Abstract
Transcriptional activation of the Cyp1B1 gene in rodents is stimulated by both polycyclic hydrocarbons and cAMP. The mouse Cyp1B1 gene structure contains three exons, of which the second nucleotide of exon 2 is the translation start site. Primer extension analysis identified a transcription start domain defining an exon 1 of 371 base pairs. The sequence 1.075 kilobases upstream of the transcription start site showed 11 xenobiotic-responsive elements (XRE) (TnGCGTG or GCGTG) that are putative aryl hydrocarbon receptor (AhR)-binding sites and three steroidogenic factor-1 motifs that are associated with cAMP-mediated transcriptional activation of genes. A transiently transfected Cyp1B1-luciferase construct, composed of exon 1 and 1.075 kilobases of 5'-flanking region, was induced by 2,3, 7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10.0 +/- 3.0-fold, n = 6) in C3H10T1/2 cells, which exclusively express Cyp1B1. The 90-base pair basal promoter contains two SP-1 sites, one SF-1 site, and a TATA-like box. TCDD induction and basal expression were dependent on positive regulatory elements present between -1075 and -810. Five XRE motifs localized in the enhancer region were completely conserved between mouse and human CYP1B1 sequences. Similar inductions were seen in Hepa-1 cells, which express Cyp1A1 but not Cyp1B1. However, basal Cyp1B1 promoter activities were 4-10-fold higher in C3H10T1/2 cells providing the enhancer region was present, partially reproducing the in vivo cell-specific expression of Cyp1B1. Gel shift experiments established that TCDD stimulates AhR binding to the downstream XRE in the enhancer region. However, oligonucleotides that encompass two other XREs show a high affinity complex of similar size that is evident even without TCDD treatment and that does not contain either the AhR or AhR nuclear translocator. The fourth XRE is immediately adjacent to an E-box, and this oligonucleotide formed a smaller complex that was dependent on this E-box sequence. Negative regulatory sequences have been located between the promoter and TCDD-responsive enhancer regions. Constitutive expression of the Cyp1B1 gene was lost in AhR-deficient cells and was restored by transfected AhR cDNA. Reporter constructs function in a parallel manner, demonstrating the key role of the AhR in constitutive as well as TCDD-induced expression of Cyp1B1 in mouse embryo fibroblasts.
Highlights
Cytochrome Cyp1B1 gene expression in rodents and humans is inducible both through activation of the AhR1 by TCDD and by a cAMP-mediated pathway (1–7)
We demonstrate for the first time a mechanism for involvement of the aryl hydrocarbon receptor (AhR) in basal expression of a gene in addition to TCDD induction and a novel protein complex formed at a pair of enhancer xenobiotic-responsive elements (XRE)
Comparison of the sequence obtained from the genomic clone with the cDNA sequence established that the gene includes sequences identical to those found in the cDNA and has allowed us to determine the mouse Cyp1B1 gene structure
Summary
Cytochrome Cyp1B1 gene expression in rodents and humans is inducible both through activation of the AhR1 by TCDD and by a cAMP-mediated pathway (1–7). For many rodent tissues (lung, liver, and kidney), there is essentially no constitutive CYP1B1, and elevated expression is only seen after administration of AhR agonists This induction through the AhR is 30 – 40 times less effective than for Cyp1A1 (1, 4). The AhR mediates gene transcription by forming a nuclear heterodimer with the related helix-loop-helix protein AhR nuclear translocator (Arnt) (13–15) The formation of this complex is stimulated by binding of agonists such as TCDD and polycyclic aromatic hydrocarbons to the AhR, which dissociates from the associated cytosolic partners such as HSP90 (16).
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