Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Estrogen (diethylstilbesterol) was administered in vivo to chicks for various time periods. Chromatin was then prepared from oviduct nuclei and assayed for its capacity to support initiation of RNA chain synthesis in vitro in the presence of saturating levels of Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6). These same nuclei were also assayed by a [3H]estradiol exchange assay for their endogenous receptor content. The number of available initiation sites for RNA synthesis on chromatin was shown to correlate with the endogenous levels of nuclear estrogen receptor. A decrease in the nuclear concentration of estrogen receptor molecules and the concentration of initiation sites for RNA synthesis occurred during withdrawal of estrogen from previously stimulated chicks. Both parameters declined with a similar half-life. When estrogen was readministered to withdrawn chicks, the number of initiation sites increased 2-fold as early as 30 min and approached a maximal level (3-fold) by 1 hr. During the same period of restimulation with estrogen, the number of estrogen receptor molecules bound to nuclei increased to a maximum at 20 min and then declined at 1 hr to a steady-state level 2-fold higher than the withdrawn chicks. Simultaneous measurements of RNA chain length and RNA chain propagation rate demonstrated that parameters remained relatively constant throughout estrogen withdrawal as well as secondary stimulation. The temporal correlation between changes in the levels of nuclear-bound estrogen receptor and the number of RNA chain initiation sites on chromatin prepared from these same nuclei strongly suggested that the hormone receptor complexes act on chromatin to mediate these changes in genetic transcriptional activity.
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PMID:Effects of estrogen on gene expression in chick oviduct: nuclear receptor levels and initiation of transcription. 17 99

Estrogen administration to chicks results in an increase in the chromatin template activity of oviduct target tissue as assayed under standard in vitro assay conditions. However, the results obtained by the simple measurement of template activity may be a complicated function of the number of available RNA polymerase initiation sites, the rate of RNA chain elongation, and the rate of reinitiation. In the present study, we have measured separately the change in both the number of chain initiations as well as the rate of RNA chain propagation under conditions in which reinitiation was eliminated. Chromatin prepared from either estrogen-treated or control oviducts both supported an RNA chain elongation rate of six nucleotides per s and a chain size of approximately 700 nucleotides. Thus, both the elongation rate and size of the average product remained relatively constant following estrogen stimulation. In contrast, within 8 hours after a single injection of estrogen to unstimulated chicks, the concentration of RNA polymerase needed to saturate chromatin binding sites was increased to 150% in comparison to control values, and by 24 hours the level of polymerase bound to chromatin was twice that of the untreated control chick chromatin. With daily injections of estrogen, polymerase binding continued to rise. Coincident with the over-all increase in chromatin-bound polymerase was an increase in rifampicin-insensitive initiation sites and newly synthesized RNA chains. Unstimulated chick oviduct chromatin initiated 10,000 RNA chains/pg of DNA, while 24 hours of steroid treatment increased the number of initiated chains to 34,000 chains. These data demonstrate that the estrogen-induced increase in chromatin transcriptive activity was due to an increased number of polymerase binding and initiation sites on the chromatin template without a detectable change in the rate of RNA chain elongation.
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PMID:Effect of estrogen on gene expression in the chick oviduct. V. Changes in the number of RNA polymerase binding and initiation sites in chromatin. 109 40

Template-engaged and total RNA polymerase II molecules were quantitated in isolated nuclei at various stages of estrogen withdrawal and secondary stimulation by using [3H]amanitin titration assays. Estrogen receptors, RNA transcriptional activity, and ovalbumin mRNA were also measured, and comparisons were made between these parameters to determine whether any significant correlations exist. In isolated nuclei, the highest positive correlations existed between template-engaged RNA polymerase II, ovalbumin mRNA synthesis in vitro, and estrogen receptor concentration. Interestingly, restimulation of estrogen-withdrawn chicks results in replenishment of RNA polymerase II activity to prewithdrawal levels within 4 h; however, the recovery of the numbers of template-engaged polymerase II molecules, ovalbumin gene transcription, and nuclear receptor binding lags behind. These findings suggest that the estrogen effect on RNA polymerase activity is more rapid than the increase in template-engaged RNA polymerase II and ovalbumin-specific gene transcription. The excellent correlation that exists between nuclear estrogen receptor concentrations, template-engaged RNA polymerase II, and ovalbumin gene transcription strongly supports the hypothesis that estrogen receptors mediate RNA polymerase II binding to sequences associated with preferential transcription of the ovalbumin gene.
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PMID:Correlation in isolated nuclei of template-engaged RNA polymerase II, ovalbumin mRNA synthesis, and estrogen receptor concentrations. 398 75

Estrogen and progesterone markedly stimulate transcription of ovalbumin and conalbumin (transferrin) genes in chick oviduct as measured by hybridization of labeled RNA synthesized in isolated nuclei to immobilized plasmid DNA containing these gene sequences. Using this direct assay for specific gene transcription, we explored the basis of previous reports indicating that steroid hormones also cause changes in oviduct chromatin structure that can be detected by Escherichia coli RNA polymerase. We observed no effect of these hormones on the ability of E. coli RNA polymerase to transcribe specifically the conalbumin and ovalbumin genes 8 1/2 h after hormone administration when transcription of these genes by endogenous RNA polymerase was elevated 5- and 30-fold, respectively. Furthermore, we were unable to detect any significant effect of either of these hormones on the total number of E. coli RNA polymerase binding sites in oviduct nuclei or chromatin. In contrast, after several days of hormone administration, we detected an apparent preferential ovalbumin RNA synthesis by E. coli RNA polymerase and this effect could be transferred to unstimulated nuclei by a 0.35 M salt extract of active nuclei. However, further experiments revealed that this preferential ovalbumin RNA synthesis is an artifact produced by transcription from contaminating ovalbumin mRNA. We conclude that E. coli RNA polymerase does not recognize steroid hormone-induced changes in oviduct chromatin.
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PMID:Regulation of gene transcription by estrogen and progesterone. Lack of hormonal effects on transcription by Escherichia coli RNA polymerase. 700 Jul 63

Estrogen receptors (ER) are ligand-inducible transcription factors regulated by Ser(Thr)-O-phosphorylation. Many transcription factors and eukaryotic RNA polymerase II itself are also dynamically modified by Ser(Thr)-O-linked N-acetylglucosamine moieties (O-GlcNAc). Here we report that subpopulations of murine, bovine, and human estrogen receptors are modified by O-GlcNAc. O-GlcNAc moieties were detected on insect cell-expressed, mouse ER (mER) by probing with bovine milk galactosyltransferase, followed by structural analysis. Wheat germ agglutinin-Sepharose affinity chromatography also readily detected terminal GlcNAc residues on subpopulations of ER purified from calf uterus, from human breast cancer cells (MCF-7), or from mER produced by in vitro translation. These data suggest that greater than 10% of these populations of estrogen receptors bear O-GlcNAc. Site mapping of insect cell expressed mER localized one major site of O-GlcNAc addition to Thr-575, within a PEST region of the carboxyl-terminal F domain. Based upon their relative resistance to both hexosaminidase and to in vitro galactosylation, O-GlcNAc moieties appear to be largely buried on native mER. This dynamic saccharide modification, like phosphorylation, may play a role in modulating the dimerization, stability, or transactivation functions of estrogen receptors.
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PMID:A subpopulation of estrogen receptors are modified by O-linked N-acetylglucosamine. 899 54

Estrogen-like chemicals are unique compared to nonestrogenic xenobiotics, because in addition to their chemical properties, the estrogenic property of these compounds allows them to act like sex hormones. Whether weak or strong, the estrogenic response of a chemical, if not overcome, will add extra estrogenic burden to the system. At elevated doses, natural estrogens and environmental estrogen-like chemicals are known to produce adverse effects. The source of extra or elevated concentration of estrogen could be either endogenous or exogenous. The potential of exposure for humans and animals to environmental estrogen-like chemicals is high. Only a limited number of estrogen-like compounds, such as diethylstilbestrol (DES), bisphenol A, nonylphenol, polychlorinated biphenyls (PCBs), and dichlorodiphenyltrichloroethane (DDT), have been used to assess the biochemical and molecular changes at the cellular level. Among them, DES is the most extensively studied estrogen-like chemical, and therefore this article is focused mainly on DES-related observations. In addition to estrogenic effects, environmental estrogen-like chemicals produce multiple and multitype genetic and/or nongenetic hits. Exposure of Syrian hamsters to stilbene estrogen (DES) produces several changes in the nuclei of target organ for carcinogenesis (kidney): (1) Products of nuclear redox reactions of DES modify transcription regulating proteins and DNA; (2) transcription is inhibited; (3) tyrosine phosphorylation of nuclear proteins, including RNA polymerase II, p53, and nuclear insulin-like growth factor-1 receptor, is altered; and (4) DNA repair gene DNA polymerase beta transcripts are decreased and mutated. Exposure of Noble rats to DES also produces several changes in the mammary gland: proliferative activity is drastically altered; the cell cycle of mammary epithelial cells is perturbed; telomeric length is attenuated; etc. It appears that some other estrogenic compounds, such as bisphenol A and nonylphenol, may also follow a similar pattern of effects to DES, because we have recently shown that these compounds alter cell cycle kinetics, produce telomeric associations, and produce chromosomal aberrations. Like DES, bisphenol A after metabolic activation is capable of binding to DNA. However, it should be noted that a particular or multitype hit(s) will depend upon the nature of the environmental estrogen-like chemical. The role of individual attack leading to a particular change is not clear at this stage. Consequences of these multitypes of attack on the nuclei of cells could be (1) nuclear toxicity/cell death; (2) repair of all the hits and then acting as normal cells; or (3) sustaining most of the hits and acting as unstable cells. Proliferation of the last type of cell is expected to result in transformed cells.
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PMID:Biochemical and molecular changes at the cellular level in response to exposure to environmental estrogen-like chemicals. 901 29

Estrogen supplements are the primary pharmacologic intervention therapy to prevent and treat loss of bone mass (osteoporosis) in postmenopausal women. Furthermore, at sites of local inflammation near bone, estrogen-deficient women are significantly more susceptible to bone loss than are estrogen-sufficient women. In the present study, we investigate whether estrogen modulates osteoblast (MG-63) production of interleukin-6 (IL-6), an osteoclast recruitment and differentiation of cytokine, in the presence of the proinflammatory cytokine, IL-1beta. Using enzyme-linked immunosorbent assay (ELISA), we demonstrate that IL-1beta significantly enhances IL-6 secretion into culture supernatants in a dose-dependent and time-dependent manner. Using reverse-transcriptase polymerase chain reaction (RT-PCR) and ELISA respectively, we demonstrate further that levels of 17beta-estradiol (active metabolite of estrogen) > or = those found in serum of estrogen-sufficient women inhibit steady-state IL-6 mRNA levels as well as inhibit secretion of IL-6 into culture supernatants. One mechanism by which estrogen therapy preserves bone mass in areas of inflammation may be via inhibition of IL-1beta-stimulated obsteoblast-derived IL-6.
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PMID:Estrogen inhibits interleukin-1beta-induced interleukin-6 production by human osteoblast-like cells. 971 63

The anterior pituitary gland produces neuronal nitric oxide synthase (nNOS) and nitric oxide regulates secretion of various anterior pituitary hormones. Estrogen has many functions in anterior pituitary cells including stimulation of prolactin (PRL) cell proliferation and secretion of various anterior pituitary hormones. However, the role of estradiol-17beta (E2) in regulating pituitary nNOS expression has not been previously examined. We studied the regulation of nNOS in normal pituitaries, and neoplastic GH3 pituitary tumors in order to analyze the effects of E2 on nNOS in pituitary cells. GH3 tumors expressed higher levels of nNOS proteins compared to normal pituitaries. Estrogen downregulated nNOS mRNA and protein in both estrogen-treated pituitaries with PRL cell hyperplasia and in GH3 tumors implanted into the flank of rats treated with E2 in silastic tubing. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated three alternatively spliced nNOS transcript isoforms--nNOSa, nNOSb, and nNOSc mRNAs--with distinct 5' untranslated first exons that arose from alternative splicing to a common second exon. All three spliced isoforms were found in the normal rat pituitary, whereas nNOSa and nNOSb, but not nNOSc, were expressed in GH3 tumors implanted into Wistar-Furth rats. E2 also downregulated the nNOSa alternative mRNA transcript isoform in vivo. These results indicate that the biological activity of nNOS in the normal rat anterior pituitary and in pituitary tumors is regulated by a complex pattern of alternative splicing and that some of these mRNA isoforms as well as nNOS protein are regulated by estrogen. Our results also indicate that the levels of nNOS and the alternatively spliced nNOS transcript between normal and GH3 pituitary tumors are different.
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PMID:Estrogen downregulates neuronal nitric oxide synthase in rat anterior pituitary cells and GH3 tumors. 1070 58

Estrogen signaling occurs through at least two distinct molecular pathways: (i) direct binding of liganded estrogen receptors (ERs) to estrogen-responsive DNA elements (EREs) (the "ER/ERE pathway") and (ii) indirect recruitment of liganded ERs to activating protein-1 (AP-1)-responsive DNA elements via heterodimers of Fos and Jun (the "ER/AP-1 pathway"). We have developed a biochemical assay for examining ligand-regulated transcription by ERs in the ER/AP-1 pathway. This assay recapitulates the altered (i.e., agonistic) pharmacology of selective estrogen receptor modulator drugs in this pathway reported previously by using various cell-based assays. We used our biochemical assay to examine the detailed mechanisms of ER/AP-1-dependent transcription. Our studies indicate that (i) ERalpha/AP-1 complexes play a critical role in promoting the formation of stable RNA polymerase II preinitiation complexes leading to transcription initiation, (ii) chromatin is a key determinant of estrogen and selective estrogen receptor modulator signaling in the ERalpha/AP-1 pathway, (iii) distinct domains of ERalpha are required for recruitment to DNA-bound Fos/Jun heterodimers and transcriptional activation at AP-1 sites, and (iv) different enhancer/activator combinations in the ERalpha and AP-1 pathways use coactivators in distinct ways. These studies have increased our understanding of the molecular mechanisms underlying ligand-dependent signaling in the ER/AP-1 pathway and demonstrate the usefulness of this biochemical approach.
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PMID:Altered pharmacology and distinct coactivator usage for estrogen receptor-dependent transcription through activating protein-1. 1564 50

Estrogen receptors activate transcription in part through direct interactions with specific DNA motifs, called estrogen response elements (EREs). Here we show that the strong and sustained induction of the gene regulated in breast cancer 1 (GREB1), a gene of unknown function that has been previously suggested to play a role in the effects of estradiol on breast cancer cell proliferation (Rae, J. M., Johnson, M. D., Scheys, J. O., Cordero, K. E., Larios, J. M., and Lippman, M. E. (2005) Breast Cancer Res. Treat 92, 141-149), is mediated by binding of estrogen receptor alpha (ERalpha) to three consensus EREs spread over approximately 20 kb of upstream flanking sequences. In addition to ERalpha, coactivator SRC-3, acetylated histones and phosphorylated RNA polymerase II (P-polII) were detected on all three EREs in the presence of estrogen, while basal recruitment of ERalpha and P-polII was observed only on the proximal element. Chromatin loops were formed between each ERE and the GREB1 transcriptional start site in the presence of estrogen but not of a total antiestrogen. Furthermore, estradiol induced physical association between EREs, suggesting that these elements function as a potent multipartite enhancer to regulate GREB1 transcription.
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PMID:Regulation of GREB1 transcription by estrogen receptor alpha through a multipartite enhancer spread over 20 kb of upstream flanking sequences. 1746


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