in response to hormonal stimulation. We have investigated if another mechanism, such as specific positioning of the genes in the nucleus, could be involved in their functional regulation. Fluorescent in situ hybridization was used to study the nuclear localization of WAP and CSN genes in mouse mammary epithelial cells (HC11) cultured in the absence and presence of lactogenic hormones. Automatic 3D image processing and analysis tools were developed to score gene positions. In the absence of lactogenic hormones, both genes are distributed non-uniformly within the nucleus: the CSN locus was located close to the nuclear periphery and the WAP gene tended to be central. Stimulation by lactogenic hormones induced a statistically significant change to their distance from the periphery, which has been described as a repressive compartment. The detection of genes in combination with the corresponding chromosome-specific probe revealed that the CSN locus is relocated outside its chromosome territory following hormonal stimulation, whereas the WAP gene, which is already sited more frequently outside its chromosome territory in the absence of hormones, is not affected. We conclude that milk protein genes are subject to nuclear repositioning when activated, in agreement with a role for nuclear architecture in gene regulation, but that they behave differently as a function of their chromosomal context.

Epigenetic modifications and chromatin loop organization explain the different expression profiles of the Tbrg4, WAP and Ramp3 genes.

Whey Acidic Protein (WAP) gene expression is specific to the mammary gland and regulated by lactogenic hormones to peak during lactation. It differs markedly from the more constitutive expression of the two flanking genes, Ramp3 and Tbrg4. Our results show that the tight regulation of WAP gene expression parallels variations in the chromatin structure and DNA methylation profile throughout the Ramp3-WAP-Tbrg4 locus. Three Matrix Attachment Regions (MAR) have been predicted in this locus. Two of them are located between regions exhibiting open and closed chromatin structures in the liver. The third, located around the transcription start site of the Tbrg4 gene, interacts with topoisomerase II in HC11 mouse mammary cells, and in these cells anchors the chromatin loop to the nuclear matrix. Furthermore, if lactogenic hormones are present in these cells, the chromatin loop surrounding the WAP gene is more tightly attached to the nuclear structure, as observed after a high salt treatment of the nuclei and the formation of nuclear halos. Taken together, our results point to a combination of several epigenetic events that may explain the differential expression pattern of the WAP locus in relation to tissue and developmental stages.

[Nuclear organization and expression of milk protein genes]. / Organisation nucléaire et expression des gènes des protéines du lait.

Milk protein gene expression varies during the pregnancy/lactation cycle under the influence of lactogenic hormones which induce the activation of several transcription factors. Beyond this activation modifying the binding properties of these factors to their consensus sequences, their interactions with DNA is regulated by variations of the chromatin structure. In the nuclei of the mammary epithelial cell, the three dimensional organisation of the chromatin loops, located between matrix attachment regions, is now being studied. The main milk components are organised in supramolecular structures. Milk fat globules are made of a triglyceride core enwrapped by a tripartite membrane originating from various intracellular compartments. The caseins, the main milk proteins, form aggregates: the casein micelles. Their gradual aggregation in the secretory pathway is initiated as soon as from the endoplasmic reticulum. The mesostructures of the milk fat globule and of the casein micelle remain to be elucidated. Our goal is to make some progress into the understanding of the molecular and cellular mechanisms involved in the formation of these milk products.

Analysis of the efficiency of the rabbit whey acidic protein gene 5' flanking region in controlling the expression of homologous and heterologous linked genes.

For 10 years, the regulatory regions of the mouse and rabbit whey acidic protein gene have been used to express heterologous proteins in the milk of transgenic mice, as well as to produce pharmaceutical proteins, on a large scale, in the milk of transgenic livestock. To date, a broad range of expression levels have been detected, and elucidation of the structure-function relationship in these regulatory regions might help to achieve high levels of expression, reproducibly. An extended 5' regulatory region (17.6 kb v. 6.3 kb) of the rabbit whey acidic promoter resulted in an increased frequency of rabbit whey acidic protein expression in transgenic mice. However, the expression levels were low compared with the high expression levels achieved in both transgenic mice and rabbits using the heterologous kappa-casein in the 6.3 kb rabbit whey acidic protein 5' regulatory region. These results underline the importance of the 3' downstream regulatory regions, which still need to be better characterized in the whey acidic protein gene.

Interactions between the rabbit CSN1 gene and the nuclear matrix of stably transfected HC11 mammary epithelial cells vary with its level of expression.

The expression of casein genes is specific to the mammary gland and maximal during lactation. However, among the numerous mammary cell lines described so far, only a few express some casein genes. The regulatory regions of casein genes have been largely described but the mechanisms explaining the mammary specific expression of these genes, and their silencing in most mammary cell lines, have not yet been fully elucidated. To test the hypothesis that the nuclear location of the casein genes may affect their expression, we transfected HC11 mouse mammary cell line with a 100 kb DNA fragment surrounding the rabbit alpha S1 casein gene. We derived stable clones which express or not the transfected rabbit casein gene, in the same cellular context, independently of the number of transgene copies. Metaphase spreads were prepared from the different clones and the transfected genes were localized. Unexpectedly, we observed that in the original HC11 cell line the number of chromosomes per metaphase spread is close to 80, suggesting that HC11 cells have undergone a duplication event, since the mouse karyotype is 2n = 40. In alpha S1 casein expressing cells, the expression level does not clearly correlate with a localization of the transfected DNA proximal to the centromeres or the telomeres. Analysis of the localization of the transfected DNA in nuclear halos allows us to conclude that when expressed, transfected DNA is more closely linked to the nuclear matrix. The next step will be to study the attachment of the endogenous casein gene in mammary nuclei during lactation.

Hormone-induced modifications of the chromatin structure surrounding upstream regulatory regions conserved between the mouse and rabbit whey acidic protein genes.

The upstream regulatory regions of the mouse and rabbit whey acidic protein (WAP) genes have been used extensively to target the efficient expression of foreign genes into the mammary gland of transgenic animals. Therefore both regions have been studied to elucidate fully the mechanisms controlling WAP gene expression. Three DNase I-hypersensitive sites (HSS0, HSS1 and HSS2) have been described upstream of the rabbit WAP gene in the lactating mammary gland and correspond to important regulatory regions. These sites are surrounded by variable chromatin structures during mammary-gland development. In the present study, we describe the upstream sequence of the mouse WAP gene. Analysis of genomic sequences shows that the mouse WAP gene is situated between two widely expressed genes (Cpr2 and Ramp3). We show that the hypersensitive sites found upstream of the rabbit WAP gene are also detected in the mouse WAP gene. Further, they encompass functional signal transducer and activator of transcription 5-binding sites, as has been observed in the rabbit. A new hypersensitive site (HSS3), not specific to the mammary gland, was mapped 8 kb upstream of the rabbit WAP gene. Unlike the three HSSs described above, HSS3 is also detected in the liver, but similar to HSS1, it does not depend on lactogenic hormone treatments during cell culture. The region surrounding HSS3 encompasses a potential matrix attachment region, which is also conserved upstream of the mouse WAP gene and contains a functional transcription factor Ets-1 (E26 transformation-specific-1)-binding site. Finally, we demonstrate for the first time that variations in the chromatin structure are dependent on prolactin alone.

Structure of the rabbit alphas1- and beta-casein gene cluster, assignment to chromosome 15 and expression of the alphas1-casein gene in HC11 cells.

Several casein (CSN) genes (CSN1, 2, 10 and alphas2-CSN) have been described and shown to be clustered in mouse, man and cattle. These genes are expressed simultaneously in the mammary gland during lactation, but they are silent in most mammary cell lines, even in the presence of lactogenic hormones. However, it has been shown that the CSN2 gene, and this gene only, can be induced in certain mammary cell lines, such as HC11. In the present paper, we describe three overlapping bacterial artificial chromosome (BAC) clones which harbor both the rabbit CSN1 and CSN2 genes. These two genes are in a convergent orientation, separated by an intergenic region of 15 kb. DNA from one of the CSN/BAC clones was used as a probe for in situ hybridization to show that the CSN1 and CSN2 gene cluster is located on chromosome 15 band q23 and not on chromosome 12 as had been previously reported. Each of the three CSN/BAC DNAs was transfected into HC11 cells. In the presence of lactogenic hormones, the rabbit CSN1 gene was clearly expressed from all three CSN/BAC DNAs, whereas the rabbit CSN2 gene, which at the most possesses a 1 kb upstream region in one of the CSN/BAC DNAs, was not expressed at detectable levels on Northern blots. The transfected HC11 cells now express both rabbit CSN1 and mouse CSN2 genes. These transfected cells will be used as a model to study the role of CSN1 in milk protein secretion.