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 P1450 gene via trans-acting regulatory factors that include the TCDD .cntdot. receptor complex. The positive control element in the P1450 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 -1647 to -611 upstream from the P1450 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 -1247 to -823 fragment or the -1051 to -823 fragment, but not the -1247 to -1052 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 P1450 were constructed. A region of 300 bases (-1218 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. A 220-bp segment (-1137 to -918) exhibits 79% similarity between mouse and human P1450 upstream sequences and includes a single GGGCGG box at -952 from the mouse P1450 mRNA cap site that is perfectly matched at position -943 from the human P1450 mRNA cap site.