2,3,7,8-Tetrachlorodibenzo-p-dioxin receptors in wild type and variant mouse hepatoma cells. Nuclear location and strength of nuclear binding

The volume of aqueous solvent present during subcellular fractionation of mouse hepatoma (Hepa 1c1c7) cells influences the distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptors between the nuclear and cytosolic fractions. When the effects of dilution are minimized, at least 80% of the receptors associate with nuclei. The receptors bind relatively strongly to nuclei, as measured by their release by KCl. TCDD-receptor complexes bind more strongly to nuclei than do unoccupied receptors. A temperature-dependent event further enhances the binding of TCDD-receptor complexes to nuclei. A class of variant cells contains receptors which bind relatively weakly to nuclei; this defect accounts for the variant phenotype. We conclude that, in the intact cell, TCDD receptors are located within the nucleus and that the temperature-dependent event in the induction of cytochrome P1-450 gene expression is one which strengthens the binding of the TCDD-receptor complex to chromatin.     

Transcriptional activation of c-fos protooncogene by 17beta-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition.

17Beta-estradiol (E2) induced c-fos protooncogene mRNA levels in MCF-7 human breast cancer cells, and maximal induction was observed within 1 h after treatment. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibited the E2-induced response within 2 h. The molecular mechanism of this response was further investigated using pFC2-CAT, a construct containing a -1400 to +41 sequence from the human c-fos protooncogene linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In MCF-7 cells transiently transfected with pFC2-CAT, 10 nM E2 induced an 8.5-fold increase of CAT activity, and cotreatment with 10 nM TCDD decreased this response by more than 45%. Alpha-Naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, blocked the inhibitory effects of TCDD; moreover, the inhibitory response was not observed in variant Ah-nonresponsive MCF-7 cells, suggesting that the AhR complex was required for estrogen receptor cross-talk. The E2-responsive sequence (-1220 to -1155) in the c-fos gene promoter contains two putative core pentanucleotide dioxin-responsive elements (DREs) at -1206 to -1202 and -1163 to -1159. In transient transfection assays using wild-type and core DRE mutant constructs, the downstream core DRE (at -1163 to -1159) was identified as a functional inhibitory DRE. The results of photo-induced cross-linking, gel mobility shift, and in vitro DNA footprinting assays showed that the AhR complex interacted with the core DRE that also overlapped the E2-responsive GC-rich site (-1168 to -1161), suggesting that the mechanism for AhR-mediated inhibitory effects may be due to quenching or masking at the Sp1-binding site.     

DNA sequence determinants for binding of transformed Ah receptor to a dioxin-responsive enhancer.

We have utilized gel retardation analysis and DNA mutagenesis to examine the specific interaction of transformed guinea pig hepatic cytosolic TCDD.AhR complex with a dioxin-responsive element (DRE). Sequence alignment of the mouse CYPIA1 upstream DREs has identified a common invariant "core" consensus sequence of TNGCGTG flanked by several variable nucleotides. Competitive gel retardation analysis using a series of DRE oligonucleotides containing single or multiple base substitutions has allowed identification of those nucleotides important for TCDD.AhR.DRE complex formation. A putative TCDD.AhR DNA-binding consensus sequence of GCGTGNNA/TNNNC/G has been derived. The four core nucleotides, CGTG, appear to be critical for TCDD-inducible protein-DNA complex formation since their substitution decreased AhR binding affinity by 100-800-fold; the remaining conserved bases are also important, albeit to a lesser degree (3-5-fold). The 5'-ward thymine, present in the invariant core sequence of all the DREs identified to date, appears not to be involved in DNA binding of the AhR. The results obtained here indicate that although the primary interaction of the TCDD.AhR complex with the DRE occurs with the conserved "core" sequence, nucleotides flanking the core also contribute to the specificity of DRE binding.     

Induction of the Cyp1a-1 dioxin-responsive enhancer in transgenic mice.

Cyp1a-1, whose product, aryl hydrocarbon hydroxylase, assists in detoxification of polycyclic aromatic hydrocarbons, is the best characterized of the murine cytochrome P450 genes. The Cyp1a-1 dioxin-responsive enhancer region has been previously analyzed in vitro and found to induce expression of heterologous genes upon exposure of transfected cells to various aromatic hydrocarbons. A 2.58 kbp DNA fragment containing the Cyp1a-1 enhancer elements and promoter region was coupled to the chloramphenicol acetyltransferase (CAT) reporter gene and used to create transgenic mice. CAT assays were performed on tissues harvested from three different lines of transgenic mice following mock-induction or induction using the aromatic hydrocarbon, 3-methylcholanthrene. Basal levels of expression were detected in the spleen and small bowel of non-induced mice, with little or no expression detected in the liver. Treatment with 3-methylcholanthrene increased hepatic expression levels by as much as 10,000-fold. More modest levels of induction were also recorded in the spleen, small bowel, kidney, and lung. The results indicate that the dioxin-responsive enhancer region functions as a strongly inducible promoter in vivo. Differences in the response to induction between male and female mice suggest that Cyp1a-1 expression may be governed in a gender related manner.     

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

The mouse cytochrome P1450 (CYP1A1) gene is responsible for the metabolism of numerous carcinogens and toxic chemicals. Induction by the environmental contaminant tetrachlorodibenzo-p-dioxin (TCDD) requires a functional aromatic hydrocarbon (Ah) receptor. We examined the 5'-flanking region of the CYP1A1 gene in mouse hepatoma Hepa-1 wild-type cells and a mutant line having a defect in chromatin binding of the TCDD-receptor complex. We identified two cis-acting elements (distal, -1071 to -901 region; proximal, -245 to -50 region) required for constitutive and TCDD-inducible CYP1A1 gene expression. Three classes of DNA-protein complexes binding to the distal element were identified: class I, found only in the presence of TCDD and a functional Ah receptor, that was heat labile and not competed against by simian virus 40 (SV40) early promoter DNA; class II, consisting of at least three constitutive complexes that were heat stable and bound to SV40 DNA; and class III, composed of at least three constitutive complexes that were thermolabile and were not competed against by SV40 DNA. Essential contacts for these proteins were centered at -993 to -990 for the class I complex, -987, -986, or both for the class II complexes, and -938 to -927 for the class III complexes. The proximal element was absolutely essential for both constitutive and TCDD-inducible CYP1A1 gene expression, and at least two constitutive complexes bound to this region. These data are consistent with the proximal element that binds proteins being necessary but not sufficient for inducible gene expression; interaction of these proteins with those at the distal element was found to be required for full CYP1A1 induction by TCDD.