Dioxin-Inducible Enhancer Region Upstream from the Mouse P₁450 Gene and Interaction with a Heterologous SV40 Promoter

Abstract

In mouse hepatoma Hepa-1 cells, polycyclic aromatic compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activate transcription of the mouse P(1)450 gene via trans-acting regulatory factors that include the TCDD X receptor complex. The positive control element in the P(1)450 5'-flanking region was examined in control and TCDD-treated Hepa-1 stable transformants that had been transfected with either of two expression vectors containing the chloramphenicol acetyltransferase (CAT) gene: pA10-cat, which has the simian virus 40 (SV40) early core promoter (without enhancers) immediately upstream from the CAT gene; and pSV0-cat, which has no promoter or enhancer. When the 1-kb DNA fragment from -1,647 to -611 upstream from the P(1)450 gene is inserted in either orientation--immediately upstream or almost 2 kb further upstream--from the SV40 promoter in pA10-cat, there is enhancement of CAT activity that can be further induced three- to fourfold by TCDD. When the same experiment is carried out with the -1,247 to -823 fragment or the -1,051 to -823 fragment, but not the -1,247 to -1,052 fragment, TCDD responsiveness is lost, or at least masked, because of a large increase in constitutive CAT activity. pSV0-cat mutants containing internal deletions in the upstream flanking sequences of P(1)450 were constructed. A region of 300 bases (-1,218 to -918) is shown to be required for TCDD responsiveness, and one TCDD-inducible element can be dissociated from an enhancer of constitutive gene expression, whereas one or more other TCDD-inducible elements cannot.(ABSTRACT TRUNCATED AT 250 WORDS)

Dioxin-dependent activation of murine Cyp1a-1 gene transcription requires protein kinase C-dependent phosphorylation.

“…Moreover, CYPlAl is an essential enzyme in the detoxification of chemicals responsible for activation of the CYPIAI gene, and paradoxically, the enzyme also potentiates promutagens and procarcinogens by converting them into reactive intermediates (cf. references 53 and 60 and references therein from the transcriptional initiation site (14,19,26,28,40,55,56). As a consequence of gene activation, the rates of CYPlAl mRNA and protein synthesis are increased 20-to 100-fold (for a review, see reference 54).…”

Regulation of mouse CYP1A1 gene expression by dioxin: requirement of two cis-acting elements during induction.

“…We previously characterized a TCDD-inducible enhancer between -1218 (1218 bases upstream from the mRNA cap site) and -918 in the mouse CYPIAI gene (27). Other laboratories, using different heterologous promoters, have also shown a TCDD-inducible enhancer at approximately the same location upstream from the mouse (18) and rat (6) CYPIAI genes.…”

“…DNA transfections. The itt and cb-cell cultures were stably transfected as previously described (27). Briefly, 25 ,ug of each test plasmid and 4 ,utg of pSV2-nieo were cotransfected by the calcium phosphate and glycerol shock method (29).…”

“…Sonic extracts were prepared from the TCDD-treated and control cells as described previously (27 When regions between -1246 and -822 were removed, the transformants showed marked decreases in both basal and TCDD-inducible CAT activities. On the other hand, deletion of the region from -245 to -50 caused a loss of all CAT activity (Fig.…”

Regulatory Xenobiotic Responsive Elements in the Distal 5′-Flanking Region of the MouseCyp1a2Gene Required for Transcriptional Activation by 3-Methylcholanthrene and 2,3,7,8-Tetrachlorodibenzo-p-dioxin

“…Because mice lacking the 1.2-kbp XRE cluster region showed partial inducible transcriptional activation of Cyp1a2 (Nukaya et al, 2009b), the involvement of a repressive element for transcriptional activation in this region is suspected; the negative regulatory region for Cyp1a1 was reported to be located in the region (Gonzalez and Nebert, 1985). The regulatory region for inducible transcriptional activation of the mouse Cyp1a1 gene was reported previously (Gonzalez and Nebert, 1985;Neuhold et al, 1986Neuhold et al, , 1989Denison et al, 1989;Fisher et al, 1990), which suggests that a single XRE was not sufficient for the inducible activation of Cyp1a1, and the regulatory region was found to contain sequences complementary to XRE13, XRE14, and XRE15 of the Cyp1a2 gene. In humans, the CYP1A1 and CYP1A2 genes are also located in a head-to-head orientation separated by a 23-kbp spacer region on chromosome 15, and the inducible transcriptional activation of human CYP1A2 and CYP1A1 is regulated via a common regulatory region, which contains XRE cluster distal to the transcriptional start site of CYP1A2 but close to that of CYP1A1, and the sequential orientation of the XRE cluster region is highly conserved between humans and mice (Ueda et al, 2006;Nukaya et al, 2009a).…”