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Polycyclic aromatic hydrocarbons like benzo[a]pyrene (BaP) are generated during incomplete combustion of organic materials. Prior research has demonstrated that BaP is a prenatal ovarian toxicant and carcinogen. However, the metabolic pathways active in the embryo and its developing gonads and the mechanisms by which prenatal exposure to BaP predisposes to ovarian tumors later in life remain to be fully elucidated. To address these data gaps, we orally dosed pregnant female mice with BaP from embryonic day (E) 6.5 to E11.5 (0, 0.2, or 2 mg/kg/day) for metabolite measurement or E9.5 to E11.5 (0 or 3.33 mg/kg/day) for embryonic gonad RNA sequencing. Embryos were harvested at E13.5 for both experiments. The sum of BaP metabolite concentrations increased significantly with dose in the embryos and placentas, and concentrations were significantly higher in female than male embryos and in embryos than placentas. RNA sequencing revealed that enzymes involved in metabolic activation of BaP are expressed at moderate to high levels in embryonic gonads and that greater transcriptomic changes occurred in the ovaries in response to BaP than in the testes. We identified 490 differentially expressed genes (DEGs) with false discovery rate P-values < 0.05 when comparing BaP-exposed to control ovaries but no statistically significant DEGs between BaP-exposed and control testes. Genes related to monocyte/macrophage recruitment and activity, prolactin family genes, and several keratin genes were among the most upregulated genes in the BaP-exposed ovaries. Results show that developing ovaries are more sensitive than testes to prenatal BaP exposure, which may be related to higher concentrations of BaP metabolites in female embryos.

Significance The documented efficacy of COX-2 inhibitors in cancer chemoprevention and in suppression of metastasis is predominantly attributed to inflammatory responses, whereas their effects on tumor-stromal interaction are poorly understood. Through single-cell transcriptome analyses in an immune-compromised mouse xenograft model and in vitro reconstitution experiments, we uncover a tumor-stromal paracrine pathway in which secretion by tumor cells of the COX-2 product prostaglandin E2 induces prolactin production by stromal cells, which activates signaling in disseminated tumor cells with upregulated prolactin receptor expression. Analysis of multiple human cancers confirms differential tumor and stromal cell expression of COX-2, prolactin, and prolactin receptor. Together, these findings may provide novel biomarkers to inform the selective application of COX-2 inhibitors and point to additional targets for suppressing metastasis recurrence., Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases. Secretion of the ligand prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E2 (PGE2). PGE2 treatment of fibroblasts activates the orphan nuclear receptor NR4A (Nur77), with prolactin as a major transcriptional target for the NR4A-retinoid X receptor (RXR) heterodimer. Ectopic expression of prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor celecoxib abrogates prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, prolactin, and prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine cross-talk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression.

Whereas in ovo exposure of genetically male (ZZ) chicken embryos to exogenous estrogens temporarily feminizes gonads at the time of hatching, the morphologically ovarian ZZ-gonads (FemZZs for feminized ZZ gonads) are masculinized back to testes within 1 year. To identify the feminization-resistant “memory” of genetic male sex, FemZZs showing varying degrees of feminization were subjected to transcriptomic, DNA methylome, and immunofluorescence analyses. Protein-coding genes were classified based on their relative mRNA expression across normal ZZ-testes, genetically female (ZW) ovaries, and FemZZs. We identified a group of 25 genes that were strongly expressed in both ZZ-testes and FemZZs but dramatically suppressed in ZW-ovaries. Interestingly, 84% (21/25) of these feminization-resistant testicular marker genes, including the DMRT1 master masculinizing gene, were located in chromosome Z. Expression of representative marker genes of germline cells (eg, DAZL or DDX4/VASA) was stronger in FemZZs than normal ZZ-testes or ZW-ovaries. We also identified 231 repetitive sequences (RSs) that were strongly expressed in both ZZ-testes and FemZZs, but these RSs were not enriched in chromosome Z. Although 94% (165/176) of RSs exclusively expressed in ZW-ovaries were located in chromosome W, no feminization-inducible RS was detected in FemZZs. DNA methylome analysis distinguished FemZZs from normal ZZ- and ZW-gonads. Immunofluorescence analysis of FemZZ gonads revealed expression of DMRT1 protein in medullary SOX9+ somatic cells and apparent germline cell populations in both medulla and cortex. Taken together, our study provides evidence that both somatic and germline cell populations in morphologically feminized FemZZs maintain significant transcriptomic and epigenetic memories of genetic sex.

Early life exposure to endocrine-disrupting chemicals (EDCs) is an emerging risk factor for the development of obesity and diabetes later in life. We previously showed that prenatal exposure to the EDC tributyltin (TBT) results in increased adiposity in the offspring. These effects linger into adulthood and are propagated through successive generations. TBT activates two nuclear receptors, the peroxisome proliferator–activated receptor (PPAR) γ and its heterodimeric partner retinoid X receptor (RXR), that promote adipogenesis in vivo and in vitro. We recently employed a mesenchymal stem cell (MSC) model to show that TBT promotes adipose lineage commitment by activating RXR, not PPARγ. This led us to consider the functional consequences of PPARγ vs RXR activation in developing adipocytes. We used a transcriptomal approach to characterize genome-wide differences in MSCs differentiated with the PPARγ agonist rosiglitazone (ROSI) or TBT. Pathway analysis suggested functional deficits in TBT-treated cells. We then compared adipocytes differentiated with ROSI, TBT, or a pure RXR agonist IRX4204 (4204). Our data show that RXR activators (“rexinoids,” 4204 and TBT) attenuate glucose uptake, blunt expression of the antidiabetic hormone adiponectin, and fail to downregulate proinflammatory and profibrotic transcripts, as does ROSI. Finally, 4204 and TBT treatment results in an inability to induce markers of adipocyte browning, in part due to sustained interferon signaling. Taken together, these data implicate rexinoids in the development of dysfunctional white adipose tissue that could potentially exacerbate obesity and/or diabetes risk in vivo. These data warrant further screening and characterization of EDCs that activate RXR.

Exposure to environmental stressors is known to increase disease susceptibility in unexposed descendants in the absence of detectable genetic mutations. The mechanisms mediating environmentally-induced transgenerational disease susceptibility are poorly understood. We showed that great-great-grandsons of female mice exposed to tributyltin (TBT) throughout pregnancy and lactation were predisposed to obesity due to altered chromatin organization that subsequently biased DNA methylation and gene expression. Here we analyzed DNA methylomes and transcriptomes from tissues of animals ancestrally exposed to TBT spanning generations, sexes, ontogeny, and cell differentiation state. We found that TBT elicited concerted alterations in the expression of “chromatin organization” genes and inferred that TBT-disrupted chromatin organization might be able to self-reconstruct transgenerationally. We also found that the location of “chromatin organization” and “metabolic” genes is biased similarly in mouse and human genomes, suggesting that exposure to environmental stressors in different species could elicit similar phenotypic effects via self-reconstruction of disrupted chromatin organization.

Germline specification underlies human reproduction and evolution, but it has proven difficult to study in humans since it occurs shortly after blastocyst implantation. This process can be modeled with human induced pluripotent stem cells (hiPSCs) by differentiating them into primordial germ cell-like cells (hPGCLCs) through an incipient mesoderm-like cell (iMeLC) state. Here, we elucidate the key transcription factors and their interactions with important signaling pathways in driving hPGCLC differentiation from iPSCs. Germline competence of iMeLCs is dictated by the duration and dosage of WNT signaling, which induces expression of EOMES to activate SOX17, a key driver of hPGCLC specification. Upon hPGCLC induction, BMP signaling activates TFAP2C in a SOX17-independent manner. SOX17 and TFAP2C then cooperatively instate an hPGCLC transcriptional program, including BLIMP1 expression. This specification program diverges from its mouse counterpart regarding key transcription factors and their hierarchies, and it provides a foundation for further study of human germ cell development.

There is evidence from longitudinal studies that being light at birth and weaning is associated with subsequent rapid weight gain in infants. This is referred to as “centile crossing”, which can lead to increased risk of lifetime obesity, glucose dysregulation and type 2 diabetes. Here, pregnant CD-1 mice were hemi-ovariectomized so that the entire litter was contained in one uterine horn to increase variability in fetal growth rate. Pregnant females were implanted on gestation day (GD) 9 with a Silastic capsule containing 6, 60 or 600 μg bisphenol A (BPA). On GD 18 the mean fetal serum unconjugated BPA concentrations were 17, 177 and 1858 pg/ml, respectively. Capsules were not removed, to avoid maternal stress, and were predicted to release BPA for at least 3 weeks. Body weight at weaning was strongly negatively correlated with post-weaning weight gain in both control and BPA-treated male mice, consistent with human data; female offspring were excluded, avoiding complications associated with postpubertal estrogens. Within each treatment group, male offspring were sorted into tertiles based on relative weight gain during the two weeks after weaning, designated as having Rapid (R), Medium (M) or Slow (S) growth rate. BPA exposure was associated with altered growth rate between weaning and postnatal week 12 (young adulthood), when a low-dose (20 mg/kg, i.p.) glucose tolerance test (GTT) was performed. We found altered glucose regulation in response to all doses of BPA. However, glucose tolerance was only significantly impaired (blood glucose levels were elevated) compared to controls in males in the rapid post-weaning growth group exposed perinatally to BPA. We conclude that male mice that are light at weaning, but then experience rapid catch-up growth immediately after weaning, represent a sensitive sub-population that is vulnerable to the metabolic disrupting effects of very low pg/ml fetal serum concentrations of BPA.

Molecular drivers underlying bone metastases in human cancer are not well understood, in part due to constraints in bone tissue sampling. Here, RNA sequencing was performed of circulating tumor cells (CTC) isolated from blood samples of women with metastatic estrogen receptor (ER)+ breast cancer, comparing cases with progression in bone versus visceral organs. Among the activated cellular pathways in CTCs from bone-predominant breast cancer is androgen receptor (AR) signaling. AR gene expression is evident, as is its constitutively active splice variant AR-v7. AR expression within CTCs is correlated with the duration of treatment with aromatase inhibitors, suggesting that it contributes to acquired resistance to endocrine therapy. In an established breast cancer xenograft model, a bone-tropic derivative displays increased AR expression, whose genetic or pharmacologic suppression reduces metastases to bone but not to lungs. Together, these observations identify AR signaling in CTCs from women with bone-predominant ER+ breast cancer, and provide a rationale for testing androgen inhibitors in this subset of patients. Implications: This study highlights a role for the AR in breast cancer bone metastasis, and suggests that therapeutic targeting of the AR may benefit patients with metastatic breast cancer. Mol Cancer Res; 16(4); 720–7. ©2018 AACR.

Circulating signals of drug resistance Cancer drugs often lose their effectiveness because tumors acquire genetic changes that confer drug resistance. Ideally, patients would be switched to a different drug before tumor growth resumes, but this requires early knowledge of how resistance arose. Miyamoto et al. have developed a non-invasive method to spot resistance by sequencing RNA transcripts in single circulating tumor cells (CTCs) (see the Perspective by Nanus and Giannakakou). For example, in prostate cancer patients, drug resistance was triggered by activation of the Wnt signaling pathway. But CTCs are rare and fragile, and the technology needs further development before it is used in clinical practice. Science , this issue p. 1351 ; see also p. 1283

Developmental exposure to environmental factors has been linked to obesity risk later in life. Nuclear receptors are molecular sensors that play critical roles during development and, as such, are prime candidates to explain the developmental programming of disease risk by environmental chemicals. We have previously characterized the obesogen tributyltin (TBT), which activates the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor (RXR) to increase adiposity in mice exposed in utero. Mesenchymal stem cells (MSCs) from these mice are biased toward the adipose lineage at the expense of the osteoblast lineage, and MSCs exposed to TBT in vitro are shunted toward the adipose fate in a PPARγ-dependent fashion. To address where in the adipogenic cascade TBT acts, we developed an in vitro commitment assay that permitted us to distinguish early commitment to the adipose lineage from subsequent differentiation. TBT and RXR activators (rexinoids) had potent effects in committing MSCs to the adipose lineage, whereas the strong PPARγ activator rosiglitazone was inactive. We show that activation of RXR is sufficient for adipogenic commitment and that rexinoids act through RXR to alter the transcriptome in a manner favoring adipogenic commitment. RXR activation alters expression of enhancer of zeste homolog 2 (EZH2) and modifies genome-wide histone 3 lysine 27 trimethylation (H3K27me3) in promoting adipose commitment and programming subsequent differentiation. These data offer insights into the roles of RXR and EZH2 in MSC lineage specification and shed light on how endocrine-disrupting chemicals such as TBT can reprogram stem cell fate.

The developing vertebrate embryo is exquisitely sensitive to retinoic acid (RA) concentration, particularly during anteroposterior patterning. In contrast to Nodal and Wnt signaling, RA was not previously considered to be an instructive signal in mesoderm formation during gastrulation. Here, we show in Xenopus that RARγ is indispensable for the expression of early mesoderm markers and is, therefore, an obligatory factor in mesodermal competence and/or maintenance. We identified several novel targets upregulated by RA receptor signaling in the early gastrula that are expressed in the circumblastoporal ring and linked to mesodermal development. Despite overlapping expression patterns of the genes encoding the RA-synthesizing enzyme Aldh1a2 and the RA-degrading enzyme Cyp26a1, RARγ1 functions as a transcriptional activator in early mesoderm development, suggesting that RA ligand is available to the embryo earlier than previously appreciated. RARγ1 is required for cellular adhesion, as revealed by spontaneous dissociation and depletion of ncam1 mRNA in animal caps harvested from RARγ1 knockdown embryos. RARγ1 knockdown obliterates somite boundaries, and causes loss of Myod protein in the presomitic mesoderm, but ectopic, persistent expression of Myod protein in the trunk. Thus, RARγ1 is required for stabilizing the mesodermal fate, myogenic commitment, somite boundary formation, and terminal skeletal muscle differentiation.

Exposure of mammalian fetuses to endocrine disruptors can increase the risk of adult-onset diseases. We previously showed that exposure of mouse fetuses to bisphenol A (BPA) caused adult-onset obesity. To examine roles of epigenetic changes in this delayed toxicity, we determined the effects of fetal mouse exposure to BPA on genome-wide DNA methylation and messenger RNA (mRNA) expression in gonadal white adipose tissues (WATs) by deep sequencing, bisulfite pyrosequencing, and real-time quantitative polymerase chain reaction. Pregnant CD-1 mice (F0) were dosed daily with 0, 5, or 500 μg/kg/d BPA during gestational days 9 to 18, and the weaned F1 animals were fed ad libitum with standard chow until they were euthanized at 19 weeks old. In the vehicle-exposed F1 animals, fggy promoter showed a clear bimodal pattern of very strong (55% to 95%) or very weak (5% to 30%) DNA methylation occurring at nearly equal incidence with no intermediate strength. Promoter hypermethylation completely suppressed mRNA expression. BPA exposure eliminated this naturally occurring dichotomy, shifting fggy promoter toward the hypomethylation state to release transcriptional suppression. The strength of Fggy mRNA expression significantly correlated with increased whole body weight and gonadal fat weight of males but not females. Bioinformatics studies showed that expression of Fggy mRNA is stronger in mouse WATs than in brown adipose tissues and enhanced in gonadal fat by diet-induced obesity. These observations suggest that prenatal exposure to BPA may disrupt the physiological bimodal nature of epigenetic regulation of fggy in mouse WATs, possibly contributing to the adult-onset obesity phenotype.

SummaryCirculating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer.

SUMMARY Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

For years, scientists from various disciplines have studied the effects of endocrine disrupting chemicals (EDCs) on the health and wellbeing of humans and wildlife. Some studies have specifically focused on the effects of low doses, i.e. those in the range that are thought to be safe for humans and/or animals. Others have focused on the existence of non-monotonic dose-response curves. These concepts challenge the way that chemical risk assessment is performed for EDCs. Continued discussions have clarified exactly what controversies and challenges remain. We address several of these issues, including why the study and regulation of EDCs should incorporate endocrine principles; what level of consensus there is for low dose effects; challenges to our understanding of non-monotonicity; and whether EDCs have been demonstrated to produce adverse effects. This discussion should result in a better understanding of these issues, and allow for additional dialogue on their impact on risk assessment.

The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.

Triple negative breast cancers (TNBCs) are highly aggressive and grow in response to sex steroid hormones despite lacking expression of the classical estrogen (E2) and progesterone (P4) receptors. Since P4 receptor membrane component 1 (PGRMC1) is expressed in breast cancer tumors and is known to mediate P4-induced cell survival, this study was designed to determine the expression of PGRMC1 in TNBC tumors and the involvement of PGRMC1 in regulating proliferation and survival of TNBC cells in vitro and the growth of TNBC tumors in vivo. For the latter studies, the MDA-MB-231 (MDA) cell line derived from TNBC was used. These cells express PGRMC1 but lack expression of the classical P4 receptor. A lentiviral-based shRNA approach was used to generate a stably transfected PGRMC1-deplete MDA line for comparison to the PGRMC1-intact MDA line. The present studies demonstrate that PGRMC1: 1) is expressed in TNBC cells; 2) mediates the ability of P4 to suppress TNBC cell mitosis in vitro; 3) is required for P4 to reduce the apoptotic effects of doxorubicin in vitro; and 4) facilitates TNBC tumor formation and growth in vivo. Taken together, these findings indicate that PGRMC1 plays an important role in regulating the growth and survival of TNBC cells in vitro and ultimately in the formation and development of these tumors in vivo. Thus, PGRMC1 may be a therapeutic target for TNBCs.

For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of “the dose makes the poison,” because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from the cell culture, animal, and epidemiology literature. We illustrate that nonmonotonic responses and low-dose effects are remarkably common in studies of natural hormones and EDCs. Whether low doses of EDCs influence certain human disorders is no longer conjecture, because epidemiological studies show that environmental exposures to EDCs are associated with human diseases and disabilities. We conclude that when nonmonotonic dose-response curves occur, the effects of low doses cannot be predicted by the effects observed at high doses. Thus, fundamental changes in chemical testing and safety determination are needed to protect human health.

SummarySterol regulatory element-binding proteins (SREBPs) activate genes involved in the synthesis and trafficking of cholesterol and other lipids and are critical for maintaining lipid homeostasis. Aberrant SREBP activity, however, can contribute to obesity, fatty liver disease, and insulin resistance, hallmarks of metabolic syndrome. Our studies identify a conserved regulatory circuit in which SREBP-1 controls genes in the one-carbon cycle, which produces the methyl donor S-adenosylmethionine (SAMe). Methylation is critical for the synthesis of phosphatidylcholine (PC), a major membrane component, and we find that blocking SAMe or PC synthesis in C. elegans, mouse liver, and human cells causes elevated SREBP-1-dependent transcription and lipid droplet accumulation. Distinct from negative regulation of SREBP-2 by cholesterol, our data suggest a feedback mechanism whereby maturation of nuclear, transcriptionally active SREBP-1 is controlled by levels of PC. Thus, nutritional or genetic conditions limiting SAMe or PC production may activate SREBP-1, contributing to human metabolic disorders.

Huntington's disease is initiated by the expression of a CAG repeat-encoded polyglutamine region in full-length huntingtin, with dominant effects that vary continuously with CAG size. The mechanism could involve a simple gain of function or a more complex gain of function coupled to a loss of function (e.g. dominant negative-graded loss of function). To distinguish these alternatives, we compared genome-wide gene expression changes correlated with CAG size across an allelic series of heterozygous CAG knock-in mouse embryonic stem (ES) cell lines (Hdh(Q20/7), Hdh(Q50/7), Hdh(Q91/7), Hdh(Q111/7)), to genes differentially expressed between Hdh(ex4/5/ex4/5) huntingtin null and wild-type (Hdh(Q7/7)) parental ES cells. The set of 73 genes whose expression varied continuously with CAG length had minimal overlap with the 754-member huntingtin-null gene set but the two were not completely unconnected. Rather, the 172 CAG length-correlated pathways and 238 huntingtin-null significant pathways clustered into 13 shared categories at the network level. A closer examination of the energy metabolism and the lipid/sterol/lipoprotein metabolism categories revealed that CAG length-correlated genes and huntingtin-null-altered genes either were different members of the same pathways or were in unique, but interconnected pathways. Thus, varying the polyglutamine size in full-length huntingtin produced gene expression changes that were distinct from, but related to, the effects of lack of huntingtin. These findings support a simple gain-of-function mechanism acting through a property of the full-length huntingtin protein and point to CAG-correlative approaches to discover its effects. Moreover, for therapeutic strategies based on huntingtin suppression, our data highlight processes that may be more sensitive to the disease trigger than to decreased huntingtin levels.

Purpose: Fulvestrant is known to promote the degradation of the estrogen receptor (ER) in the nucleus. However, fulvestrant also promotes the aggregation of the newly synthesized ER in the cytoplasm. Accumulation of protein aggregates leads to cell death but this effect is limited as a result of their elimination by the proteasome. We tested whether combining fulvestrant with the proteasome inhibitor, bortezomib, could enhance the accumulation of ER aggregates and cause apoptotic cell death. Experimental Design: The rate of aggregation of the ER was monitored in ER+ breast cancer cells lines, T47D, ZR-75.1, BT474, MDA-MB-361, MCF-7, fulvestrant resistance MCF-7, and tamoxifen-resistant T47D-cyclin D1 cells. Activation of the unfolded protein response, apoptosis, and metabolic rate were also monitored in these cell lines following treatment with fulvestrant, bortezomib, or bortezomib in combination with fulvestrant. Results: We found that bortezomib enhances the fulvestrant-mediated aggregation of the ER in the cytoplasm without blocking the degradation of the ER in the nucleus. Further, these aggregates activate a sustained unfolded protein response leading to apoptotic cell death. Further, we show that the combination induced tumor regression in a breast cancer mouse model of tamoxifen resistance. Conclusions: Adding bortezomib to fulvestrant enhances its efficacy by taking advantage of the unique ability of fulvestrant to promote cytoplasmic aggregates of the ER. As this effect of fulvestrant is independent of the transcriptional activity of the ER, these results suggest that this novel combination may be effective in breast cancers that are ER+ but estrogen independent. Clin Cancer Res; 17(8); 2292–300. ©2011 AACR.

TOX3 is a nuclear protein containing a high mobility group (HMG)-box domain, which regulates Ca2+-dependent transcription in neurons through interaction with the cAMP-response-element-binding protein (CREB). TOX3 appears to be associated with breast cancer susceptibility and was previously shown to be expressed downstream of a cytoprotective cascade together with CITED1, a transcriptional regulator that does not bind directly to DNA. In the present study we show that TOX3 is predominantly expressed in the brain, forms homodimers and interacts with CITED1. TOX3 overexpression protects neuronal cells from cell death caused by endoplasmic reticulum stress or BAX overexpression through the induction of anti-apoptotic transcripts and repression of pro-apoptotic transcripts, which correlates with enhanced transcription involving isolated estrogen-responsive elements and estrogen-responsive promoters. However, both functions cannot be inhibited with the anti-estrogen fulvestrant and are only attenuated by mutation of estrogen-responsive elements. TOX3 also interacts with native CREB and induces the CREB-responsive BCL-2 promoter, which can be inhibited by coexpression of CITED1. Coexpression of CREB, by contrast, abolishes TOX3-mediated transcription from the estrogen-responsive complement C3 promoter. Our results suggest that TOX3 can enhance transcriptional activation from different cytoprotective promoters and that this is dependent on the predominance of either phosphorylated CREB or CITED1 within the transcriptionally active complex.

Genetic sex typing of vertebrate animals is an essential technique for research on reproductive phenomena such as sex determination of embryonic tissues. Polymerase chain reaction amplification of genomic DNA segments in the Z and W sex chromosomes has been widely used as a standard laboratory method to determine genetic sex of the chicken (Gallus gallus domesticus). However, conventional protocols for PCR determination of avian sex typically involve tedious steps of genomic DNA isolation, which often require relatively large amounts of tissue samples, and the purity of genomic DNA specimens significantly affects PCR efficiency. Moreover, detection of sex chromosome-specific PCR products by gel electrophoresis is prone to misjudgment caused by amplification of contaminating genomic DNA segments derived from tissue or DNA samples as well as previously generated PCR products. Thus, the credibility of genetic sex typing by conventional PCR-based methods that measure the relative amounts of the end product DNA amplicons critically depends on several experimental steps that are potentially vulnerable to errors. Here, we describe an optimized protocol of chicken genetic sex typing by TaqMan real-time quantitative PCR amplification of markers on the sex chromosomes. This TaqMan sex typing method accurately quantifies relative amounts of the Z and W sex chromosome markers directly from only 0.5 to 2 μL of total blood lysate without nucleic acid purification. The real-time amplification curves of the quantitative PCR reaction readily distinguishe truly homozygous (ZZ) and heterozygous (ZW) sex chromosomes from contamination of the sex chromosomal DNA, ensuring highly credible sex determination. Thus, the TaqMan typing of chicken genetic sex has several advantageous features for high-throughput operation compared with conventional methods.

miR-33 in Cholesterol Control With the well-established link between serum cholesterol levels and cardiovascular disease and the availability of effective cholesterol-lowering drugs, cholesterol screening has rapidly become a routine part of health care. Yet, much remains to be learned about how cholesterol levels are regulated at the cellular level (see the Perspective by Brown et al. ). Now, Najafi-Shoushtari et al. (p. 1566 , published online 13 May) and Rayner et al. (p. 1570 , published online 13 May) have discovered a new molecular player in cholesterol control—a small noncoding RNA that, intriguingly, is embedded within the genes coding for sterol regulatory element-binding proteins (SREBPs), transcription factors already known to regulate cholesterol levels. This microRNA, called miR-33, represses expression of the adenosine triphosphate–binding cassette transporter A1, a protein that regulates synthesis of high-density lipoprotein (HDL, or “good” cholesterol) and that helps to remove “bad” cholesterol from the blood. Reducing the levels of miR-33 in mice boosted serum HDL levels, suggesting that manipulation of this regulatory circuit might be therapeutically useful.

Induced pluripotent stem cells (iPSCs) have been generated by enforced expression of defined sets of transcription factors in somatic cells. It remains controversial whether iPSCs are molecularly and functionally equivalent to blastocyst-derived embryonic stem cells (ESCs). By comparing genetically identical mouse ESCs and iPSCs, we show here that the overall mRNA and miRNA expression patterns of these cell types are indistinguishable with the exception of a few transcripts and miRNAs encoded on chromosome 12qF1. Specifically, maternally expressed imprinted genes in the Dlk1-Dio3 cluster including Gtl2, Rian and Mirg as well as a larger number of miRNAs encoded within this region were aberrantly silenced in the majority of iPSC clones, irrespective of their cell type of origin. Consistent with a developmental role of the Dlk1-Dio3 gene cluster, iPSC clones with repressed Gtl2 contributed poorly to chimeras and failed to support the development of entirely iPSC-derived animals (“all-iPSC mice”). In contrast, iPSC clones with normal expression levels of these genes contributed to high-grade chimeras and generated viable all-iPSC mice. Importantly, treatment of an iPSC clone that had silenced Dlk1-Dio3 and failed to give rise to all-iPSC animals with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of exclusively iPSC-derived mice. Thus, the expression state of a single imprinted gene cluster distinguishes most murine iPSCs from ESCs and allows for the prospective identification of iPSC clones that have the full development potential of ESCs.

The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5'-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (

The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK1epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.

Epithelial-to-mesenchymal transition (EMT) has been implicated in models of tumor cell migration, invasion, and metastasis. In a search for candidate therapeutic targets to reverse this process, nontumorigenic MCF10A breast epithelial cells were infected with an arrayed lentiviral kinome shRNA library and screened for either suppression or enhancement of a 26-gene EMT RNA signature. No individual kinase gene knockdown was sufficient to induce EMT. In contrast, grouped epithelial markers were induced by knockdown of multiple kinases, including mitogen activated protein kinase 7 (MAPK7). In breast cancer cells, suppression of MAPK7 increased E-cadherin (CDH1) expression and inhibited cell migration. In an orthotopic mouse model, MAPK7 suppression reduced the generation of circulating tumor cells and the appearance of lung metastases. Together, these observations raise the possibility that targeting kinases that maintain mesenchymal cell properties in cancer cells, such as MAPK7, may lessen tumor invasiveness. Implications: Suppression of MAPK7 induces epithelial markers, reduces generation of circulating tumor cells and appearance of lung metastases. Mol Cancer Res; 13(5); 934–43. ©2015 AACR.

Endometrial cancer is the leading gynecologic cancer in women in the United States with 52,630 women predicted to be diagnosed with the disease in 2014. The objective of this study was to determine if progesterone (P4) receptor membrane component 1 (PGRMC1) influenced endometrial cancer cell viability in response to chemotherapy in vitro and in vivo. A lentiviral-based shRNA knockdown approach was used to generate stable PGRMC1-intact and PGRMC1-deplete Ishikawa endometrial cancer cell lines that also lacked expression of the classical progesterone receptor (PGR). Progesterone treatment inhibited mitosis of PGRMC1-intact, but not PGRMC1-deplete cells, suggesting that PGRMC1 mediates the anti-mitotic actions of P4. To test the hypothesis that PGRMC1 attenuates chemotherapy-induced apoptosis, PGRMC1-intact and PGRMC1-deplete cells were treated in vitro with vehicle, P4 (1 mu M), doxorubicin (Dox, 2 mu g/ml), or P4 + Dox for 48 h. Doxorubicin treatment of PGRMC1-intact cells resulted in a significant increase in cell death; however, co-treatment with P4 significantly attenuated Dox-induced cell death. This response to P-4 was lost in PGRMC1-deplete cells. To extend these observations in vivo, a xenograft model was employed where PGRMC1-intact and PGRMC1-deplete endometrial tumors were generated following subcutaneous and intraperitoneal inoculation of immunocompromised NOD/SCID and nude mice, respectively. Tumors derived from PGRMC1-deplete cells grew slower than tumors from PGRMC1-intact cells. Mice harboring endometrial tumors were then given three treatments of vehicle (1:1 cremophor EL: ethanol + 0.9% saline) or chemotherapy [Paditaxel (15 mg/kg, i.p.) followed after an interval of 30 minutes by CARBOplatin (50 mg/kg)] at five day intervals. In response to chemotherapy, tumor volume decreased approximately four-fold more in PGRMC1-deplete tumors when compared with PGRMC1-intact control tumors, suggesting that PGRMC1 promotes tumor cell viability during chemotherapeutic stress. In sum, these in Vitro and in vivo findings demonstrate that PGRMC1 plays a prominent role in the growth and chemoresistance of human endometrial tumors. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

Tumors can become lethal when they progress from preinvasive lesions to invasive carcinomas. Here, we identify candidate tumor progression genes using gene array analysis of preinvasive and invasive tumors from mice, which were then evaluated in human cancers. Immediate early response protein IEX-1, small stress protein 1 (HSPB8), and tumor necrosis factor-associated factor–interacting protein mRNAs displayed higher expression levels in invasive lesions than in preinvasive lesions using samples obtained by laser capture microdissection (LCM) from transgenic erbB2, ras, and cyclin D1 mice. LCM-isolated tissues from patient-matched normal, ductal carcinoma in situ, and invasive ductal carcinoma revealed similar increased expression in invasive human cancers compared with preinvasive and normal samples. These genes induced anchorage independence, increased cell proliferation, and protected against apoptosis, singly or in collaboration with erbB2. Surprisingly, they were all up-regulated by 17β-estradiol and cyclin D1, and cyclin D1 overexpression increased p300/CBP binding to their promoters, supporting the model that cyclin D1-estrogen receptor (ER) coactivator interactions may be important to its role in ER-positive breast cancer. Additionally, an irreversible dual kinase inhibitor of ErbB signaling inhibited expression of the same genes. The up-regulation of genes contributing to increased invasiveness of ER-positive cancers offers a novel explanation for the contribution of cyclin D1 to a worse prognosis in ER-positive cancers. As targets of estrogen, cyclin D1, and erbB2 signaling, these candidates offer insights into the nature of the second events involved in breast cancer progression, regulatory events contributing to invasion, and potential targets of combined inhibition of hormone and growth factor signaling pathways. (Cancer Res 2006; 66(24): 11649-58)

Expression microarray analysis identified CITED1 among a group of genes specifically upregulated in the pubertal mouse mammary gland. At puberty, CITED1 localizes to the luminal epithelial cell population of the mammary ducts and the body cells of the terminal end buds. Generation of CITED1 gene knockout mice showed that homozygous null mutants exhibit retarded mammary ductal growth at puberty and, in addition, dilated ductal structures with a lack of spatial restriction of the subtending branches. Analysis of CITED1 homozygous null and heterozygous null mammary gland gene expression using microarrays suggested that the mammary-specific phenotype seen in the homozygous null females is due to a disturbance in the transcription of a number of key mediators of pubertal ductal morphogenesis. These include estrogen and TGFbeta responsive genes, such as the EGFR/ErbB2 ligand, amphiregulin, whose transcription we suggest is directly or indirectly regulated by CITED1.

Chicken CITED3 (cCITED3) is a novel gene, which is expressed in the pre-somitic mesoderm, the mesonephric tubules, the Wolffian ducts and collecting tubules of the developing urogenital system and in the cranial sensory ganglia. Sequence analysis revealed that cCITED3 encodes a protein which contains two conserved domains that have been described for members of the CITED family.

Transforming growth factor-beta (TGF-beta) family members signal through a unique set of intracellular proteins called Smads. Smad4, previously identified as the tumor suppressor DPC4, is functionally distinct among the Smad family, and is required for the assembly and transcriptional activation of diverse, Smad-DNA complexes. We previously identified a 48-amino acid proline-rich regulatory element within the middle linker domain of this molecule, the Smad4 activation domain (SAD), which is essential for mediating these signaling activities. We now characterize the functional activity of the SAD. Mutants lacking the SAD are still able to form complexes with other Smad family members and associated transcription factors, but cannot activate transcription in these complexes. Furthermore, the SAD itself is able to activate transcription in heterologous reporter assays, identifying it as a proline-rich transcriptional activation domain, and indicating that the SAD is both necessary and sufficient to activate Smad-dependent transcriptional responses. We show that transcriptional activation by the SAD is p300-dependent, and demonstrate that this activity is associated with a physical interaction of the SAD with the amino terminus of p300. These data identify a novel function of the middle linker region of Smad4, and define the role of the SAD as an important locus determining the transcriptional activation of the Smad complex.

The C-terminal cytoplasmic tail of chemokine receptors is important for their internalization upon ligand binding. We generated several deletion mutants of the C-terminal cytoplasmic tail of CXCR-4, a co-receptor for T cell line tropic strains of human immunodeficiency virus type 1 (HIV-1), to know whether or not co-receptor internalization is associated with HIV-1 entry. Our data showed that the removal of C-terminal 15 amino acid residues of the cytoplasmic tail from CXCR-4 completely abolished its internalization, but did not affect the co-receptor activity at all. Co-receptor activity was fully retained even when all 45 amino acid residues in the C-terminal cytoplasmic tail had been deleted. These data indicated that no cytoplasmic tail nor internalization of CXCR-4 is required for its co-receptor activity for HIV-1 entry.

MSG1 is a recently described melanocyte-specific nuclear protein whose biochemical function is unknown [Shioda et al. (1996) Proc. Natl. Acad. Sci. USA 93, 12298–12303]. Two human cDNA sequences found in the EST (expressed sequence tag) database were predicted to encode a small peptide (45 aa) that showed 69% identity to the C-terminal sequence of MSG1, suggesting the existence of a novel MSG1-related protein. Based on these EST sequences, we isolated a novel gene, MRG1 ( MSG1 -Related Gene 1), by the 5′-RACE (rapid amplification of cDNA ends) technique. The MRG1 mRNA transcript is expressed widely and encodes a nuclear protein that share two highly conserved domains, CR1 (14 aa) and CR2 (approx. 50 aa), with MSG1. The CR2 domain is significantly acidic and activates transcription in yeast cells. The full-length MSG1 and MRG1 fused to GAL4 DNA-binding domain activates transcription in mammalian cells, and this is dependent on the presence of the CR2 domain. These results suggest that MRG1 and MSG1 may function as transcription activators.

International audience; Exposure of rodent fetuses to low doses of the endocrine disruptor bisphenol A (BPA) causes subtle morphological changes in the prenatal mammary gland and results in pre-cancerous and cancerous lesions during adulthood. To examine whether the BPA-induced morphological alterations of the fetal mouse mammary glands are a) associated with changes in mRNA expression reflecting estrogenic actions and/or b) dependent on the estrogen receptor α (ERα), we compared the transcriptomal effects of BPA and the steroidal estrogen ethinylestradiol (EE2) on fetal mammary tissues of wild type and ERα knock-out mice. Mammary glands from fetuses of dams exposed to vehicle, 250 ng BPA/kg BW/d or 10 ng EE2/kg BW/d from embryonic day (E) 8 were harvested at E19. Transcriptomal analyses on the ductal epithelium and periductal stroma revealed altered expression of genes involved in the focal adhesion and adipogenesis pathways in the BPA-exposed stroma while genes regulating the apoptosis pathway changed their expression in the BPA-exposed epithelium. These changes in gene expression correlated with previously reported histological changes in matrix organization, adipogenesis, and lumen formation resulting in enhanced maturation of the fat-pad and delayed lumen formation in the epithelium of BPA-exposed fetal mammary glands. Overall similarities in the transcriptomal effects of BPA and EE2 were more pronounced in the epithelium, than in the stroma. In addition, the effects of BPA and EE2 on the expression of various genes involved in mammary stromal-epithelial interactions were suppressed in the absence of ERα. These observations support a model whereby BPA and EE2 act directly on the stroma, which expresses ERα, ERβ and GPR30 in fetal mammary glands, and that the stroma, in turn, affects gene expression in the epithelium, where ERα and ERβ are below the level of detection at this stage of development.

Sex of birds is genetically determined through inheritance of the ZW sex chromosomes (ZZ males and ZW females). Although the mechanisms of avian sex determination remains unknown, the genetic sex is experimentally reversible by in ovo exposure to exogenous estrogens (ZZ-male feminization) or aromatase inhibitors (ZW-female masculinization). Expression of various testis- and ovary-specific marker genes during the normal and reversed gonadal sex differentiation in chicken embryos has been extensively studied, but the roles of sex-specific epigenetic marks in sex differentiation are unknown. In this study, we show that a 170-nt region in the promoter of CYP19A1/aromatase, a key gene required for ovarian estrogen biosynthesis and feminization of chicken embryonic gonads, contains highly quantitative, nucleotide base-level epigenetic marks that reflect phenotypic gonadal sex differentiation. We developed a protocol to feminize ZZ-male chicken embryonic gonads in a highly quantitative manner by direct injection of emulsified ethynylestradiol into yolk at various developmental stages. Taking advantage of this experimental sex reversal model, we show that the epigenetic sex marks in the CYP19A1/aromatase promoter involving DNA methylation and histone lysine methylation are feminized significantly but only partially in sex-converted gonads even when morphological and transcriptional marks of sex differentiation show complete feminization, being indistinguishable from gonads of normal ZW females. Our study suggests that the epigenetic sex of chicken embryonic gonads is more stable than the morphologically or transcriptionally characterized sex differentiation, suggesting the importance of the nucleotide base-level epigenetic sex in gonadal sex differentiation.

Huntington's disease (HD) involves marked early neurodegeneration in the striatum, whereas the cerebellum is relatively spared despite the ubiquitous expression of full-length mutant huntingtin, implying that inherent tissue-specific differences determine susceptibility to the HD CAG mutation. To understand this tissue specificity, we compared early mutant huntingtin-induced gene expression changes in striatum to those in cerebellum in young Hdh CAG knock-in mice, prior to onset of evident pathological alterations. Endogenous levels of full-length mutant huntingtin caused qualitatively similar, but quantitatively different gene expression changes in the two brain regions. Importantly, the quantitatively different responses in the striatum and cerebellum in mutant mice were well accounted for by the intrinsic molecular differences in gene expression between the striatum and cerebellum in wild-type animals. Tissue-specific gene expression changes in response to the HD mutation, therefore, appear to reflect the different inherent capacities of these tissues to buffer qualitatively similar effects of mutant huntingtin. These findings highlight a role for intrinsic quantitative tissue differences in contributing to HD pathogenesis, and likely to other neurodegenerative disorders exhibiting tissue-specificity, thereby guiding the search for effective therapeutic interventions.

We present a protocol for in vitro production of recombinant lentiviruses using COS-1 African green monkey kidney epithelial cells and HEK293T human embryonic kidney epithelial cells as packaging cells. COS-1 and HEK293T express SV40 large T antigen, amplifying transfected circular plasmids harboring SV40 replication origin. Support protocols for evaluation of transfection efficiency by in situ β-galactosidase enzyme activity assay and titer of infection-capable virions are also provided. Advantages of using COS-1 packaging cells over the standard HEK293T cells for contamination-sensitive applications or automated processing are discussed. Curr. Protoc. Cell Biol. 50:26.7.1-26.7.15. © 2011 by John Wiley & Sons, Inc. Keywords: lentivirus; COS-1; packaging; HEK293T

The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD+-dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in diet-induced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBP-dependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis.

To determine whether progesterone receptor membrane component 1 (PGRMC1) regulates the development and cisplatin (CDDP)-sensitivity of human ovarian tumors, PGRMC1 was depleted from a human ovarian cancer cell line, dsRed-SKOV-3 cells, using a short hairpin RNA knockdown approach. Compared with parental dsRed-SKOV-3 cells, the PGRMC1-deplete cells grew slower in vitro and did not show progesterone’s (P4) antiapoptotic effect. In fact, P4 induced apoptosis in PGRMC1-deplete cells in a dose-dependent manner. When transplanted into the peritoneum of athymic nude mice, parental dsRed-SKOV-3 cells developed numerous tumors, which were classified as either typical or oxyphilic clear cell tumors. CDDP increased the percentage of apoptotic nuclei in typical clear cell tumors and P4 attenuated CDDP-induced apoptosis. In contrast, the percentage of apoptotic nuclei in oxyphilic clear cell tumors was low (≤1%) and was not significantly affected by CDDP and/or P4. Compared with tumors derived from parental dsRed SKOV-3 cells, PGRMC1-deplete tumors: 1) developed in fewer mice, 2) formed less frequently, 3) appeared smaller, and 4) resulted in fewer oxyphilic clear cell tumors. These PGRMC1-deplete tumors were not responsive to CDDP’s apoptotic effects. The failure to respond to CDDP could be due to their poorly developed microvasculature system as judged by percentage of CD31-stained endothelial cells and/or their increased expression of ATP-binding cassette transporters, which are involved in drug resistance. Taken together, these findings indicate that PGRMC1 plays an essential role in the development and CDDP sensitivity of human ovarian tumors.

The HEK293T human embryonic kidney cells have been used widely as a packaging host for transfection-based production of recombinant lentiviruses. The present study describes advantages of using COS-1 African green monkey kidney cells versus HEK293T cells as a packaging host for small-volume production of high-quality recombinant lentiviruses. The particle performance index, defined as the ratio of infection-competent viral particles to the total number of particles, was three- to four-fold greater in transfection supernatants generated using COS-1 cells than that generated using HEK293T cells. Adhesion of HEK293T cells to the cell culture-treated plastic surface was weak, causing significant HEK293T cell contamination in the transfection supernatants produced by laboratory automation using the 96-well cell culture plates. In contrast, COS-1 cells adhered strongly to the plastic surface, and cell contamination was not detected in the transfection supernatants. These results suggest that COS-1 cells may be a useful alternative packaging host for use for automated generation of large numbers of high-quality lentivirus reagents, particularly because they eliminate the need for additional purification steps to remove viral particles from cell culture supernatant.

PTH regulates osteoblastic differentiation and activity and exerts different overall skeletal effects in vivo, depending on the schedule and dose of administration. In clonal Wt9 murine osteoblastic cells, mRNA and protein levels of CITED1 transcriptional coactivator were strongly up-regulated by human (h) PTH(1-34). Stimulation of CITED1 mRNA by PTH was transient, peaking at 4 h, concentration dependent, and blocked by actinomycin D but not cycloheximide. The stimulation was mimicked by forskolin, phorbol ester, and the cAMP-selective PTH analog [G(1),R(19)] hPTH (1-28) and inhibited completely by the protein kinase A inhibitor, H89 and partially by phorbol ester-induced protein kinase C depletion. Increased CITED1 expression was not maintained during persistent (24 h) PTH exposure. Cultured primary calvarial osteoblasts from neonatal homozygous or hemizygous CITED1-knockout (KO) mice achieved 2-fold greater mineralized nodule formation in comparison with wild type (WT) osteoblasts. This effect was blocked by restoration of CITED1 expression via adenoviral gene transfer. Intermittent administration of hPTH(1-34) (10 nm, for 4 h every 48 h) for 3-6 wk increased mineralization up to 2-fold over basal levels in both WT and CITED1 KO mouse calvarial cell cultures. Whereas the cAMP-selective [G(1),R(19)]hPTH(1-28) analog [at 100 nm, equivalent to 10 nm hPTH(1-34)] did not stimulate mineralization in WT cultures, it was twice as effective as hPTH(1-34) in CITED1 KO cultures. Thus, CITED1 negatively regulates osteoblastic differentiation in vitro and inhibits the cAMP-dependent stimulation of differentiation by intermittent PTH. We conclude also that PTH receptor signaling pathways independent of cAMP restrain osteoblastic differentiation, an effect normally obscured in the presence of CITED1 but revealed in its absence.

Early kidney development in mammals is characterized by reciprocal tissue interaction between the ureteric bud and the metanephric mesenchyme. The coordinated response to this interaction is regulated largely at the transcriptional level. Here, we investigate the expression and function of Cited1, a transcriptional cofactor that we have previously implicated in kidney development. We show that Cited1 is expressed in the metanephric mesenchyme after invasion of the ureteric bud and that its expression is limited to the cap mesenchyme, those cells that aggregate most tightly around the tip of the ureteric bud and give rise to nephronic epithelium of the adult kidney. Cited1 is down-regulated during the initial stages of epithelial conversion and is not expressed past this progenitor stage. Despite its unique expression pattern, deletion of Cited1 does not disrupt kidney development. We hypothesized that this finding was due to functional redundancy with other members of this gene family. The expression pattern of Cited2 overlaps that of Cited1, but its deletion, either alone or in combination with Cited1, does not disrupt epithelial differentiation of the metanephric mesenchyme. From these studies, we conclude that Cited1 and 2 are dynamically expressed during kidney development, but are not required for nephrogenesis. Developmental Dynamics 236:2321–2330, 2007. © 2007 Wiley-Liss, Inc.

Transcriptomal changes in the uterine endometrium induced in response to the implanting embryo remain largely unknown. In this study, using Affymetrix mRNA expression microarray analysis, we identified genes differentially expressed in the murine endometrium in the presence or absence of the embryo. Compared with the pseudopregnant deciduoma induced by a mechanical stimulus in the absence of an embryo, approximately 1500 genes (753 up-regulated, 686 down-regulated; P < 0.05) were differentially expressed by at least 1.2-fold in the uterine decidua of pregnancy. Most of these genes fall into five major biological categories that include binding (45%), catalysis (24%), signal transduction (10%), transcriptional regulators (5%), and transporters (5%). This strong, embryo-induced transcriptomal impact represented approximately 10% of the total number of genes expressed in the decidualizing endometrium. Validation studies with mRNA and protein confirmed existence of the phylogenetically conserved, embryo-regulated genes involved in the following: 1) hemostasis and inflammation; 2) interferon signaling; 3) tissue growth and remodeling; and 4) natural killer cell function. Interestingly, whereas expression of many growth factors and their cognate receptors were not different between the decidual and deciduomal endometria, a number of proteases that degrade growth factors were selectively up-regulated in the decidual tissue. Increased expression of IGF and activin A neutralizing factors (i.e. HtrA1 and Fstl3) correlated with reduced stromal cell mitosis, tissue growth, and mitogenic signaling in the decidual endometrium. These results support the hypothesis that the implanting murine embryo takes a proactive role in modulating endometrial gene expression and development during early gestation.

Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.

In a screen to identify factors that regulate the conversion of mesenchyme to epithelium during the early stages of nephrogenesis, it was found that the Smad4-interacting transcriptional cofactor, Cited1, is expressed in the condensed cap mesenchyme surrounding the tip of the ureteric bud (UB), is downregulated after differentiation into epithelia, and has the capacity to block UB branching and epithelial morphogenesis in cultured metanephroi. Cited1 represses Wnt/beta-catenin but activates Smad4-dependent transcription involved in TGF-beta and Bmp signaling. By modifying these pathways, Cited1 may coordinate cellular differentiation and survival signals that regulate nephronic patterning in the metanephros.

DNA microarray technology is becoming increasingly popular in the field of toxicological research. Information on the global gene expression profile may provide clues to understanding biological actions of toxic substances at the molecular, cellular, tissue, and individual animal levels. This review focuses on the practical aspects of DNA microarray experiments and their applications to relatively small sizes of research projects in toxicology. First, different types of DNA microarrays and their characters are reviewed. Then, two fundamental concepts of DNA microarray data analysis--supervised and unsupervised methods--and their applications to toxicological research projects are discussed. Typical pitfalls of DNA microarray experiments are examined. Finally, strategies of experimental design and data analysis of minimum scale DNA microarray project are provided.

Cited1 is a transcriptional cofactor that interacts with Smad4, estrogen receptors alpha and beta, TFAP2, and CBP/p300. It is expressed in a restricted manner in the embryo as well as in extraembryonic tissues during embryonic development. In this study we report the engineering of a loss-of-function Cited1 mutation in the mouse. Cited1 null mutants show growth restriction at 18.5 days postcoitum, and most of them die shortly after birth. Half the heterozygous females, i.e., those that carry a paternally inherited wild-type Cited1 allele, are similarly affected. Cited1 is normally expressed in trophectoderm-derived cells of the placenta; however, in these heterozygous females, Cited1 is not expressed in these cells. This occurs because Cited1 is located on the X chromosome, and thus the wild-type Cited1 allele is not expressed because the paternal X chromosome is preferentially inactivated. Loss of Cited1 resulted in abnormal placental development. In mutants, the spongiotrophoblast layer is irregular in shape and enlarged while the labyrinthine layer is reduced in size. In addition, the blood spaces within the labyrinthine layer are disrupted; the maternal sinusoids are considerably larger in mutants, leading to a reduction in the surface area available for nutrient exchange. We conclude that Cited1 is required in trophoblasts for normal placental development and subsequently for embryo viability.

The CITED family proteins bind to CBP/p300 transcriptional integrators through their conserved C-terminal acidic domain and function as coactivators. The 21-kDa mouse Cited4 protein, a novel member of the CITED family, interacted with CBP/p300 as well as isoforms of the TFAP2 transcription factor, coactivating TFAP2-dependent transcription. The cited4 gene consisted of only a single exon located on chromosome 4 at 56.5-56.8 cM flanked by marker genes kcnq4 and scml1. Expression of Cited4 protein was strong and selective in embryonic hematopoietic tissues and endothelial cells. In adult animals, Cited4 showed strong milk cycle-dependent induction in pregnant and lactating mammary epithelial cells. Strong induction of Cited4 expression was also observed in SCp2 mouse mammary epithelial cells during their prolactin-dependent in vitro differentiation. These results implied possible roles for Cited4 in regulation of gene expression during development and differentiation of blood cells, endothelial cells, and mammary epithelial cells.