Enhanced bovine genome annotation through integration of transcriptomics and epi-transcriptomics datasets facilitates genomic biology.

BACKGROUND: The accurate identification of the functional elements in the bovine genome is a fundamental requirement for high-quality analysis of data informing both genome biology and genomic selection. Functional annotation of the bovine genome was performed to identify a more complete catalog of transcript isoforms across bovine tissues. RESULTS: A total of 160,820 unique transcripts (50% protein coding) representing 34,882 unique genes (60% protein coding) were identified across tissues. Among them, 118,563 transcripts (73% of the total) were structurally validated by independent datasets (PacBio isoform sequencing data, Oxford Nanopore Technologies sequencing data, de novo assembled transcripts from RNA sequencing data) and comparison with Ensembl and NCBI gene sets. In addition, all transcripts were supported by extensive data from different technologies such as whole transcriptome termini site sequencing, RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin using sequencing. A large proportion of identified transcripts (69%) were unannotated, of which 86% were produced by annotated genes and 14% by unannotated genes. A median of two 5' untranslated regions were expressed per gene. Around 50% of protein-coding genes in each tissue were bifunctional and transcribed both coding and noncoding isoforms. Furthermore, we identified 3,744 genes that functioned as noncoding genes in fetal tissues but as protein-coding genes in adult tissues. Our new bovine genome annotation extended more than 11,000 annotated gene borders compared to Ensembl or NCBI annotations. The resulting bovine transcriptome was integrated with publicly available quantitative trait loci data to study tissue-tissue interconnection involved in different traits and construct the first bovine trait similarity network. CONCLUSIONS: These validated results show significant improvement over current bovine genome annotations.

Noncanonical role of singleminded-2s in mitochondrial respiratory chain formation in breast cancer.

Dysregulation of cellular metabolism is a hallmark of breast cancer progression and is associated with metastasis and therapeutic resistance. Here, we show that the breast tumor suppressor gene SIM2 promotes mitochondrial oxidative phosphorylation (OXPHOS) using breast cancer cell line models. Mechanistically, we found that SIM2s functions not as a transcription factor but localizes to mitochondria and directly interacts with the mitochondrial respiratory chain (MRC) to facilitate functional supercomplex (SC) formation. Loss of SIM2s expression disrupts SC formation through destabilization of MRC Complex III, leading to inhibition of electron transport, although Complex I (CI) activity is retained. A metabolomic analysis showed that knockout of SIM2s leads to a compensatory increase in ATP production through glycolysis and accelerated glutamine-driven TCA cycle production of NADH, creating a favorable environment for high cell proliferation. Our findings indicate that SIM2s is a novel stabilizing factor required for SC assembly, providing insight into the impact of the MRC on metabolic adaptation and breast cancer progression.

SIM2s directed Parkin-mediated mitophagy promotes mammary epithelial cell differentiation.

The functionally differentiated mammary gland adapts to extreme levels of stress from increased demand for energy by activating specific protective mechanisms to support neonatal health. Here, we identify the breast tumor suppressor gene, single-minded 2 s (SIM2s) as a novel regulator of mitophagy, a key component of this stress response. Using tissue-specific mouse models, we found that loss of Sim2 reduced lactation performance, whereas gain (overexpression) of Sim2s enhanced and extended lactation performance and survival of mammary epithelial cells (MECs). Using an in vitro model of MEC differentiation, we observed SIM2s is required for Parkin-mediated mitophagy, which we have previously shown as necessary for functional differentiation. Mechanistically, SIM2s localizes to mitochondria to directly mediate Parkin mitochondrial loading. Together, our data suggest that SIM2s regulates the rapid recycling of mitochondria via mitophagy, enhancing the function and survival of differentiated MECs.

EZH2 and Endometrial Cancer Development: Insights from a Mouse Model.

Enhancer of zeste homolog 2 (EZH2), a core component of polycomb repressive complex 2, plays an important role in cancer development. As both oncogenic and tumor suppressive functions of EZH2 have been documented in the literature, the objective of this study is to determine the impact of Ezh2 deletion on the development and progression of endometrial cancer induced by inactivation of phosphatase and tensin homolog (PTEN), a tumor suppressor gene frequently dysregulated in endometrial cancer patients. To this end, we created mice harboring uterine deletion of both Ezh2 and Pten using Cre recombinase driven by the progesterone receptor (Pgr) promoter. Our results showed reduced tumor burden in Ptend/d; Ezh2d/d mice compared with that of Ptend/d mice during early carcinogenesis. The decreased Ki67 index in EZH2 and PTEN-depleted uteri versus that in PTEN-depleted uteri indicated an oncogenic role of EZH2 during early tumor development. However, mice harboring uterine deletion of both Ezh2 and Pten developed unfavorable disease outcome, accompanied by exacerbated epithelial stratification and heightened inflammatory response. The observed effect was non-cell autonomous and mediated by altered immune response evidenced by massive accumulation of intraluminal neutrophils, a hallmark of endometrial carcinoma in Ptend/d; Ezh2d/d mice during disease progression. Hence, these results reveal dual roles of EZH2 in endometrial cancer development.

Autophagy regulates functional differentiation of mammary epithelial cells.

Mitochondria operate as a central hub for many metabolic processes by sensing and responding to the cellular environment. Developmental cues from the environment have been implicated in selective autophagy, or mitophagy, of mitochondria during cell differentiation and tissue development. Mitophagy occurring in this context, termed programmed mitophagy, responds to cell state rather than mitochondrial damage and is often accompanied by a metabolic transition. However, little is known about the mechanisms that engage and execute mitophagy under physiological or developmental conditions. As the mammary gland undergoes post-natal development and lactation challenges mitochondrial homeostasis, we investigated the contribution of mitochondria to differentiation of mammary epithelial cells (MECs). Using lactogenic differentiation of the HC11 mouse MEC line, we demonstrated that HC11 cells transition to a highly energetic state during differentiation by engaging both oxidative phosphorylation and glycolysis. Interestingly, this transition was lost when autophagy was inhibited with bafilomycin A1 or knockdown of Atg7 (autophagy related 7). To evaluate the specific targeting of mitochondria, we traced mitochondrial oxidation and turnover in vitro with the fluorescent probe, pMitoTimer. Indeed, we found that differentiation engaged mitophagy. To further evaluate the requirement of mitophagy during differentiation, we knocked down the expression of Prkn/parkin in HC11 cells. We found that MEC differentiation was impaired in shPrkn cells, implying that PRKN is required for MEC differentiation. These studies suggest a novel regulation of MEC differentiation through programmed mitophagy and provide a foundation for future studies of development and disease associated with mitochondrial function in the mammary gland.Abbreviations: AA: antimycin A; ATG5: autophagy related 5; BAF: bafilomycin A1; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; COX8A: cytochrome c oxidase subunit 8A; CQ: chloroquine; CSN2: casein beta; ECAR: extracellular acidification rate; FCCP: trifluoromethoxy carbonylcyanide phenylhydrazone; FUNDC1: FUN14 domain containing 1; HIF1A: hypoxia inducible factor 1 subunit alpha; L1: lactation day 1; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEC: mammary epithelial cell; mitoQ: mitoquinol; mROS: mitochondrial reactive oxygen species; OCR: oxygen consumption rate; P: priming; P16: pregnancy day 16; PARP1: poly(ADP-ribose) polymerase 1; PINK1: PTEN induced kinase 1; PPARGC1A: PPARG coactivator 1 alpha; PRKN: parkin RBR E3 ubiquitin protein ligase; shNT: short hairpin non-targeting control; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; TEM: transmission electron microscopy; TFAM: transcription factor A, mitochondrial; U: undifferentiated.

Transforming growth factor beta signaling and decidual integrity in mice†.

Transforming growth factor beta (TGFß) signaling regulates multifaceted reproductive processes. It has been shown that the type 1 receptor of TGFß (TGFBR1) is indispensable for female reproductive tract development, implantation, placental development, and fertility. However, the role of TGFß signaling in decidual development and function remains poorly defined. Our objective is to determine the impact of uterine-specific deletion of Tgfbr1 on decidual integrity, with a focus on the cellular and molecular properties of the decidua during development. Our results show that the developmental dynamics of the decidua is altered in TGFBR1 conditionally depleted uteri from embryonic day (E) 5.5 to E8.5, substantiated by downregulation of genes associated with inflammatory responses and uterine natural killer cell abundance, reduced presence of nondecidualized fibroblasts in the antimesometrial region, and altered decidual cell development. Notably, conditional ablation of TGFBR1 results in the formation of decidua containing more abundant alpha smooth muscle actin (ACTA2)-positive cells at the peripheral region of the antimesometrial side versus controls at E6.5-E8.5. This finding is corroborated by upregulation of a subset of smooth muscle marker genes in Tgfbr1 conditionally deleted decidua at E6.5 and E8.5. Moreover, increased cell proliferation and enhanced decidual ERK1/2 signaling were found in Tgfbr1 conditional knockout mice upon decidual regression. In summary, conditional ablation of TGFBR1 in the uterus profoundly impacts the cellular and molecular properties of the decidua. Our results suggest that TGFBR1 in uterine epithelial and stromal compartments is important for the integrity of the decidua, a transient but crucial structure that supports embryo development.

Loss of SIM2s inhibits RAD51 binding and leads to unresolved replication stress.

BACKGROUND: Mutations in genes associated with homologous recombination (HR) increase an individual's risk of developing triple-negative breast cancer (TNBC). Although known for their role in repairing dsDNA breaks, HR repair elements also stabilize and restart stalled replication forks. Essential to these functions are RAD51 and its paralogs, each of which has a unique role in preventing replication fork collapse and restart. However, progress toward understanding the regulation of these factors has been slow. With such a pivotal role in the maintenance of genomic integrity, furthering our understanding of this pathway through the discovery of new factors involved in HR is important. Recently, we showed that singleminded-2s (SIM2s) is stabilized in response to dsDNA breaks and is required for effective HR. METHODS: Initial analysis of the effect loss of SIM2s has on replication stress resolution was conducted using DNA combing assays in established breast cancer cell lines. Further analysis was conducted via immunostaining to determine the effect loss of SIM2s has on factor recruitment. In vivo confirmation was achieved through the use of a mammary epithelial cell conditional knockout mouse model before SIM2s' role in RAD51 recruitment was determined by immunoblotting. RESULTS: Here, we show loss of SIM2s decreases replication fork stability, leading to fork collapse in response to genotoxic stress. Furthermore, loss of SIM2s results in aberrant separation of sister chromatids during mitosis, which has been previously shown to result in chromosomal fragmentation and aneuploidy. Interestingly, loss of SIM2s was shown to result in failure of RAD51 to localize to sites of replication stress in both breast cancer cell lines and primary mammary epithelial cells. Finally, we observed SIM2 is stabilized in response to genotoxic stress and interacts with RAD51, which is necessary for RAD51-DNA binding. CONCLUSIONS: Together, these results show a role for SIM2s in the resolution of replication stress and further characterize the necessity of SIM2s for effective RAD51 loading in response to DNA damage or stress, ultimately promoting genomic integrity and thus preventing the accumulation of cancer-promoting mutations.

Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit Is Required for Uterine Epithelial Integrity.

Normal proliferation and differentiation of uterine epithelial cells are critical for uterine development and function. Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a core component of polycomb repressive complexes 2, possesses histone methyltransferase activity that catalyzes the trimethylation of lysine 27 of histone H3. EZH2 has been involved in epithelial-mesenchymal transition, a key event in development and carcinogenesis. However, its role in uterine epithelial cell function remains unknown. To determine the role of uterine EZH2, Ezh2 was conditionally deleted using progesterone receptor Cre recombinase, which is expressed in both epithelial and mesenchymal compartments of the uterus. Loss of EZH2 promoted stratification of uterine epithelium, an uncommon and detrimental event in the uterus. The abnormal epithelium expressed basal cell markers, including tumor protein 63, cytokeratin 5 (KRT5), KRT6A, and KRT14. These results suggest that EZH2 serves as a guardian of uterine epithelial integrity, partially via inhibiting the differentiation of basal-like cells and preventing epithelial stratification. The observed epithelial abnormality was accompanied by fertility defects, altered uterine growth and function, and the development of endometrial hyperplasia. Thus, the Ezh2 conditional knockout mouse model may be useful to explore mechanisms that regulate endometrial homeostasis and uterine function.

ATM-dependent activation of SIM2s regulates homologous recombination and epithelial-mesenchymal transition.

There is increasing evidence that genomic instability is a prerequisite for cancer progression. Here we show that SIM2s, a member of the bHLH/PAS family of transcription factors, regulates DNA damage repair through enhancement of homologous recombination (HR), and prevents epithelial-mesenchymal transitions (EMT) in an Ataxia-telangiectasia mutated (ATM)-dependent manner. Mechanistically, we found that SIM2s interacts with ATM and is stabilized through ATM-dependent phosphorylation in response to IR. Once stabilized, SIM2s interacts with BRCA1 and supports RAD51 recruitment to the site of DNA damage. Loss of SIM2s through the introduction of shSIM2 or the mutation of SIM2s at one of the predicted ATM phosphorylation sites (S115) reduces HR efficiency through disruption of RAD51 recruitment, resulting in genomic instability and induction of EMT. The EMT induced by the mutation of S115 is characterized by a decrease in E-cadherin and an induction of the basal marker, K14, resulting in increased invasion and metastasis. Together, these results identify a novel player in the DNA damage repair pathway and provides a link in ductal carcinoma in situ progression to invasive ductal carcinoma through loss of SIM2s, increased genomic instability, EMT, and metastasis.

In silico mapping of quantitative trait loci (QTL) regulating the milk ionome in mice identifies a milk iron locus on chromosome 1.

The breast-feeding neonate depends on mother's milk for both macronutrients and micronutrients including minerals. The goals of the present study were to document the effects of genetic background in mice on milk concentrations of select minerals and to use genome-wide association study (GWAS) to identify quantitative trait loci (QTL) regulating milk mineral concentrations. Milk samples from lactating mice in each of 31 different inbred strains of the mouse diversity panel (MDP) were analyzed by inductively coupled plasma-optical emission spectroscopy to determine the concentrations of calcium (Ca), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), phosphorus (P), sulfur (S), and zinc (Zn). GWAS identified a single pleiotropic milk mineral concentration QTL (Mmcq) on chromosome 3 for Ca, Mg, and P. For the remaining minerals, six QTL were detected for Fe, four for K, three for Zn, and one for S. Intersecting the Mmcq with published chromatin immunoprecipitation sequence data identified 15 out of 4633 high-linkage disequilibrium single-nucleotide polymorphisms that resided in signal transducer and activation of transcription 5 (STAT5) binding regions. A milk Fe-associated locus (Mmcq9) on chromosome 1 contained an SNP that localized to a STAT5 binding region and intersected with a HOMER motif predicted to bind the transcriptional regulator E74-Like ETS transcription factor 5. This locus also contained the genes for solute carrier family (Slc) members Slc9a2, Slc9a4, Slc39a10, and Slc40a1. Expression analysis of these transporters supports the conclusion that Slc9a2 and Slc40a1 within the mammary gland could mediate the effect of Mmcq9 on milk Fe concentration.

PER2 regulation of mammary gland development.

The molecular clock plays key roles in daily physiological functions, development and cancer. Period 2 (PER2) is a repressive element, which inhibits transcription activated by positive clock elements, resulting in diurnal cycling of genes. However, there are gaps in our understanding of the role of the clock in normal development outside of its time-keeping function. Here, we show that PER2 has a noncircadian function that is crucial to mammalian mammary gland development. Virgin Per2-deficient mice, Per2-/- , have underdeveloped glands, containing fewer bifurcations and terminal ducts than glands of wild-type mice. Using a transplantation model, we show that these changes are intrinsic to the gland and further identify changes in cell fate commitment. Per2-/- mouse mammary glands have a dual luminal/basal phenotypic character in cells of the ductal epithelium. We identified colocalization of E-cadherin and keratin 14 in luminal cells. Similar results were demonstrated using MCF10A and shPER2 MCF10A human cell lines. Collectively this study reveals a crucial noncircadian function of PER2 in mammalian mammary gland development, validates the Per2-/- model, and describes a potential role for PER2 in breast cancer.

Genetic variation in sensitivity to estrogens and breast cancer risk.

Breast cancer risk is intimately intertwined with exposure to estrogens. While more than 160 breast cancer risk loci have been identified in humans, genetic interactions with estrogen exposure remain to be established. Strains of rodents exhibit striking differences in their responses to endogenous ovarian estrogens (primarily 17ß-estradiol). Similar genetic variation has been observed for synthetic estrogen agonists (ethinyl estradiol) and environmental chemicals that mimic the actions of estrogens (xenoestrogens). This review of literature highlights the extent of variation in responses to estrogens among strains of rodents and compiles the genetic loci underlying pathogenic effects of excessive estrogen signaling. Genetic linkage studies have identified a total of the 35 quantitative trait loci (QTL) affecting responses to 17ß-estradiol or diethylstilbestrol in five different tissues. However, the QTL appear to act in a tissue-specific manner with 9 QTL affecting the incidence or latency of mammary tumors induced by 17ß-estradiol or diethylstilbestrol. Mammary gland development during puberty is also exquisitely sensitive to the actions of endogenous estrogens. Analysis of mammary ductal growth and branching in 43 strains of inbred mice identified 20 QTL. Regions in the human genome orthologous to the mammary development QTL harbor loci associated with breast cancer risk or mammographic density. The data demonstrate extensive genetic variation in regulation of estrogen signaling in rodent mammary tissues that alters susceptibility to tumors. Genetic variants in these pathways may identify a subset of women who are especially sensitive to either endogenous estrogens or environmental xenoestrogens and render them at increased risk of breast cancer.

In-silico QTL mapping of postpubertal mammary ductal development in the mouse uncovers potential human breast cancer risk loci.

Genetic background plays a dominant role in mammary gland development and breast cancer (BrCa). Despite this, the role of genetics is only partially understood. This study used strain-dependent variation in an inbred mouse mapping panel, to identify quantitative trait loci (QTL) underlying structural variation in mammary ductal development, and determined if these QTL correlated with genomic intervals conferring BrCa susceptibility in humans. For about half of the traits, developmental variation among the complete set of strains in this study was greater (P < 0.05) than that of previously studied strains, or strains in current common use for mammary gland biology. Correlations were also detected with previously reported variation in mammary tumor latency and metastasis. In-silico genome-wide association identified 20 mammary development QTL (Mdq). Of these, five were syntenic with previously reported human BrCa loci. The most significant (P = 1 × 10(-11)) association of the study was on MMU6 and contained the genes Plxna4, Plxna4os1, and Chchd3. On MMU5, a QTL was detected (P = 8 × 10(-7)) that was syntenic to a human BrCa locus on h12q24.5 containing the genes Tbx3 and Tbx5. Intersection of linked SNP (r(2) > 0.8) with genomic and epigenomic features, and intersection of candidate genes with gene expression and survival data from human BrCa highlighted several for further study. These results support the conclusion that mammary tumorigenesis and normal ductal development are influenced by common genetic factors and that further studies of genetically diverse mice can improve our understanding of BrCa in humans.

From genes to milk: genomic organization and epigenetic regulation of the mammary transcriptome.

Even in genomes lacking operons, a gene's position in the genome influences its potential for expression. The mechanisms by which adjacent genes are co-expressed are still not completely understood. Using lactation and the mammary gland as a model system, we explore the hypothesis that chromatin state contributes to the co-regulation of gene neighborhoods. The mammary gland represents a unique evolutionary model, due to its recent appearance, in the context of vertebrate genomes. An understanding of how the mammary gland is regulated to produce milk is also of biomedical and agricultural importance for human lactation and dairying. Here, we integrate epigenomic and transcriptomic data to develop a comprehensive regulatory model. Neighborhoods of mammary-expressed genes were determined using expression data derived from pregnant and lactating mice and a neighborhood scoring tool, G-NEST. Regions of open and closed chromatin were identified by ChIP-Seq of histone modifications H3K36me3, H3K4me2, and H3K27me3 in the mouse mammary gland and liver tissue during lactation. We found that neighborhoods of genes in regions of uniquely active chromatin in the lactating mammary gland, compared with liver tissue, were extremely rare. Rather, genes in most neighborhoods were suppressed during lactation as reflected in their expression levels and their location in regions of silenced chromatin. Chromatin silencing was largely shared between the liver and mammary gland during lactation, and what distinguished the mammary gland was mainly a small tissue-specific repertoire of isolated, expressed genes. These findings suggest that an advantage of the neighborhood organization is in the collective repression of groups of genes via a shared mechanism of chromatin repression. Genes essential to the mammary gland's uniqueness are isolated from neighbors, and likely have less tolerance for variation in expression, properties they share with genes responsible for an organism's survival.

Epigenetic modifications unlock the milk protein gene loci during mouse mammary gland development and differentiation.

BACKGROUND: Unlike other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organization plays a key role in transcriptional regulation and underlies epigenetic regulation during development and differentiation. However, the role of chromatin organization in mammary gland development and differentiation is less well-defined. Here, we have studied the changes in chromatin organization at the milk protein gene loci (casein, whey acidic protein, and others) in the mouse mammary gland before and after functional differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Distal regulatory elements within the casein gene cluster and whey acidic protein gene region have an open chromatin organization after pubertal development, while proximal promoters only gain open-chromatin marks during pregnancy in conjunction with the major induction of their expression. In contrast, other milk protein genes, such as alpha-lactalbumin, already have an open chromatin organization in the mature virgin gland. Changes in chromatin organization in the casein gene cluster region that are present after puberty persisted after lactation has ceased, while the changes which occurred during pregnancy at the gene promoters were not maintained. In general, mammary gland expressed genes and their regulatory elements exhibit developmental stage- and tissue-specific chromatin organization. CONCLUSIONS/SIGNIFICANCE: A progressive gain of epigenetic marks indicative of open/active chromatin on genes marking functional differentiation accompanies the development of the mammary gland. These results support a model in which a chromatin organization is established during pubertal development that is then poised to respond to the systemic hormonal signals of pregnancy and lactation to achieve the full functional capacity of the mammary gland.

G-NEST: a gene neighborhood scoring tool to identify co-conserved, co-expressed genes.

BACKGROUND: In previous studies, gene neighborhoods-spatial clusters of co-expressed genes in the genome-have been defined using arbitrary rules such as requiring adjacency, a minimum number of genes, a fixed window size, or a minimum expression level. In the current study, we developed a Gene Neighborhood Scoring Tool (G-NEST) which combines genomic location, gene expression, and evolutionary sequence conservation data to score putative gene neighborhoods across all possible window sizes simultaneously. RESULTS: Using G-NEST on atlases of mouse and human tissue expression data, we found that large neighborhoods of ten or more genes are extremely rare in mammalian genomes. When they do occur, neighborhoods are typically composed of families of related genes. Both the highest scoring and the largest neighborhoods in mammalian genomes are formed by tandem gene duplication. Mammalian gene neighborhoods contain highly and variably expressed genes. Co-localized noisy gene pairs exhibit lower evolutionary conservation of their adjacent genome locations, suggesting that their shared transcriptional background may be disadvantageous. Genes that are essential to mammalian survival and reproduction are less likely to occur in neighborhoods, although neighborhoods are enriched with genes that function in mitosis. We also found that gene orientation and protein-protein interactions are partially responsible for maintenance of gene neighborhoods. CONCLUSIONS: Our experiments using G-NEST confirm that tandem gene duplication is the primary driver of non-random gene order in mammalian genomes. Non-essentiality, co-functionality, gene orientation, and protein-protein interactions are additional forces that maintain gene neighborhoods, especially those formed by tandem duplicates. We expect G-NEST to be useful for other applications such as the identification of core regulatory modules, common transcriptional backgrounds, and chromatin domains. The software is available at http://docpollard.org/software.html.

Lactation and neonatal nutrition: defining and refining the critical questions.

This paper resulted from a conference entitled "Lactation and Milk: Defining and refining the critical questions" held at the University of Colorado School of Medicine from January 18-20, 2012. The mission of the conference was to identify unresolved questions and set future goals for research into human milk composition, mammary development and lactation. We first outline the unanswered questions regarding the composition of human milk (Section I) and the mechanisms by which milk components affect neonatal development, growth and health and recommend models for future research. Emerging questions about how milk components affect cognitive development and behavioral phenotype of the offspring are presented in Section II. In Section III we outline the important unanswered questions about regulation of mammary gland development, the heritability of defects, the effects of maternal nutrition, disease, metabolic status, and therapeutic drugs upon the subsequent lactation. Questions surrounding breastfeeding practice are also highlighted. In Section IV we describe the specific nutritional challenges faced by three different populations, namely preterm infants, infants born to obese mothers who may or may not have gestational diabetes, and infants born to undernourished mothers. The recognition that multidisciplinary training is critical to advancing the field led us to formulate specific training recommendations in Section V. Our recommendations for research emphasis are summarized in Section VI. In sum, we present a roadmap for multidisciplinary research into all aspects of human lactation, milk and its role in infant nutrition for the next decade and beyond.

The chromatin landscape of the casein gene locus.

For several decades, the regulation of casein gene expression by the lactogenic hormones, prolactin and glucocorticoids, has provided an excellent model system in which to study how steroid and peptide hormones regulate gene expression. Early studies of casein gene regulation defined conserved sequence elements in the 5' flanking region of these genes, including one of which was identified as a γ-interferon activation sequence (GAS). Although this site was thought to interact with a mammary gland-specific factor, purification and cloning of this factor by Bernd Groner and his colleagues revealed it was instead a new member of the signal transducers and activators of transcription family, Stat5, which was expressed in many tissues. The exquisite tissue-specific expression of the casein genes was subsequently shown to depend not on a single transcription factor but on composite response elements that interacted with a number of ubiquitous transcription factors in response to the combinatorial effects of peptide and steroid hormone signaling. More recent studies have defined cooperative effects of prolactin and glucocorticoids as well as antagonistic effects of progesterone on the chromatin structure of both the casein gene proximal promoter region as well as a distal enhancer. Local chromatin modifications as well as long-range interactions facilitated by DNA looping are required for the hormonal regulation of ß-casein gene expression. The casein genes are part of a large gene cluster, and the chromatin landscape of the entire cluster is regulated in a tissue-specific and developmental manner. Finally, newly discovered large non coding RNAs, such as the pregnancy-induced non coding RNA (PINC) may play an important role in the epigenetic regulation of mammary gland differentiation.

Short-term administration of rhGH increases markers of cellular proliferation but not milk protein gene expression in normal lactating women.

Growth hormone is one of few pharmacologic agents known to augment milk production in humans. We hypothesized that recombinant human GH (rhGH) increases the expression of cell proliferation and milk protein synthesis genes. Sequential milk and blood samples collected over four days were obtained from five normal lactating women. Following 24 h of baseline milk and blood sampling, rhGH (0.1 mg/kg/day) was administered subcutaneously once daily for 3 days. Gene expression changes were determined by microarray studies utilizing milk fat globule RNA isolated from each milk sample. Following rhGH administration, DNA synthesis and cell cycle genes were induced, while no significant changes were observed in the expression of milk synthesis genes. Expression of glycolysis and citric acid cycle genes were increased by day 4 compared with day 1, while lipid synthesis genes displayed a circadian-like pattern. Cell cycle gene upregulation occurred after a lag of ∼2 days, likely explaining the failure to increase milk production after only 3 days of rhGH treatment. We conclude that rhGH induces expression of cellular proliferation and metabolism genes but does not induce milk protein gene expression, as potential mechanisms for increasing milk production and could account for the known effect of rhGH to increase milk production following 7-10 days.

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.