Regulatory DNA elements localized remotely upstream from the drug-metabolizing cytochrome P-450c gene

Fujisawa‐Sehara, Atsuko; Sogawa, Kazuhiro; Nishi, Carolina N.; Fujii‐Kuriyama, Yoshiaki

doi:10.1093/nar/14.3.1465

Cited by 126 publications

(65 citation statements)

References 26 publications

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“…Structure qf'chimeric expression plasmids. The construction of pMC6.3k (A) was described previously [5]. In the P-45Oc plasmid, the P-45Oc sequence from -6.3 kb to 2566 bp relative to the transcription start site is fused to a bacterial CAT gene.…”

Section: Resultsmentioning

confidence: 99%

“…These results suggest that the repression of P-45Oc gene expression by the Ela gene was not a tissue-or species-specific phenomena. To determine whether or not the reduction in CAT activity in cells cotransfected with the plasmids containing the Ela sequence was at the transcriptional level, we measured the amount of the P-45Oc-CAT chimeric mRNA from HeLa cells cotransfected with 12SEla DNA by the primer extension method [5]. A synthetic primer described previously [5] was used for the experiment.…”

Section: Resultsmentioning

confidence: 99%

“…The construction of plasmids containing the P-45Oc gene sequence, pMC6.3k and NhFn, was described previously [5,71. The two constructions are represented schematically in Figs 1 and 6 A, respectively.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

“…Primer extension analysis was carried out using a 5'-end-labelled synthetic polynucleotide (35-mer) as a primer, as described previously [5]. Chloramphenicol acetyltransferase (CAT) activity was determined by the method of Gorman et al [6, 321.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

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Repression of cytochrome P‐450c gene expression by cotransfection with adenovirus E1a DNA

Sogawa

Handa

Fujisawa‐Sehara

et al. 1989

European Journal of Biochemistry

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Gene expression of rat cytochrome P-45Oc (P-45Oc) depends upon inducible enhancers scattered in the 5'-upstream region of the gene. We show that expression of the P-45Oc gene is repressed by contransfection with adenovirus Ela DNA, regardless of the presence or absence of inducers, in a transient expression system of HeLa cells. Since cotransfection of either 13s or 12s Ela cDNA was effective in the repression, the region necessary for repression could be separated from that of transactivation of other adenovirus early genes. Moreover, we investigated the regions responsible for the inhibitory activity using in-frame deletion mutants lacking internal or external portions of the Ela proteins. The sequence responsible for the repression was located in the aminoterminal half of the Ela proteins. The inducible expression of the chimeric plasmid containing a 24-base-pair enhancer sequence of the P-45Oc gene placed in a heterologous promoter of SV40 was repressed by cotransfection with Ela DNA, suggesting strongly that the inhibitory effect of the Ela proteins upon P-45Oc gene expression was caused by blocking the enhancer activity.Cytochrome P-450 (P-450) constitutes a superfamily and plays an important role in oxidative metabolism of exogenous and endogenous substrates as a terminal oxidase in an NADPH-dependent electron pathway of liver microsomes [l]. Of these P-450s, P-45Oc (new nomenclature: P-450IA1 [2]) is distributed fairly ubiquitously in various tissues in rats and is induced by the administration of inducers such as 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin [3,4]. Severallaboratories have attempted to reveal the molecular mechanisms of the induction phenomena, using the DNA transfection technique [5 -91. From these studies, a cis-acting DNA element, responsible for the inducible expression of the gene, was identified within short segments in the upstreamflanking region of the P-45Oc gene and designated XRE (xenobiotic responsive element) [7], although the element is also indispensable for the basal level expression of the P-45Oc gene. Furthermore, this element was found to operate also on a heterologous promoter of SV40 in a manner relatively independent of distance and orientation and is, therefore, classified into the category of inducible enhancers. Several lines of evidence strongly suggest that the inducible expression of the P-45Oc gene after administration of inducers is mediated Correspondence to K. Sogawa, Department of Chemistry, Faculty of Science, Tohoku University, Aobayarna, Sendai-shi Miyagi-ken, Japan 980Abbreviations. P-450c, cytochrome P-45Oc; CAT, chloramphenicol acetyltransferase; XRE, xenobiotic responsive element; Ad, adenovirus.Enzymes. T4 DNA ligase (EC 6.5. [18], were activated by the Ela proteins, while activities of viral enhancers, including SV40 and polyoma virus and cellular enhancer elements such as immunoglobulin-heavychain and insulin genes, were repressed [19-221. We are interested in the mechanism whereby the inducible enhancer of the P-45Oc gene is infl...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Construction Of Plasmidsmentioning

confidence: 99%

Section: Cell Culture and Transfectionmentioning

confidence: 99%

See 2 more Smart Citations

Repression of cytochrome P‐450c gene expression by cotransfection with adenovirus E1a DNA

Sogawa

Handa

Fujisawa‐Sehara

et al. 1989

European Journal of Biochemistry

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“…The isolated RNA (20 pg) was then hybridized with 2 pmol 32P-labeled primer at the 5' end in 22 pl Hepes buffer (6.6 mM, pH 7.5) containing 180 mM NaCl at 55°C for 5 min. The primer is a 35mer synthetic oligonucleotide in the sequence of the structural region of the CAT gene [3]. The location of the primer is shown in the map by a solid bar (Fig.…”

Section: Si Mappingmentioning

confidence: 99%

The 5′‐flanking region of the human P‐450(SCC) gene shows responsiveness to CAMP‐dependent regulation in a transient gene‐expression system of Y‐1 adrenal tumor cells

Inoue

Higashi

Morohashi

et al. 1988

European Journal of Biochemistry

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The chronic effect of CAMP‐dependent regulation on adrenocortical steroidogenesis is known to be revealed in the stimulation of the biosynthesis of steroidogenic enzymes. P‐450(SCC), one of the enzymes, catalyzes the first and the rate‐limiting reaction in steroidogenesis from cholesterol and its synthesis is regulated by CAMP. In order to investigate cis‐acting DNA elements of this gene in response to CAMP‐dependent regulation, we have constructed a fusion gene (pSCC5.4k) by ligating the 5′‐flanking and the upstream untranslated region (5.4 kb) of the human P‐450(SCC) gene to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected it into various culture cells including Y‐1 (mouse adrenal tumor), L929 (mouse fibroblast), HTC (rat hepatoma) and Hepa‐1 (mouse hepatoma). Only Y‐1 cells transfected with pSCC5.4k were found to express transiently the enhanced CAT activity in response to the cAMP analogue, cyclic dibutyryl‐AMP (Bt2cAMP). Primer‐extension analysis of RNA prepared from the cells treated with or without Bt2cAMP showed that the enhanced CAT activity was due to an increase in the CAT mRNA and that the transcription start site, determined here with the human P‐450 gene in the adrenal cortex, was correctly utilized with the fusion gene in the transient expression system. Forskolin and cholera toxin, activators of adenylate cyclase, also increased the expression of the CAT activity in the Y‐1 cells. It has been demonstrated, therefore, that the CAMP‐dependent regulation of the P‐450(SCC) gene in adrenal cortex is faithfully reflected in the transient expression system using Y‐1 cells and the fusion gene and that a cis‐acting DNA element(s) in response to cAMP is present within the 5′‐flanking sequence (5.4 kb) of the P‐45O(SCC) gene.

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The specific DNA binding activity of the dioxin receptor is modulated by the 90 kd heat shock protein.

et al. 1990

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The dioxin receptor is a gene regulatory protein which exhibits many structural and functional similarities to steroid hormone receptors. In this study we compare the subunit composition of two forms of the dioxin receptor, sedimenting at approximately 9S and approximately 6S respectively, which are present in nuclear extract from wild‐type Hepa 1c1c7 mouse hepatoma cells following treatment in vivo with dioxin. The nuclear approximately 9S receptor form contained the 90 kd heat shock protein, hsp90. As assessed by a gel mobility shift assay, this receptor form did not bind to the xenobiotic response element (XRE) of the target gene cytochrome P‐450 IA1. In contrast, the smaller approximately 6S receptor form did not contain any immunochemically detectable hsp90. Moreover, this receptor form specifically bound to the XRE recognition sequence. Thus, the specific DNA binding activity of the dioxin receptor was inhibited by association with hsp90, and the approximately 9S dioxin receptor species could be regarded as a nonactive receptor form. Neither the approximately 9S nor the approximately 6S receptor forms were detected in nuclear extract from a dioxin treated mutant clone of Hepa 1 that expresses a nuclear translocation deficient receptor phenotype. We conclude that activation of the dioxin receptor is, at least, a two step process involving binding of the ligand and dissociation of hsp90 from the ligand‐binding receptor protein. Inhibition of the DNA binding activity of transcription factors by protein‐‐protein interaction has also been described for several steroid hormone receptors and for the NF kappa B factor.(ABSTRACT TRUNCATED AT 250 WORDS)

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Regulatory DNA elements localized remotely upstream from the drug-metabolizing cytochrome P-450c gene

Cited by 126 publications

References 26 publications

Repression of cytochrome P‐450c gene expression by cotransfection with adenovirus E1a DNA

Repression of cytochrome P‐450c gene expression by cotransfection with adenovirus E1a DNA

The 5′‐flanking region of the human P‐450(SCC) gene shows responsiveness to CAMP‐dependent regulation in a transient gene‐expression system of Y‐1 adrenal tumor cells

The specific DNA binding activity of the dioxin receptor is modulated by the 90 kd heat shock protein.

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