Orphan quality control shapes network dynamics and gene expression.

All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.

Assembly and function of branched ubiquitin chains.

Post-translational modification with ubiquitin is required for cell division, differentiation, and survival in all eukaryotes. As part of an intricate signaling code, ubiquitin is attached to its targets as single molecules or polymeric chains, with the distinct modifications encoding a wide range of outcomes. After early work focused on homotypic ubiquitin chains, such as the K48-linked polymers that drive proteasomal degradation, recent studies noted abundant conjugates that contained ubiquitin molecules modified on two or more sites. Such branched ubiquitin chains are produced in response to specific signals and they exert functions that are critical for cellular and organismal homeostasis. In this review, we will discuss our rapidly evolving understanding of the assembly and function of branched ubiquitin chains.

The Mouse Mammary Gland: a Tool to Inform Adolescents About Environmental Causes of Breast Cancer.

Adolescence is a vulnerable period of breast development, and environmental chemical exposures that occur during this period can increase the risk of breast cancer in adulthood. Discussing breast health with adolescent girls can be difficult for several reasons. In this project, we worked to not only inform adolescent researchers about environmental risks for breast cancer but to also involve them in research studies. We taught adolescents about the stages of mammary gland development using samples collected from mice, with a specific focus on pre-pubertal and pubertal stages of development. Our analysis shows that adolescent researchers, with relatively modest training, can collect reliable and reproducible data on aspects of mammary gland biology that are known to be disrupted by environmental chemicals, with coefficients of variation < 2.5% for basic mammary gland parameters and 5-7% for more complex measures. Finally, we provided these adolescents with information about environmental risk factors for breast cancer that they could share with their peers and community and action items to potentially modify their individual risk. We hope that researchers working in this field will engage adolescent researchers in projects to evaluate chemicals that influence breast cancer risk. Summer research programs that inform young adolescents about breast cancer risk factors not only benefit these novice researchers individually but also benefit their communities when they are encouraged to talk about the value of basic science studies, discuss vulnerable periods of mammary gland development, and share what they have learned about cancer and the environment.

Bisphenol S alters development of the male mouse mammary gland and sensitizes it to a peripubertal estrogen challenge.

Humans are exposed to estrogenic chemicals in food and food packaging, personal care products, and other industrial and consumer goods. Bisphenol A (BPA), a well-studied xenoestrogen, is known to alter development of estrogen-sensitive organs including the brain, reproductive tract, and mammary gland. Bisphenol S (BPS; 4,4'-sulfonyldiphenol), which has a similar chemical structure to BPA, is also used in many consumer products, but its effects on estrogen-sensitive organs in mammals has not been thoroughly examined. Here, we quantified the effects of perinatal exposures to BPS on the male mouse mammary gland. In our first study, pregnant CD-1 mice were orally exposed to BPS (2 or 200 µg/kg/day) starting on pregnancy day 9 through lactation day 20, and male mammary glands were evaluated on embryonic day 16, prior to puberty, and in early adulthood. We observed modest changes in tissue organization in the fetal gland, and significant increases in growth of the gland induced by developmental BPS exposure in adulthood. In our second study, pregnant CD-1 mice were orally exposed to BPS (2, 200 or 2000 µg/kg/day) starting on pregnancy day 9 through lactational day 2. After weaning, the male pups were administered either oil (vehicle) or an estrogen challenge (1 µg ethinyl estradiol/kg/day) for ten days starting prior to puberty. After the 10-day estrogen challenge, we examined hormone-sensitive outcomes including anogenital index (AGI), weight of the seminal vesicles, and morphological parameters of the mammary gland. Although AGI and seminal vesicle weight were not affected by BPS, we observed dose-specific effects on the response of male mammary glands to the peripubertal estrogen challenge. Because male mammary glands are structurally less developed compared to females, they may provide a simple model tissue to evaluate the effects of putative xenoestrogens.

Developmental Exposure to 2,2',4,4'-Tetrabromodiphenyl Ether Permanently Alters Blood-Liver Balance of Lipids in Male Mice.

Polybrominated diphenyl ethers (PBDEs) were used as flame-retardant additives starting 1965 and were recently withdrawn from commerce in North America and Europe. Approximately 1/5 of the total U.S. population were born when environmental concentrations of PBDE plateaued at their maximum. Accumulating evidence suggests that developmental exposures to PBDE may result in long-lasting programming of liver metabolism. In this study, CD-1 mice were exposed prenatally or neonatally to 1 mg/kg body weight of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), and changes in liver histology, transcriptome, and liver-blood balance of triglycerides were analyzed in 10 months old male offspring. In both exposure groups, long-term reprogramming of lipid metabolism was observed, including increased liver triglycerides and decreased blood triglycerides, and altered expression of metabolic genes in the liver. Significant upregulation of lipid influx transporter Cd36 2.3- and 5.7-fold in pre- and neonatal exposure groups, respectively was identified as a potential mechanism of blood/liver imbalance of triglycerides. Analysis of our and previously published all-genome gene expression data identified changes in expression of ribosomal protein genes as a transcriptomic signature of PBDE exposure. Further comparison of our new data and published data demonstrate that low doses (0.2 mg/kg body weight) of PBDE induce long-lasting up-regulation of ribosomal genes, suppression of Cd36 in liver and increase circulating triglycerides in blood, while moderated doses (≥1 mg/kg body weight) produce opposite long-lasting effects. To conclude, this study shows that an environmentally relevant developmental exposures to BDE-47 permanently alter lipid uptake and accumulation in the liver, with low and moderate doses having opposite effect on liver transcriptomics and triglyceride balance. Similar effects of pre- and neonatal exposures point at hepatocyte maturation as a sensitive window of the liver metabolism programming. These results suggest that PBDE exposure may be an important factor increasing risks of cardio-vascular disease and non-alcoholic fatty liver disease via modulation of liver/blood balance of lipids. The translational relevance of these findings for human remain to be studied.

Asymmetric development of the male mouse mammary gland and its response to a prenatal or postnatal estrogen challenge.

The CD-1 mouse mammary gland is sexually dimorphic, with males lacking nipples. Recent studies have revealed that the underlying epithelium in the male mammary gland is sensitive to estrogenic environmental chemicals. In ongoing investigations, we observed asymmetric morphology in the left and right male mouse mammary glands. Here, we quantified these asymmetries in the embryonic, prepubertal, pubertal and adult male mammary gland. We found that the right gland was typically larger with more branching points compared to the left gland. We next evaluated the response of the left and right glands to 17α-ethinyl estradiol (EE2) after perinatal or peripubertal exposures. We found that the right gland was more responsive to EE2 than the left at both periods of exposure. These results reveal novel aspects of male mammary gland biology and suggest that future studies should control for laterality in the evaluation of hazards associated with exposures to estrogenic chemicals.

Low dose bisphenol S or ethinyl estradiol exposures during the perinatal period alter female mouse mammary gland development.

Throughout life, mammary tissue is strongly influenced by hormones. Scientists have hypothesized that synthetic chemicals with hormonal activities could disrupt mammary gland development and contribute to breast diseases and dysfunction. Bisphenol S (BPS) is an estrogenic compound used in many consumer products. In this study, CD-1 mice were exposed to BPS (2 or 200 µg/kg/day) during pregnancy and lactation. Mice exposed to 0.01 or 1 µg/kg/day ethinyl estradiol (EE2), a pharmaceutical estrogen, were also evaluated. Mammary glands from female offspring were collected prior to the onset of puberty, during puberty, and in early adulthood. Growth parameters, histopathology, cell proliferation and expression of hormone receptors were quantified. Our evaluations revealed age- and dose-specific effects of BPS that were different from the effects of EE2, and distinct from the effects of BPA that have been reported previously. These assessments suggest that individual xenoestrogens may have unique effects on this sensitive tissue.

The mouse mammary gland as a sentinel organ: distinguishing 'control' populations with diverse environmental histories.

BACKGROUND: There are numerous examples of laboratory animals that were inadvertently exposed to endocrine disrupting chemicals (EDCs) during the process of conducting experiments. Controlling contaminations in the laboratory is challenging, especially when their source is unknown. Unfortunately, EDC contaminations can interfere with the interpretation of data during toxicological evaluations. We propose that the male CD-1 mouse mammary gland is a sensitive bioassay to evaluate the inadvertent contamination of animal colonies. METHODS: We evaluated mammary glands collected from two CD-1 mouse populations with distinct environmental histories. Population 1 was born and raised in a commercial laboratory with unknown EDC exposures; Population 2 was the second generation raised in an animal facility with limited exposures to xenoestrogens from caging, feed, etc. Mammary glands were collected from all animals and evaluated using morphometric techniques to quantify morphological characteristics of the mammary gland. RESULTS: Population 1 (with suspected history of environmental chemical exposure) and Population 2 (with known limited history of xenoestrogen exposure) were morphologically distinguishable in adult males, prepubertal females, and pubertal females. Mammary glands from males raised in the commercial animal facility were significantly more developed, with larger ductal trees and more branching points. The appearance of these mammary glands was consistent with prior reports of male mice exposed to low doses of bisphenol A (BPA) during early development. In females, the two populations were morphologically distinct at both prepuberty and puberty, with the most striking differences observed in the number, size, and density of terminal end buds, e.g. highly proliferative structures found in the developing mammary gland. CONCLUSIONS: Collectively, these results suggest that the mouse mammary gland has the potential to be used as a sentinel organ to evaluate and distinguish animal colonies raised in different environmental conditions including potential EDC exposures. Our findings could help researchers that wish to perform a posteriori evaluations to determine whether inadvertent contamination with xenoestrogens (and potentially other EDCs) has occurred in their animal colonies, especially after new materials (feed, caging, water bottles) have been introduced. Finally, our results challenge the relatively common practice of using historical controls in toxicological experiments.