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V X Jin, H Sun, T T Pohar, S Liyanarachchi, S K Palaniswamy, T H-M Huang and R V Davuluri

The estrogen receptor (ER) plays an important role in several physiologic functions of both the reproductive and non-reproductive systems. Malignancies of the ER have been associated with the development of cancers, including those of the prostate and breast. Hence it has become of significant importance to characterize the transcriptional regulation of ER target genes. We have created ERTargetDB in order to integrate the previously published ER target gene information that is available in various publications and databases. This information resource provides researchers with an easy access to ER target genes and the regulatory mechanisms in the corresponding promoters. The current version contains 40 genes with experimentally verified estrogen response elements (EREs), 32 experimentally verified ERE tethering sites, 40 genes identified by the chromatin immunoprecipitation microarray, 381 genes from gene expression microarray and 2948 genes from computational prediction. ERTargetDB provides an integral information resource for direct target genes of ERs for the endocrinology research community. It should prove useful in the investigation of gene regulation and aid the development of computational tools for the prediction of ER target genes.

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R Betney and IJ McEwan

The intracellular androgen receptor (AR) is a ligand-activated transcription factor. Upon binding the steroids testosterone or dihydrotestosterone, the activated receptor translocates to the nucleus, binds to specific DNA response elements and interacts with the transcription machinery in order to regulate gene transcription. In the present study, we have described a highly conserved region (amino acids 224-258) within the AR AF-1 domain and have investigated the role of conserved bulky hydrophobic residues in gene regulation. Mutating pairs of residues (I229A/L236A; V240A/V242A; L251A/L254A) reduced transactivation activity by 25-40%. Mutating residues M244, L246 and V248 to alanines had a more dramatic affect on receptor activity, disrupting activity by at least 60%. The latter mutations also disrupted binding to the RNA polymerase-associated protein 74 subunit of the general transcription factor TFIIF. The protein conformation and stability of the mutant polypeptide in vitro was not significantly different from the wild type. None of the mutations tested disrupted binding of the AF-1 domain with the coactivator protein steroid receptor coactivator-1a. Thus we have concluded that conserved hydrophobic residues are important for receptor-dependent gene transcription and that M244, L246 and V248 are part of the binding interface for TFIIF.

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PR Holden and JD Tugwood

Peroxisome proliferators (PPs) are chemicals of industrial and pharmaceutical importance that elicit liver carcinogenesis by a non-genotoxic mechanism. One of the intriguing properties of PPs is that the pleiotropic effects of these compounds (including increased DNA synthesis and peroxisome proliferation) are seen in rats and mice only, but not humans. It is important to determine the risks to humans of environmental and therapeutic exposure to these compounds by understanding the mechanisms of non-genotoxic hepatocarcinogenesis in rodents. To understand this apparent lack of human susceptibility, attention has focused on the peroxisome proliferator-activated receptor alpha (PPARalpha), which appears to mediate the effects of PPs in rodents. It is also known to mediate the hypolipidaemic effects that fibrate drugs exert on humans with elevated plasma cholesterol and triglyceride levels. Human PPARalphas share many functional characteristics with the rodent receptors, in that they can be transcriptionally activated by PPs and regulate specific gene expression. However, one key difference is that PPARalpha is less abundant in human than in rodent liver, which has led to the suggestion that species differences result from quantitative differences in gene expression. In this review we describe the effects of PPs and what is known of the molecular mechanisms of action and species differences with respect to rodents and man. Attention will be given to differences in the amounts of PPARalpha between species as well as the 'qualitative' aspects of PPARalpha-mediated gene regulation which might also explain the activation of some genes and not of others in human liver by PPs.

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Tomoko Kakizawa, Shin-ichi Nishio, Gerard Triqueneaux, Stephanie Bertrand, Juliette Rambaud and Vincent Laudet

The orphan nuclear receptor Rev-erbα (NR1D1) plays an important role in the regulation of the circadian pacemaker and its expression has been shown to be regulated with a robust circadian rhythm in zebrafish and mammals. In addition, in zebrafish its expression has been shown to be developmentally regulated. In order to analyze the mechanisms of the zfRev-erbα gene regulation, we have isolated its 5′-upstream region. We found that two promoters control the zfRev-erbα expression. The first one (ZfP1) is characterized by a very high degree of sequence identity with the mammalian P1 promoter and contains, as the mammalian P1, a functional Rev-erbα-binding site (RevDR2). Inhibition of zfRev-erbα activity in zebrafish embryos using antisense-morpholino knockdown results in an increase of zfRev-erbα gene expression suggesting that zfRev-erbα is repressing its own transcription in vivo. In addition, we show that ROR orphan receptors also regulate in vitro and in vivo zfRev-erbα gene expression through the same RevDR2 element. In contrast, the second promoter ZfP2 is strikingly different from the mammalian P2: its sequence is not conserved between zebrafish and mammals and is not regulated by the same transcription factors. Together, these data suggest that ZfP1 is orthologous to the mammalian P1 promoter, whereas zebrafish ZfP2 has no mammalian ortholog and does not function like ZfP1 to control Rev-erbα expression.

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S W Curtis, H Shi, C Teng and K S Korach

ABSTRACT

Certain types of estrogenic compounds have been shown to have tissue-specific actions. In addition, some tissues may exhibit differential gene regulation by agonists and antagonists. Our previous studies using structurally modified estrogenic molecules had indicated differential effects on specific estrogen responses, indicating that the activity of the estrogen receptor protein can be altered depending not only upon the structure of the bound ligand but also the regulated gene itself.

The mechanism of differential induction, however, was not determined, and might involve altered binding to the estrogen response element (ERE), altered transcription, or post-transcriptional modification of gene products. Our previous studies indicated that differential induction by modified diethylstilbestrol (DES) agonists could not be accounted for by differences in ligand affinity for the estrogen receptor (ER) or differential binding of the ER to a consensus vitellogenin A2 (vit A2) ERE. To determine if this differential hormonal responsiveness was reflected at the level of transcription, we analyzed mouse uterine mRNA of several estrogen-responsive genes, including glucose-6-phosphate dehydrogenase (G6PD), ornithine decarboxylase (ODC) and lactoferrin, by Northern blot following injection with the modified agonists DES, indenestrol A (IA), indenestrol B (IB) and Z-pseudo DES (ZPD). All compounds induced the G6PD message, although IB and ZPD induced expression only transiently, while DES and IA maintained the message for 24 h. No difference in induction was seen for ODC message, which was induced equally by all the compounds. In contrast, lactoferrin, a highly estrogen-responsive gene, was induced only by DES and IA and not by the other agonists IB or ZPD, showing that the lactoferrin gene was differentially regulated by these compounds. To determine whether this difference was due to altered transcriptional activity, the mouse lactoferrin estrogen-responsive module (mERM) linked to a chloramphenicol acetyl transferase (CAT) reporter gene was tested in transfected cells. Using the mouse estrogen receptor in RL95 cells, DES and IA induced expression of CAT, but IB did not, confirming the differential response seen in vivo. To show whether this difference in transcription occurred because of altered binding to the lactoferrin ERE, which is not a perfect consensus ERE, a gel shift assay was used to examine DNA binding of ER bound to the agonists. All ligands produced equivalent binding to the lactoferrin ERE suggesting that differential regulation was not a result of altered DNA binding. Taken together, these observations indicate that the differential induction of lactoferrin by these compounds occurs via altered activation of the transcriptional components unique to lactoferrin and is likely to involve altered interaction with co-activators. Surprisingly, unlike the mouse ER, the human estrogen receptor activated and induced expression of lactoferrin estrogen-responsive module-CAT with all the compounds. Mouse ER is also known to vary from the human ER in its activity with the triphenylethylene estrogen tamoxifen, which has agonist activity with the mouse ER but mixed antagonist/agonist activity with the human ER. The data show that human and mouse estrogen receptors are activated differently by this group of stilbestrol estrogen ligands when assayed on the lactoferrin response element, which is the first description of this type of gene and species specific difference. Lactoferrin gene regulation by estrogen receptor can be used as a model to study the mechanism of differential gene activation by different estrogen agonists and antagonists using a more physiological situation than commonly used with in vitro gene reporter systems.

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LK Beitel, YA Elhaji, R Lumbroso, SS Wing, V Panet-Raymond, B Gottlieb, L Pinsky and MA Trifiro

The androgen receptor (AR) N-terminal domain plays a critical role in androgen-responsive gene regulation. A novel AR N-terminal-interacting protein (ARNIP) was isolated using the yeast two-hybrid system and its interaction with amino acids 11-172 of the normal or corresponding region of the polyglutamine-expanded human AR confirmed by glutathione S-transferase pulldown assays. ARNIP cDNAs cloned from NSC-34 (mouse neuroblastoma/spinal cord) or PC-3 (human prostate adenocarcinoma) mRNA encoded highly homologous 30 kDa (261 amino acids) cysteine-rich proteins with a RING-H2 (C3H2C3 zinc finger) domain; this motif is highly conserved in predicted ARNIP-homologous proteins from several other species. Expression of the approximately 1.7 kb ARNIP mRNA was detected in various tissues by Northern blotting, but was highest in mouse testes, kidney and several neuronal cell lines. In addition, the human ARNIP protein was found to be encoded by nine exons spanning 32 kb on chromosome 4q21. In COS-1 cells, coexpression of ARNIP and AR did not affect AR ligand-binding kinetics, nor did ARNIP act as a coactivator or corepressor in transactivation assays. However, AR N-terminal:C-terminal interaction was reduced in the presence of ARNIP. Intriguingly, ARNIP, and in particular its RING-H2 domain, functioned as a ubiquitin-protein ligase in vitro in the presence of a specific ubiquitin-conjugating enzyme, Ubc4-1. Mutation of a single cysteine residue in the ARNIP RING-H2 domain (Cys145Ala) abolished this E3 ubiquitin ligase activity. Fluorescent protein tagging studies revealed that AR-ARNIP interaction was hormone-independent in COS-1 cells, and suggest that colocalization of both AR and ARNIP to the nucleus upon androgen addition may allow ARNIP to play a role in nuclear processes. Thus, identification of a novel AR-interacting protein with ubiquitin ligase activity will stimulate further investigation into the role of ubiquitination and the ubiquitin-proteasome system in AR-mediated cellular functions.

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Ralph A Zirngibl, Janet S M Chan and Jane E Aubin

We previously demonstrated that the orphan nuclear receptor, estrogen receptor-related receptor α (ERRα) is highly expressed in osteoblasts and osteoclasts, regulates osteogenesis and expression of osteoblast-associated markers in the rat calvaria cell differentiation system, and is dysregulated in the rat ovariectomy model of postmenopausal osteoporosis. There are conflicting published data on the transcriptional regulation by ERRα of the gene for osteopontin (OPN), an extracellular matrix protein required in bone remodeling, and a potential direct target mediating ERRα effects in bone. We therefore readdressed OPN gene regulation by ERRα in both osteoblastic (rat osteosarcoma ROS17/2.8 cells) and non-osteoblastic (HeLa) cell lines using a mouse proximal 2 kb OPN promoter fragment. A minimal OPN promoter fragment spanning from −56 to +9 bp is activated in HeLa cells but repressed it in ROS17/2.8 cells. Adenine scanning mutagenesis revealed the presence of a non-canonical ERRα response element in this minimal promoter. Surprisingly, prototypical inactivating mutations in the activation function 2 (AF2) domain or a naturally occurring allelic variant of ERRα (ERRαH408) were all better activators than wild-type ERRα in HeLa cells, activities that were generally paralleled by repression in ROS17/2.8 cells. Finally, we found that the N-terminus of ERRα harbors a repressor domain that acts in a cell context-dependent manner. We conclude that OPN is an ERRα target gene whose promoter is regulated by ERRα in a cell context-dependent manner and that a predicted silencing mutation in AF2 or a more flexible helix 12 increases ERRα transcriptional activity, effects with implications for ERRα as a therapeutic target in bone.

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Seung-Chul Kim, Jae-Eon Lee, Seong Soo Kang, Hoe-Saeng Yang, Sun Suk Kim and Beum-Soo An

Oxytocin (OXT) is a peptide hormone that plays a central role in the regulation of parturition and lactation. OXT signaling is mediated by OXT receptor (OXTR), which shows species- and tissue-specific expressions and gene regulation. In the present study, we examined the synthesis of OXT and OXTR in human placenta tissue according to gestational age. A total of 48 placentas were divided into early preterm, late preterm and term groups depending on gestational age, and expression of OXT and OXTR was evaluated. First, OXT and OXTR mRNA and protein were detected in normal placenta tissue via Q-PCR, Dot-blot and Western blot assay. Both OXT and OXTR levels in normal placenta increased gradually in the late stage of pregnancy, suggesting that local OXT may play a critical role in the function of the placenta. To determine the regulatory mechanism of OXT, placental BeWo cells were administrated estrogen (E2) or progesterone (P4), and expression of OXT and OXTR was tested. The mRNA and protein levels of OXT and OXTR were upregulated by E2 but blocked by co-treatment with P4. In order to confirm the estrogen receptor (ESR)-mediated signaling, we administrated ESR antagonists together with E2 to BeWo cells. As a result, both OXT and OXTR were significantly altered by ESR1 antagonist (MPP) while moderately regulated by ESR2 antagonist (PHTPP). These results suggest that OXT and OXTR are controlled mainly by E2 in the placenta via ESR1 and thus may play physiological functions in the human placenta during the late stage of pregnancy.

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Kjersti M Aagaard-Tillery, Kevin Grove, Jacalyn Bishop, Xingrao Ke, Qi Fu, Robert McKnight and Robert H Lane

Chromatin structure is epigenetically altered via covalent modifications of histones to allow for heritable gene regulation without altering the nucleotide sequence. Multiple lines of evidence from rodents have established a role for epigenetic remodeling in regulating gene transcription in response to an altered gestational milieu. However, to date, it is unknown whether variations in the intrauterine environment in primates similarly induce changes in key determinants of hepatic chromatin structure. We hypothesized that a maternal high-fat diet would alter the epigenomic profile of the developing offspring, which would result in alterations in fetal gene expression. Age- and weight-matched adult female Japanese macaques were placed on control (13% fat) or high-fat (35% fat) breeder diets and mated annually over a 4-year interval. Fetuses in successive years were delivered near term (e130 of 167 days) and underwent necropsy with tissue harvest. Fetal histones were acid extracted for characterization of H3 modification and chromatin immunoprecipitation (ChIP) with differential display PCR; fetal RNA, DNA, and cytoplasmic and nuclear protein extracts were similarly extracted for comparison. Chronic consumption of a maternal high-fat diet results in a threefold increase in fetal liver triglycerides and histologic correlates of non-alcoholic fatty liver disease. These gross changes in the fetal liver are accompanied by a statistically significant hyperacetylation of fetal hepatic tissue at H3K14 (199.85±9.64 vs 88.8±45.4; P=0.038) with a trend towards the increased acetylation at H3K9 (140.9±38.7 vs 46.6±6.53; P=0.097) and at H3K18 (69.0±3.54 vs 58.0±4.04; P=0.096). However, epigenetic modifications on fetal hepatic H3 associated with gene repression were absent or subtle (P>0.05). Subsequent characterization of key epigenetic determinants associated with H3 acetylation marks revealed similar significant alterations in association with a high-fat maternal diet (e.g., relative fetal histone deacetylase 1 (HDAC1) gene expression 0.61±0.25; P=0.011). Consistent with our mRNA expression profile, fetal nuclear extracts from offspring of high-fat diet animals were observed to be significantly relatively deplete of HDAC1 protein (36.07±6.73 vs 83.18±7.51; P=0.006) and in vitro HDAC functional activity (0.252±0.03 vs 0.698±0.02; P<0.001). We employ these observations in ChIP differential display PCR to attempt to identify potential fetal genes whose expression is reprogramed under conditions of a high-fat maternal diet. We quantitatively confirm a minimum of a 40% alteration in the expression of several genes of interest: glutamic pyruvate transaminase (alanine aminotransferase) 2 (GPT2) (1.59±0.23-fold; P=0.08), DNAJA2 (1.36±0.21; P=0.09), and Rdh12 (1.88±0.15; P=0.01) are appreciably increased in fetal hepatic tissue from maternal caloric-dense diet animals when compared with control while Npas2, a peripheral circadian regulator, was significantly downmodulated in the offspring of high-fat diet animals (0.66±0.08; P=0.03). In this study, we show that a current significant in utero exposure (caloric-dense high-fat maternal diet) induces site-specific alterations in fetal hepatic H3 acetylation. Employing ChIP, we extend these observations to link modifications of H3 acetylation with alterations in gene-specific expression. These results suggest that a caloric-dense maternal diet leading to obesity epigenetically alters fetal chromatin structure in primates via covalent modifications of histones and hence lends a molecular basis to the fetal origins of adult disease hypothesis.

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Adrian J L Clark and Anna Spada

organism level. Journal of Molecular Endocrinology – Focus on molecular and cellular mechanisms in endocrinology, including gene regulation, cell biology and signalling. The journal considers basic and pathophysiological studies at the molecular and