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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Aromatase, a cytochrome P-450, catalyzes the formation of aromatic C18 estrogenic steroids from C19 androgens. DNA sequence analysis of the human aromatase gene has revealed that a putative promoter sequence exists immediately up-stream of the second exon. Chloramphenicol acetyltransferase functional analyses of cells transfected with chloramphenicol acetyltransferase expression plasmids containing various DNA fragments derived from the 3'-end of the first intron of the aromatase gene were performed to show that a promoter indeed exists in this region. However, in all of the cell lines used in this study, MCF-7, JAR, OVCAR-3, and skin fibroblast, the function of this promoter was inhibited by a negative regulatory element situated up-stream from the promoter. The results further suggest that this inhibitory element behaves as a silencer element, in that it could inhibit a simian virus-40 promoter from a distance of several kilobases. This negative element worked in both orientations and inhibited the functions of several promoters, including the newly identified promoter situated in the 3'-end of the first intron of the human aromatase gene. Primer extension analysis has been performed to determine the potential transcription start site. The mechanism of the regulation of aromatase expression is known to be very complex. The presence of a promoter and a silencer at the 3'-end of the first intron may represent one additional way that aromatase expression is controlled in estrogen-producing cells.
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PMID:Identification of a promoter and a silencer at the 3'-end of the first intron of the human aromatase gene. 133 79

The gene encoding aromatase P-450 (CYP XIX) has been isolated from two types of human genomic DNA libraries. It spans at least 70 kb and consists of 10 exons. The translational initiation site and the termination site are located in exon 2 and exon 10, respectively. The promoter region of the gene contains a TATA box, a CAAT box and two putative AP-1 binding sites beginning at -28, -83, -55 and -68 bp, respectively, from the transcriptional initiation site. In addition, a palindromic nucleotide sequence is observed between -209 and -196 and two types of repetitious hexanucleotide (consensus: AATGAA and CCATAAGG) are also present within the regions between -485 and -433 and between -358 and -331. Transient expression studies of chloramphenicol acetyltransferase constructs bearing various lengths of 5'-flanking region of the gene show that the region between -500 and -243 contains negative cis-acting element(s), whereas the region between -242 and -183 is required for efficient transcriptional activity. Northern blot analysis demonstrates that the expression of aromatose P-450 gene is remarkably stimulated by treatment of cells with 12-O-tetradecanoyl-phorbol 13-acetate. By chloramphenicol acetyltransferase assay the region up to nucleotide position -242 relative to the transcriptional initiation site is shown to participate in the transcriptional responsiveness to this phorbol ester.
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PMID:Structural and functional characterization of human aromatase P-450 gene. 217 39

In the present report we describe a heretofore unrecognized role for a Jak/STAT signaling pathway, namely the stimulation of expression of the aromatase P450 (CYP19) gene, and hence of estrogen biosynthesis, in human adipose tissue. Expression of this gene in adipose tissue as well as in adipose stromal cells maintained in the presence of serum and glucocorticoids is regulated by a distal TATA-less promoter, I.4, which contains a glucocorticoid response element, an Sp1 binding site, and an interferon-gamma activation site (GAS) element. The stimulatory action of serum (in the presence of dexamethasone) can be replaced by interleukin (IL)-11, leukemia inhibitory factor, and oncostatin-M, as well as by IL-6, providing the IL-6 soluble receptor is also present. Stimulation of the cells by these factors led to rapid phosphorylation of Jak1, but not Jak2 or Jak3, on tyrosine residues. STAT3 but not STAT1 was also phosphorylated and bound to the GAS element in the I.4 promoter region. When regions of this promoter were fused upstream of the chloramphenicol acetyltransferase reporter gene and transfected into the cells, mutagenesis or deletion of the GAS element led to complete loss of reporter gene expression. Since adipose tissue is the major site of estrogen biosynthesis in men and in postmenopausal women, this pathway involving a Jak/STAT signaling mechanism acting together with glucocorticoids and Sp1 appears to be the principal means whereby estrogen biosynthesis is regulated in the elderly.
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PMID:Aromatase P450 gene expression in human adipose tissue. Role of a Jak/STAT pathway in regulation of the adipose-specific promoter. 760 17

The biosynthesis of estrogens is catalyzed by aromatase P450 (P450arom), the product of the CYP19 gene. The tissue-specific expression of the CYP19 gene is regulated by means of tissue-specific promoters through the use of alternative splicing mechanisms. Thus, transcripts containing various 5'-untranslated termini are present in human placenta and other fetal tissues, ovary, brain, and adipose stromal cells. Sequence corresponding to untranslated exon 1.4 is present in 5'-termini of transcripts expressed in adipose tissue and fetal liver, as well as adipose stromal cells in primary culture in the presence of dexamethasone and fetal calf serum (FCS). Identification of hormone-responsive, tissue-specific promoter regions, as well as growth factor-response elements upstream of exon 1.4, may provide insight into the regulation of estrogen biosynthesis in adipose tissue, which is implicated in the development of breast and endometrial cancer. The goals of the present study were to define the 1.4 promoter region with respect to the start of transcription and to characterize the region(s) responsible for conferring glucocorticoid responsiveness on aromatase expression. The transcription initiation site was identified by means of primer extension and S1 nuclease protection analyses. No TATA-like sequence was evident upstream of this site. Various deletion mutations of the upstream flanking region of exon 1.4 and including part of exon 1.4 were made using polymerase chain reaction or restriction enzyme digestion. The genomic fragments were fused upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These constructs were transfected into adipose stromal cells and fetal hepatocytes in primary culture in medium containing FCS with or without dexamethasone. The -560/+10 base pair (bp) construct expressed CAT activity after a putative silencer element was deleted, and expression was induced by dexamethasone about 3-fold. Transfection of the -330/+170 bp construct, which contains an upstream glucocorticoid response element (GRE) as well as an Sp1-like sequence in untranslated exon 1.4, resulted in an 8-fold stimulation of expression of CAT activity by dexamethasone. The upstream GRE as well as the Sp1-like sequence in untranslated exon 1.4 were mutated separately, and together, to further confirm whether the GRE or Sp1 binding site play a role in the regulation of promoter 1.4-driven transcription. Mutation of either the GRE or Sp1 binding site, or both, in the -330/+170 bp construct, resulted in loss of dexamethasone-induced CAT reporter gene expression.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of the sequences of the human CYP19 (aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes. 777 80

CYP19, the human aromatase cytochrome P-450 (P450arom) gene, encodes an enzyme which converts androgens to estrogens by three successive hydroxylation reactions by coupling with NADPH-cytochrome P-450 reductase. In the present study, we have characterized two cis-acting transcriptional regulatory elements of CYP19, termed as hATRE-1 (human aromatase cytochrome P-450 gene transcriptional regulatory element-1) ([sequence: see text]) and hATRE-2 ([sequence: see text]). These sequences are located between -2238 and -2214, and between -2141 and -2098 relative to the major cap site of the gene, respectively. Transient expression analysis in human BeWo choriocarcinoma cells, in which CYP19 is expressed, shows that hATRE-1 represses the expression of the bacterial chloramphenicol acetyltransferase reporter gene driven by the promoter of CYP19, whereas hATRE-2 enhances the reporter gene expression in response to 12-O-tetradecanoylphorbol 13-acetate. Electrophoretic mobility shift analysis indicates that nuclear binding factors specific to hATRE-1 are present in BeWo cells, but not in HeLa cells nor in TYK-nu cells that lack the expression of CYP19. In contrast, nuclear binding factors to hATRE-2 are present not only in BeWo cells but also in the latter two types of cells. Nevertheless, hATRE-2 does not affect the reporter gene expression in HeLa cells and TYK-nu cells. These results indicate that hATRE-1 and hATRE-2 are cis-acting transcriptional regulatory elements involving in the regulation of the cell type-specific expression of CYP19.
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PMID:Identification and characterization of cis-acting regulatory elements for the expression of the human aromatase cytochrome P-450 gene. 813 35

The proximal promoter of the rat aromatase CYP19 gene contains two functional regions that, by 5'-deletion analyses, have been shown to confer hormone/ cAMP inducibility to chimeric genes in primary cultures of rat granulosa cells and constitutive expression in R2C Leydig cells. Promoter region A binds Steroidogenic Factor-1 (SF-1); region B binds cAMP-regulatory element binding protein (CREB) and two other factors (designated X and Y). Mutations were generated within the context of the intact promoter to selectively eliminate the binding of either SF-1, CREB, CREB plus factors X and Y, or all of the above. When expression vectors that failed to bind either CREB alone or CREB plus factors X and Y were transfected into granulosa cells, cAMP-dependent chloramphenicol acetyltransferase (CAT) activity was reduced 65% indicating that CREB alone, and not factors X and Y, mediates the cAMP response of this cAMP response element-like domain. Similarly, cAMP-dependent CAT activity was reduced 50% in constructs that failed to bind SF-1 and was abolished with vectors that were unable to bind either factor. In R2C Leydig cells, the absence of either CREB or SF-1 binding resulted in an almost complete loss in CAT activity. Both immunoreactive CREB and phosphorylated CREB (phospho-CREB) were present in extracts and nuclei of R2C cells. Immunoreactive phosphoCREB was low in granulosa cell extracts and nuclei but increased rapidly (90 min) in response to FSH/cAMP and was highest at 48 h, at a time when SF-1 was also phosphorylated and expression of the endogenous gene was elevated. Although the amount of CREB and SF-1 remained unchanged in response to FSH, LH mediated a rapid decrease in the amount of SF-1 (but not CREB) that is coincident with decreased aromatase mRNA in luteinizing granulosa cells. Taken together, the data indicate that expression of the aromatase gene is dependent on the additive interactions of regions A and B of the aromatase promoter in granulosa cells and the synergistic interactions of these same regions in R2C cells and that these interactions are dependent, in turn, on the phosphorylation of CREB and SF-1 and the content of these factors, as well as the presence of putative coregulatory molecules.
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PMID:Functional interactions, phosphorylation, and levels of 3',5'-cyclic adenosine monophosphate-regulatory element binding protein and steroidogenic factor-1 mediate hormone-regulated and constitutive expression of aromatase in gonadal cells. 905 76

The expression of aromatase in human breast tumors was studied using the reverse transcription-polymerase chain reaction (RT-PCR) method on 70 breast tissue specimens. An RT-PCR analysis using two oligonucleotide primers derived from exon II of the human aromatase gene revealed that aromatase mRNA was detected in all but three tissue specimens. Furthermore, primer-directed RT-PCR was performed to determine the exon I usage in aromatase mRNA in these breast tumor specimens. The analysis revealed that exons I.3 and PII are the two major exons I present in aromatase mRNA isolated from breast tumors, suggesting that promoters I.3 and II are the major promoters driving aromatase expression in breast cancer and surrounding adipose stromal cells (ASCs). Recently, the regulatory properties of a 696-base pair region that contains promoter II, and is situated immediately upstream of exon II of the human aromatase gene, were investigated. Detailed DNase 1 footprinting analysis, DNA mobility shift assays, and chloramphenicol acetyltransferase (CAT) functional studies of this genomic region were performed and led to the identification of a segment (B1) that could act as a promoter (probably promoter I.3) in adipose stromal and breast cancer cells. The study further revealed that the B1 region could be divided into two domains which were designated RE1 and RE2. RE1 was found to have the promoter activity, and RE2 was found to regulate the promoter activity of RE1, but in different manners in MCF-7 cells (as an example of breast cancer cells) and in ASCs. RE2 was found to function as a positive regulatory element in MCF-7 cells and as a negative regulatory element in ASCs, respectively. It was also found that in several breast cancer cell lines, including MCF-7, the promoter activities of both promoter II and promoter I.3 were found to be suppressed by a negative regulatory element, a silencer, present in the 162 bp fragment which is located upstream from promoter II and downstream from promoter I.3. The precise position of the silencer element (termed S1) was localized by deletion mutation and DNase 1 footprinting analysis, and the silencing activity of S1 on promoter I.3 (in B1 fragment) was confirmed by CAT plasmid transfection experiments. UV crosslinking experiments are being performed to examine the regulatory proteins interacting with the silencer element. These studies serve as the basis for the further characterization of the regulatory mechanism of aromatase expression in human breast cancer and ASCs.
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PMID:Gene regulation studies of aromatase expression in breast cancer and adipose stromal cells. 936 1

Among multiple exons 1 of the mouse aromatase gene, brain-specific exon 1 is only utilized in the hypothalamus and amygdala regions. In this study, identification of the promoter region necessary for basal transcription of the aromatase gene in the brain was undertaken. Deletions of various lengths were introduced into the overall promoter region, which was fused to the chloramphenicol acetyltransferase gene. The resulting reporters were transfected into cultured neurons from the diencephala of fetal mouse brains on embryonic day 13 and then their CAT mRNA levels were determined. The reporter plasmid containing the promoter region 202 bp upstream from the transcriptional initiation site gave the greatest expression. Then binding of trans-acting factors in a nuclear extract of the diencephala to the -202 bp promoter region was investigated by DNase I footprint analysis, multiple protected areas, referred to as Arom-Aalpha, Abeta, Agamma, B and C, being found. Gel shift assays, performed with oligonucleotides corresponding to the protected areas, showed that nuclear DNA binding factors form specific complexes exhibiting different mobilities. Substitution in the Arom-Aalpha or -B sequence in the promoter region in the CAT reporters decreased the CAT mRNA expression levels to about one-fifth the wild type one. These results suggest that multiple nuclear factors bound to the core promoter region participate in the expression of the aromatase gene in mouse brain neurons.
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PMID:Identification of cis-acting elements in the proximal promoter region for brain-specific exon 1 of the mouse aromatase gene. 1009 84

Estrogen-related receptor (ERR) alpha-1 shares a high amino acid sequence homology with estrogen receptor alpha. Although estrogens are not ligands of ERR alpha-1, our recent results suggest that toxaphene and chlordane, two organochlorine pesticides with estrogen-like activity, behave as antagonists for this orphan nuclear receptor. The two compounds increased ERR alpha-1-mediated expression of the reporter enzyme beta-galactosidase in a yeast-based assay. The screen was developed by expressing the hERR alpha-1-yeast Gal 4 activation domain fusion protein in yeast cells carrying the beta-galactosidase reporter plasmid, which contains an ERR alpha-1-binding element. In transfection experiments using mammalian cell lines, such as the SK-BR-3 breast cancer cell line, the compounds were found to have an antagonist activity against ERR alpha-1-mediated expression of the reporter chloramphenicol acetyltransferase. In contrast to the findings with ERR alpha-1, the two compounds were found to slightly induce the estrogen receptor a-mediated expression of chloramphenicol acetyltransferase in SK-BR-3 cells. In a ligand-independent manner, the ERR alpha-1 activity in SK-BR-3 cells was induced 3-fold by cotransfection with the GRIP1 coactivator expression plasmid. Toxaphene was found to be capable of suppressing the GRIP1 coactivator-induced ERR alpha-1 activity in SK-BR-3 cells. In addition, a stable ERR alpha-1 expressing HepG2 hepatoma cell line was generated, and the aromatase activity in the transfected cell line was found to be twice that in the untransfected cell line. The enzyme aromatase converts androgens to estrogens, and aromatase expression in HepG2 cells is regulated in part by an ERR alpha-1-modulating promoter. A 24-h incubation of an ERR alpha-1-transfected HepG2 cell line with 10 microM toxaphene reduced its aromatase activity to the level in the untransfected cell line. Because toxaphene is not an inhibitor of aromatase, it is thought that the decrease of the aromatase activity in ERR alpha-1 transfected HepG2 cells following toxaphene treatment resulted from a suppression of the aromatase expression by toxaphene acting as the antagonist of ERR alpha-1. Toxaphene and chlordane are among the 12 persistent organic pollutants identified by the United Nations Environment Programme as requiring urgent attention. Their antagonistic effects on ERR alpha-1 should not be overlooked.
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PMID:Two organochlorine pesticides, toxaphene and chlordane, are antagonists for estrogen-related receptor alpha-1 orphan receptor. 1049 99

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