Characterization of the Mouse Cyp1B1 Gene

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) (T n GCGTG 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 TCDDresponsive 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.

Transcriptional Regulation of the Human CYP1B1 Gene

“…These factors may act by binding to cis-acting regulatory regions and functioning as transcriptional enhancers or repressors directly or by interacting with DNA or DNA-binding proteins that modulate higher order chromatin structure. These studies also demonstrate that chromatin structure likely plays a major role in transcriptional regulation of the human CYP1B1 gene, as has been suggested for the mouse gene (29,30).…”

“…ARNT, on the other hand, is known to partner with other transcription factors and activate genes in an aryl hydrocarbon-independent manner (37,38). It has been shown that complexes other than AhR/ARNT bind in vitro to two of the analogous DREs in this region in the mouse Cyp1B1 gene in two different cell types (29,30). Therefore, because evidence suggests there may be a role for the DREs in constitutive gene expression, we analyzed the involvement of these elements in constitutive expression of the human CYP1B1 gene.…”

“…Studies of Cyp1B1 regulation in the mouse have shown a certain degree of cell specificity regarding the role of AhR. Primary bone marrow stromal cells prepared from AhR null mice constitutively express functional CYP1B1 (31), whereas fibroblasts from the same AhR Ϫ/Ϫ genotype do not express basal CYP1B1 (29). Recently, ARNT has been implicated as a repressor of Cyp1B1 basal expression in murine Hepa-1 cells, but the results are complicated by the finding that an ARNT-like protein may also be involved (30).…”

Cytochrome P450 CYP1B1 determines susceptibility to 7,12-dimethylbenz[ a ]anthracene-induced lymphomas

“…The gene structure is distinct from other mammalian P450s comprising only three exons and having a very long 3Ј untranslated region in exon 3 that results in a 5.2-kilobase (kb) mRNA (11,12). CYP1B1 has a pattern of expression that differs from the other two CYP1 family P450s; it is expressed constitutively in steroidogenic tissues like the adrenal, ovary, and testes and is inducible by adrenocorticotropin, cAMP, peptide hormones, and aryl hydrocarbon receptor ligands (4,8,10,13,14).…”

Ah Receptor and NF-κB Interactions, a Potential Mechanism for Dioxin Toxicity

“…The heterodimer binds to the xenobiotic response element (XRE) (12) and alters expression of genes controlled by enhancer XREs. XREs, with the conserved core sequences "GCGTG", are found in the promoter regions of several genes involved in the metabolism of xenobiotics, including cytochromes P-450 (CYP1A1, CYP1A2, and CYP1B1) (13)(14)(15)(16), and NAD(P)H-quinone oxidoreductase (17). Studies of the regulation of these genes, especially the regulation of CYP1A1, have provided a basis for understanding the mode of action of dioxin and related compounds (18,19).…”