Dioxin-Inducible Enhancer Region Upstream from the Mouse P 1 450 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 · 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 pSVO-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.