Publications by authors named "Monique Rijnkels"

28 Publications

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

Biol Reprod 2020 12;103(6):1186-1198

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA.

Transforming growth factor beta (TGFβ) signaling regulates multifaceted reproductive processes. It has been shown that the type 1 receptor of TGFβ (TGFBR1) is indispensable for female reproductive tract development, implantation, placental development, and fertility. However, the role of TGFβ 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.
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http://dx.doi.org/10.1093/biolre/ioaa155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711917PMC
December 2020

Autophagy regulates functional differentiation of mammary epithelial cells.

Autophagy 2021 02 5;17(2):420-438. Epub 2020 Feb 5.

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA.

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 A or knockdown of (). To evaluate the specific targeting of mitochondria, we traced mitochondrial oxidation and turnover with the fluorescent probe, . Indeed, we found that differentiation engaged mitophagy. To further evaluate the requirement of mitophagy during differentiation, we knocked down the expression of in HC11 cells. We found that MEC differentiation was impaired in 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.: AA: antimycin A; ATG5: autophagy related 5; BAF: bafilomycin A; 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; : 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.
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http://dx.doi.org/10.1080/15548627.2020.1720427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007166PMC
February 2021

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

Breast Cancer Res 2019 11 27;21(1):125. Epub 2019 Nov 27.

Department of Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, TX, 77843, USA.

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.
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http://dx.doi.org/10.1186/s13058-019-1207-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882179PMC
November 2019

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

Am J Pathol 2019 06 5;189(6):1212-1225. Epub 2019 Apr 5.

Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ajpath.2019.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547058PMC
June 2019

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

Oncogene 2019 04 10;38(14):2611-2626. Epub 2018 Dec 10.

Department of Integrative Biosciences, College of Veterinary Medicine, Texas A&M University, College Station, TX, 77843, USA.

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.
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http://dx.doi.org/10.1038/s41388-018-0622-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450754PMC
April 2019

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

Mamm Genome 2018 10 2;29(9-10):632-655. Epub 2018 Aug 2.

USDA/ARS Red River Valley Agricultural Research Center, Fargo, ND, USA.

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.
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http://dx.doi.org/10.1007/s00335-018-9762-7DOI Listing
October 2018

PER2 regulation of mammary gland development.

Development 2018 03 14;145(6). Epub 2018 Mar 14.

Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA

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 -deficient mice, , 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. 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 MCF10A human cell lines. Collectively this study reveals a crucial noncircadian function of PER2 in mammalian mammary gland development, validates the model, and describes a potential role for PER2 in breast cancer.
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http://dx.doi.org/10.1242/dev.157966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897596PMC
March 2018

Genetic variation in sensitivity to estrogens and breast cancer risk.

Mamm Genome 2018 02 27;29(1-2):24-37. Epub 2018 Feb 27.

Department of Population Health Sciences and the Carbone Cancer Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA.

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 17β-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 17β-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 17β-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.
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http://dx.doi.org/10.1007/s00335-018-9741-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936622PMC
February 2018

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

Mamm Genome 2015 Feb 1;26(1-2):57-79. Epub 2015 Jan 1.

Department of Pediatrics, USDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, 1100 Bates St. Suite 10072, Mail Stop: BCM-320, Houston, TX, 77030-2600, USA,

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.
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http://dx.doi.org/10.1007/s00335-014-9551-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4548818PMC
February 2015

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

PLoS One 2013 26;8(9):e75030. Epub 2013 Sep 26.

Genome Center, University of California Davis, Davis, California, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075030PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784412PMC
August 2014

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

PLoS One 2013 2;8(1):e53270. Epub 2013 Jan 2.

United States Department of Agriculture/Agriculture Research Service Children's Nutrition Research Center, Department of Pediatrics-Nutrition, Baylor College of Medicine, Houston, Texas, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053270PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3534698PMC
August 2013

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

BMC Bioinformatics 2012 Sep 28;13:253. Epub 2012 Sep 28.

Genome Center, University of California Davis, 451 Health Science Dr, Davis, CA, 95616, United States of America.

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.
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http://dx.doi.org/10.1186/1471-2105-13-253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575404PMC
September 2012

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

J Mammary Gland Biol Neoplasia 2012 Jun 1;17(2):167-88. Epub 2012 Jul 1.

University of Colorado School of Medicine, Aurora, CO 80045, USA.

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.
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http://dx.doi.org/10.1007/s10911-012-9261-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428522PMC
June 2012

The chromatin landscape of the casein gene locus.

Horm Mol Biol Clin Investig 2012 Mar;10(1):201-205

Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA.

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 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 () may play an important role in the epigenetic regulation of mammary gland differentiation.
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http://dx.doi.org/10.1515/hmbci-2012-0004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3729923PMC
March 2012

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

Physiol Genomics 2011 Apr 4;43(8):381-91. Epub 2011 Jan 4.

Department of Pediatrics - Nutrition, Baylor College of Medicine, Children's Nutrition Research Center, Houston, Texas, USA.

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.
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http://dx.doi.org/10.1152/physiolgenomics.00079.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3092339PMC
April 2011

The epigenetic landscape of mammary gland development and functional differentiation.

J Mammary Gland Biol Neoplasia 2010 Mar 17;15(1):85-100. Epub 2010 Feb 17.

USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA.

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.
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http://dx.doi.org/10.1007/s10911-010-9170-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3006238PMC
March 2010

Epigenetics in mammary gland biology and cancer.

J Mammary Gland Biol Neoplasia 2010 Mar;15(1):1-4

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http://dx.doi.org/10.1007/s10911-010-9171-3DOI Listing
March 2010

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

J Mammary Gland Biol Neoplasia 2010 Mar 10;15(1):73-83. Epub 2010 Feb 10.

UR1196 Génomique et Physiologie de la Lactation, INRA, Domaine de Vilvert, F-78352, Jouy-en-Josas, France.

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.
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http://dx.doi.org/10.1007/s10911-010-9169-xDOI Listing
March 2010

Lactogenic hormonal induction of long distance interactions between beta-casein gene regulatory elements.

J Biol Chem 2009 Aug 19;284(34):22815-24. Epub 2009 Jun 19.

Departments of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Mail Box BCM-130, Houston, TX 77030, USA.

Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of beta-casein gene expression involves two principal regulatory regions, a proximal promoter and a distal enhancer located in the mouse approximately -6 kb upstream of the transcription start site. Using a chromosome conformation capture assay and quantitative real time PCR, we demonstrate that a chromatin loop is created in conjunction with the recruitment of specific transcription factors and p300 in HC11 mammary epithelial cells. Stimulation with both prolactin and hydrocortisone is required for the induction of these long range interactions between the promoter and enhancer, and no DNA looping was observed in nontreated cells or cells treated with each of the hormones separately. The lactogenic hormone-induced interaction between the proximal promoter and distal enhancer was confirmed in hormone-treated primary three-dimensional mammary acini cultures. In addition, the developmental regulation of DNA looping between the beta-casein regulatory regions was observed in lactating but not in virgin mouse mammary glands. Furthermore, beta-casein mRNA induction and long range interactions between these regulatory regions were inhibited in a progestin-dependent manner following stimulation with prolactin and hydrocortisone in HC11 cells expressing human PR-B. Collectively, these data suggest that the communication between these regulatory regions with intervening DNA looping is a crucial step required to both create and maintain active chromatin domains and regulate transcription.
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http://dx.doi.org/10.1074/jbc.M109.032490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755689PMC
August 2009

Lessons from the bovine genome: implications for human nutrition and research.

J Nutr 2009 Jul 6;139(7):1271-2. Epub 2009 May 6.

Department of Food Science and Technology, University of California, Davis, CA 95616, USA.

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http://dx.doi.org/10.3945/jn.109.107656DOI Listing
July 2009

The bovine lactation genome: insights into the evolution of mammalian milk.

Genome Biol 2009 24;10(4):R43. Epub 2009 Apr 24.

Department of Food Science and Technology, University of California Davis, One Shields Avenue, Davis, CA 95616, USA.

Background: The newly assembled Bos taurus genome sequence enables the linkage of bovine milk and lactation data with other mammalian genomes.

Results: Using publicly available milk proteome data and mammary expressed sequence tags, 197 milk protein genes and over 6,000 mammary genes were identified in the bovine genome. Intersection of these genes with 238 milk production quantitative trait loci curated from the literature decreased the search space for milk trait effectors by more than an order of magnitude. Genome location analysis revealed a tendency for milk protein genes to be clustered with other mammary genes. Using the genomes of a monotreme (platypus), a marsupial (opossum), and five placental mammals (bovine, human, dog, mice, rat), gene loss and duplication, phylogeny, sequence conservation, and evolution were examined. Compared with other genes in the bovine genome, milk and mammary genes are: more likely to be present in all mammals; more likely to be duplicated in therians; more highly conserved across Mammalia; and evolving more slowly along the bovine lineage. The most divergent proteins in milk were associated with nutritional and immunological components of milk, whereas highly conserved proteins were associated with secretory processes.

Conclusions: Although both copy number and sequence variation contribute to the diversity of milk protein composition across species, our results suggest that this diversity is primarily due to other mechanisms. Our findings support the essentiality of milk to the survival of mammalian neonates and the establishment of milk secretory mechanisms more than 160 million years ago.
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http://dx.doi.org/10.1186/gb-2009-10-4-r43DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2688934PMC
September 2009

The genome sequence of taurine cattle: a window to ruminant biology and evolution.

Science 2009 Apr;324(5926):522-8

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.
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http://dx.doi.org/10.1126/science.1169588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943200PMC
April 2009

Gene expression in the human mammary epithelium during lactation: the milk fat globule transcriptome.

Physiol Genomics 2009 Mar 18;37(1):12-22. Epub 2008 Nov 18.

Department of Pediatrics-Nutrition, Baylor College of Medicine, Children's Nutrition Research Center, Houston, Texas 77030, USA.

The molecular physiology underlying human milk production is largely unknown because of limitations in obtaining tissue samples. Determining gene expression in normal lactating women would be a potential step toward understanding why some women struggle with or fail at breastfeeding their infants. Recently, we demonstrated the utility of RNA obtained from breast milk fat globule (MFG) to detect mammary epithelial cell (MEC)-specific gene expression. We used MFG RNA to determine the gene expression profile of human MEC during lactation. Microarray studies were performed using Human Ref-8 BeadChip arrays (Illumina). MFG RNA was collected every 3 h for 24 h from five healthy, exclusively breastfeeding women. We determined that 14,070 transcripts were expressed and represented the MFG transcriptome. According to GeneSpring GX 9, 156 ontology terms were enriched (corrected P < 0.05), which include cellular (n = 3,379 genes) and metabolic (n = 2,656) processes as the most significantly enriched biological process terms. The top networks and pathways were associated primarily with cellular activities most likely involved with milk synthesis. Multiple sampling over 24 h enabled us to demonstrate core circadian clock gene expression and the periodicity of 1,029 genes (7%) enriched for molecular functions involved in cell development, growth, proliferation, and cell morphology. In addition, we found that the MFG transcriptome was comparable to the metabolic gene expression profile described for the lactating mouse mammary gland. This paper is the first to describe the MFG transcriptome in sequential human samples over a 24 h period, providing valuable insights into gene expression in the human MEC.
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http://dx.doi.org/10.1152/physiolgenomics.90341.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2661101PMC
March 2009

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

Exp Cell Res 2008 Mar 12;314(5):975-87. Epub 2008 Jan 12.

UR1196, Unité de Génomique et Physiologie de la Lactation, INRA, Jouy en Josas, France.

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.
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http://dx.doi.org/10.1016/j.yexcr.2008.01.001DOI Listing
March 2008

Integration of prolactin and glucocorticoid signaling at the beta-casein promoter and enhancer by ordered recruitment of specific transcription factors and chromatin modifiers.

Mol Endocrinol 2006 Oct 13;20(10):2355-68. Epub 2006 Jun 13.

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent system in which to perform kinetic studies of chromatin remodeling and transcriptional activation. Using HC11 cells as a model, we have investigated the effects of prolactin (Prl) and glucocorticoids both singly and in combination at different time points after hormone treatment. Using chromatin immunoprecipitation analysis, we have determined the dynamics of assembly and disassembly of signal transducer and activator of transcription 5, glucocorticoid receptor, CCAAT enhancer binding protein beta, and Ying Yang-1 at the hormonally activated beta-casein proximal promoter as well as the distal mouse beta-casein enhancer located approximately -6 kb upstream of the transcription start site. Prl alone resulted in a rapid recruitment of both signal transducer and activator of transcription 5 and histone deacetylase 1 to the beta-casein promoter and enhancer, and reciprocally the dissociation of Ying Yang-1 from the proximal promoter. In addition, we have examined the recruitment of coactivator p300 and determined chromatin acetylation status as a function of hormonal treatment. Finally, we have established the time course of RNA polymerase II and phospho-RNA polymerase II accumulation at the beta-casein promoter and enhancer after stimulation with hydrocortisone and Prl. Although glucocorticoids alone led to a rapid increase in histone H3 acetylation, treatment with both hormones was required for stable association of p300 and phospho-RNA polymerase II at both the promoter and enhancer. Collectively, these data suggest a model for the assembly of a multiprotein complex that helps to define how the signaling pathways controlled by these lactogenic hormones are integrated to regulate beta-casein gene expression.
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http://dx.doi.org/10.1210/me.2006-0160DOI Listing
October 2006

A noncoding RNA is a potential marker of cell fate during mammary gland development.

Proc Natl Acad Sci U S A 2006 Apr 30;103(15):5781-6. Epub 2006 Mar 30.

Department of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA.

PINC is a large, alternatively spliced, developmentally regulated, noncoding RNA expressed in the regressed terminal ductal lobular unit-like structures of the parous mammary gland. Previous studies have shown that this population of cells possesses not only progenitor-like qualities (the ability to proliferate and repopulate a mammary gland) and the ability to survive developmentally programmed cell death but also the inhibition of carcinogen-induced proliferation. Here we report that PINC expression is temporally and spatially regulated in response to developmental stimuli in vivo and that PINC RNA is localized to distinct foci in either the nucleus or the cytoplasm in a cell-cycle-specific manner. Loss-of-function experiments suggest that PINC performs dual roles in cell survival and regulation of cell-cycle progression, suggesting that PINC may contribute to the developmentally mediated changes previously observed in the terminal ductal lobular unit-like structures of the parous gland. This is one of the first reports describing the functional properties of a large, developmentally regulated, mammalian, noncoding RNA.
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http://dx.doi.org/10.1073/pnas.0600745103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1420634PMC
April 2006

Multispecies comparative analysis of a mammalian-specific genomic domain encoding secretory proteins.

Genomics 2003 Oct;82(4):417-32

Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

The mammalian-specific casein gene cluster comprises 3 or 4 evolutionarily related genes and 1 physically linked gene with a functional association. To gain a better understanding of the mechanisms regulating the entire casein cluster at the genomic level we initiated a multispecies comparative sequence analysis. Despite the high level of divergence at the coding level, these studies have identified uncharacterized family members within two species and the presence at orthologous positions of previously uncharacterized genes. Also the previous suggestion that the histatin/statherin gene family, located in this region, was primate specific was ruled out. All 11 genes identified in this region appear to encode secretory proteins. Conservation of a number of noncoding regions was observed; one coincides with an element previously suggested to be important for beta-casein gene expression in human and cow. The conserved regions might have biological importance for the regulation of genes in this genomic "neighborhood."
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http://dx.doi.org/10.1016/s0888-7543(03)00114-9DOI Listing
October 2003

Multispecies comparison of the casein gene loci and evolution of casein gene family.

Authors:
Monique Rijnkels

J Mammary Gland Biol Neoplasia 2002 Jul;7(3):327-45

Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.

Caseins, the major milk proteins, are present in a genomic cluster spanning 250-350 kb. The divergence at the coding level between human, rodent, and cattle sequences is rather extensive for most of the genes in this region. Nevertheless, comparative analysis of genomic sequences harboring the casein gene cluster region of these species (with equal evolutionary distances 79-88 Myr) shows that the organization and orientation of the genes is highly conserved. The conserved gene structure indicates that the molecular diversity of the casein genes is achieved through variable use of exons in different species and high evolutionary divergence. Comparative analysis also revealed the presence within two species of uncharacterized casein family members and ruled out the previously held notion that another gene family, located in this region, is primate-specific. Several other new genes as well as conserved noncoding sequences with potential regulatory functions were identified. All genes identified in this region are, or are predicted to be, secreted proteins involved in mineral homeostasis, nutrition, and/or host defense, and are mostly expressed in the mammary and/or salivary glands. These observations suggest a possible common ancestry for the genes in this region.
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http://dx.doi.org/10.1023/a:1022808918013DOI Listing
July 2002