Identification Of Estrogen-responsive Genes Research Articles

Identification of estrogen responsive genes using esophageal squamous cell carcinoma (ESCC) as a model

BackgroundEstrogen therapy has positively impact the treatment of several cancers, such as prostate, lung and breast cancers. Moreover, several groups have reported the importance of estrogen induced gene regulation in esophageal cancer (EC). This suggests that there could be a potential for estrogen therapy for EC. The efficient design of estrogen therapies requires as complete as possible list of genes responsive to estrogen. Our study develops a systems biology methodology using esophageal squamous cell carcinoma (ESCC) as a model to identify estrogen responsive genes. These genes, on the other hand, could be affected by estrogen therapy in ESCC.ResultsBased on different sources of information we identified 418 genes implicated in ESCC. Putative estrogen responsive elements (EREs) mapped to the promoter region of the ESCC genes were used to initially identify candidate estrogen responsive genes. EREs mapped to the promoter sequence of 30.62% (128/418) of ESCC genes of which 43.75% (56/128) are known to be estrogen responsive, while 56.25% (72/128) are new candidate estrogen responsive genes. EREs did not map to 290 ESCC genes. Of these 290 genes, 50.34% (146/290) are known to be estrogen responsive. By analyzing transcription factor binding sites (TFBSs) in the promoters of the 202 (56+146) known estrogen responsive ESCC genes under study, we found that their regulatory potential may be characterized by 44 significantly over-represented co-localized TFBSs (cTFBSs). We were able to map these cTFBSs to promoters of 32 of the 72 new candidate estrogen responsive ESCC genes, thereby increasing confidence that these 32 ESCC genes are responsive to estrogen since their promoters contain both: a/mapped EREs, and b/at least four cTFBSs characteristic of ESCC genes that are responsive to estrogen. Recent publications confirm that 47% (15/32) of these 32 predicted genes are indeed responsive to estrogen.ConclusionTo the best of our knowledge our study is the first to use a cancer disease model as the framework to identify hormone responsive genes. Although we used ESCC as the disease model and estrogen as the hormone, the methodology can be extended analogously to other diseases as the model and other hormones. We believe that our results provide useful information for those interested in genes responsive to hormones and in the design of hormone-based therapies.

Identification of estrogen-responsive genes in the parenchyma and fat pad of the bovine mammary gland by microarray analysis

Identification of estrogen-responsive genes is an essential step toward understanding mechanisms of estrogen action during mammary gland development. To identify these genes, 16 prepubertal heifers were used in a 2 x 2 factorial experiment, with ovarian status (intact or ovariectomized) as the first factor and estrogen treatment as the second (control or estradiol). Heifers were ovariectomized at approximately 4.5 mo of age, and estrogen treatments were initiated 1 mo later. After 3 days of treatment, gene expression was analyzed in the parenchyma and fat pad of the bovine mammary gland using a high-density oligonucleotide microarray. Oligonucelotide probes represented 40,808 tentative consensus sequences from TIGR Bos taurus Gene Index and 4,575 singleton expressed sequence tags derived from libraries of pooled mammary gland and gut tissues. Microarray data were analyzed by use of the SAS mixed procedure, with an experiment-wide permutation-based significance level of P < 0.1. Considerable differences in basal gene expression were noted between mammary parenchyma and fat pad. A total of 124 estrogen-responsive genes were identified, with most responding only in the parenchyma or the fat pad. The majority of genes identified were not previously reported to be estrogen responsive. These undoubtedly include genes that are regulated indirectly but also include known estrogen-targeted genes and novel genes with potential estrogen-responsive elements in their promoter regions. The distinctive expression patterns regulated by estrogen in parenchyma and fat pad shed light on the need for both tissues to obtain normal mammary development.

Identification of Estrogen-responsive Genes Using a Genome-wide Analysis of Promoter Elements for Transcription Factor Binding Sites

We developed a pipeline to identify novel genes regulated by the steroid hormone-dependent transcription factor, estrogen receptor, through a systematic analysis of upstream regions of all human and mouse genes. We built a data base of putative promoter regions for 23,077 human and 19,984 mouse transcripts from National Center for Biotechnology Information annotation and 8793 human and 6785 mouse promoters from the Data Base of Transcriptional Start Sites. We used this data base of putative promoters to identify potential targets of estrogen receptor by identifying estrogen response elements (EREs) in their promoters. Our program correctly identified EREs in genes known to be regulated by estrogen in addition to several new genes whose putative promoters contained EREs. We validated six genes (KIAA1243, NRIP1, MADH9, NME3, TPD52L, and ABCG2) to be estrogen-responsive in MCF7 cells using reverse transcription PCR. To allow for extensibility of our program in identifying targets of other transcription factors, we have built a Web interface to access our data base and programs. Our Web-based program for Promoter Analysis of Genome, PAGen@UIC, allows a user to identify putative target genes for vertebrate transcription factors through the analysis of their upstream sequences. The interface allows the user to search the human and mouse promoter data bases for potential target genes containing one or more listed transcription factor binding sites (TFBSs) in their upstream elements, using either regular expression-based consensus or position weight matrices. The data base can also be searched for promoters harboring user-defined TFBSs given as a consensus or a position weight matrix. Furthermore, the user can retrieve putative promoter sequences for any given gene together with identified TFBSs located on its promoter. Orthologous promoters are also analyzed to determine conserved elements.

Identification of Estrogen-responsive Genes in p53+/- Knockout and Isogenic Wild-type Parent Strain Mice by CDNA Macroarray Analysis

Identification of Estrogen-responsive Genes in p53+/- Knockout and Isogenic Wild-type Parent Strain Mice by CDNA Macroarray Analysis

Identification of estrogen-responsive genes in the GH3 cell line by cDNA microarray analysis

To identify estrogen-responsive genes in somatolactotrophic cells of the pituitary gland, a rat pituitary cell line GH3 was subjected to cDNA microarray analysis. GH3 cells respond to estrogen by growth as well as prolactin synthesis. RNAs extracted from GH3 cells treated with 17β-estradiol (E2) at 10 −9 M for 24 h were compared with the control samples. The effect of an antiestrogen ICI182780 was also examined. The array analysis indicated 26 genes to be up-regulated and only seven genes down-regulated by E2. Fourteen genes were further examined by real-time RT-PCR quantification and 10 were confirmed to be regulated by the hormone in a dose-dependent manner. Expression and regulation of these genes were then examined in the anterior pituitary glands of female F344 rats ovariectomized and/or treated with E2 and 8 out of 10 were again found to be up-regulated. Interestingly, two of the most estrogen-responsive genes in GH3 cells were strongly dependent on E2 in vivo. #1 was identified as calbindin-D9k mRNA, with 80- and 118-fold induction over the ovariectomized controls at 3 and 24 h, respectively, after E2 administration. #2 was found to be parvalbumin mRNA, with 30-fold increase at 24 h. Third was c- myc mRNA, with 4.5 times induction at 24 h. The levels were maintained after one month of chronic E2 treatment. Identification of these estrogen-responsive genes should contribute to understating of estrogen actions in the pituitary gland.

Identification of Estrogen-Responsive Genes by Complementary Deoxyribonucleic Acid Microarray and Characterization of a Novel Early Estrogen-Induced Gene:EEIG1

Estrogen receptors (ERs) are nuclear transcription factors that regulate gene expression in response to estrogen and estrogen-like compounds. Identification of estrogen-regulated genes in target cells is an essential step toward understanding the molecular mechanisms of estrogen action. Using cDNA microarray examinations, 19 genes were identified as induced by 17 beta-estradiol in MCF-7 cells, 10 of which have been reported previously to be estrogen responsive or to be linked with ER status. Five known estrogen-regulated genes, E2IG4, IGFBP4, SLC2A1, XBP1 and B4GALT1, and AFG3L1, responded quickly to estrogen treatment. A novel estrogen-responsive gene was identified and named EEIG1for early estrogen-induced gene 1. EEIG1 was clearly induced by 17 beta-estradiol within 2 h of treatment, and was widely responsive to a group of estrogenic compounds including natural and synthetic estrogens and estrogenic environmental compounds. EEIG1 was expressed in ER-positive but not in ER-negative breast cancer cell lines. EEIG1 expression was repressed by antiestrogens 4-OH-tamoxifen and ICI 182,780 but not by protein synthesis inhibitors cycloheximide and puromycin. These results provide evidence that some estrogenic compounds differentially enhance the transcription of estrogen-regulated genes and suggest a role for EEIG1 in estrogen action.

Identification of estrogen-responsive genes in chick liver.

Identification of targets of estrogen is an important step in understanding the mechanisms of estrogen action. A two-step strategy was developed to identify estrogen-responsive genes (ERGs) in chick liver. Initially, differential-display, reverse transcription polymerase chain reaction (DDRT-PCR) was introduced to isolate ERGs. Isolated ERGs were then analyzed using Northern blot hybridization. A number of differentially expressed cDNA fragments were isolated following estrogen exposure. Four cDNA fragments that displayed dramatic change after estrogen administration were identified as liver adenylosuccinate lyase (ADL), phenobarbital-inducible cytochrome P-450 (CYP450), ovoinhibitor, and glutathione-dependent prostaglandin D2 synthase (PGDS). Time sequence analysis showed that estrogen-induced alteration occurred as early as 0.5 h and peaked between 1 and 4 h after estrogen exposure. Nuclear runoff assay indicated that estrogen significantly increased the transcription rate of these genes. To determine whether the observed alteration was due to the direct effect of estrogen, protein synthesis was inhibited by cycloheximide (CHX) during stimulation by estradiol, Estrogen-mediated upregulation of PGDS was completely abolished by a concurrent treatment with CHX, suggesting that its activation requires the participation of some newly synthesized factor(s). In contrast, CHX did not affect the expression of other genes, indicating the alteration is a direct response to estrogen. In conclusion, ADL, CYP450, ovoinhibitor, and PGDS represent the novel targets of estrogen, which regulates the transcriptional activity of these genes.

Estrogen modulates the expression of L-arginine:glycine amidinotransferase in chick liver.

Identification of estrogen-responsive genes is important to understand the molecular mechanisms of estrogen action. Suppression subtractive hybridization was employed to screen estrogen-responsive genes in chick liver. A single injection of estrogen into 6-week-old chick induced up-regulation of several known genes encoded for yolk proteins, such as Vitellogenin I and II and very low density lipoprotein II (apo-VLDL II). One novel sequence displayed a dramatic change (3-fold increase) in response to estrogen treatment. This cDNA fragment was extended and the resultant sequence was analyzed. Translated amino acid sequence was 90, 88, 83 and 87% identical to the L-arginine:glycine amidinotransferase of pig, rat, frog and human, respectively. The sequence has a conservative catalytic site of L-arginine: glycine amidinotransferase. The expression pattern of this gene in organs is consistent with previous reports of L-arginine:glycine amidinotransferase in chick. Thus, this clone represented the chicken L-arginine:glycine amidinotransferase. It appeared that estrogen-induced alteration of arginine:glycine amidinotransferase was not dependent on protein synthesis, because concurrent administration of cycloheximide did not affect the estrogen-mediated expression pattern. This is the first study demonstrating that L-arginine:glycine amidinotransferase is a target of the estrogen receptor.

Estrogen stimulates expression of adenine nucleotide translocator ANT1 messenger RNA in female rat hearts

The identification of estrogen-responsive genes in the heart, is necessary to understand estrogen-induced changes in cardiac function. Using Delta RNA fingerprinting, we demonstrate that a single injection of estradiol benzoate (50 μg, s.c.) revealed mRNA species that were elevated, down-regulated, or were unaffected in the heart tissue of ovariectomized female rats. One of the upregulated genes was identified, by cloning and sequencing, to have 95.8% (230/240) identity with the 3′ end of the rat ant1 gene encoding the mitochondrial adenine nucleotide translocator, ANT1. Using the isolated ANT1 cDNA (280 bp) as a probe in Northern analysis, estrogen was shown to upregulate the expression of cardiac ANT1, by at least 3-fold in female rats, from as early as 1 h to as long as 24 h. In contrast, estrogen treatment had no effect on ANT1 expression in heart tissue from male rats. RNA yields were low in rat atria and no transcript was detectable by Northern analysis. Using primers specific to the known rat ANT1 gene, the estrogen upregulation of the cardiac ANT1 transcript in female rat was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR); a predicted product of 249 bp was obtained and this was stimulated by at least 3-fold upon estrogen treatment for 24 h.

Identification of Estrogen-Responsive Genes in Neuroblastoma SK-ER3 Cells

To evaluate the role of estrogen receptor in the differentiation of cells of neural origin, we developed a molecular approach aimed at the identification of estrogen target genes by mRNA differential display PCR (ddPCR) in human neuroblastoma SK-ER3 cells. More than 3000 RNAs were examined, a few of which displayed a differential regulation pattern in response to 17beta-estradiol (E2). Sequence analysis of three differentially amplified ddPCR products showed homology with the growth-associated nuclear protein prothymosin-alpha (PTMA), the Bcl2-interacting protein Nip2, and one mRNA previously described by others in fetal human brain. Two ddPCR products, referred to as P4 and P10, corresponded to new DNA sequences. Northern analysis confirmed that estrogen treatment of SK-ER3 cells resulted in the upregulation and downregulation of expression of these messages. In particular, PTMA was found to accumulate at both 1 and 17 hr after E2 treatment, whereas P10 product accumulated only at 1 hr. Conversely, P4, Nip2, and the fetal brain-related mRNAs were significantly decreased by the treatment. Further time course analysis of PTMA and Nip2 mRNAs levels indicated that the hormone exerted a marked biphasic regulatory effect on expression of both messages during the course of cell differentiation. In the present study we report for the first time the identification of a panel of estrogen target genes in neural cells that provide new insights in the molecular mechanism of action of E2 in cells of neural origin.