Functional analysis of the dioxin response elements (DREs) of the murine CYP1A1 gene promoter: beyond the core DRE sequence

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological and toxicological effects of halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). When activated by dioxin, the cytosolic AhR protein complex translocates into the nucleus and dimerizes with the ARNT (Ah receptor nuclear translocator) protein. The heteromeric ligand:AhR/Arnt complex then recognizes and binds to its specific DNA recognition site, the dioxin response element (DRE). DREs are located upstream of cytochrome P4501A1 (CYP1A1) and other AhR-responsive genes, and binding of the AhR complex stimulates their transcription. Although CYP1A1 expression has been used as the model system to define the biochemical and molecular mechanism of AhR action, there is still limited knowledge about the roles of each of the seven DREs located in the CYP1A1 promoter. These seven DREs are conserved in mouse, human and rat. Deletion analysis showed that a single DRE at -488 was enough to activate the transcription. Truncation analysis demonstrated that the DRE at site -981 has the highest transcriptional efficiency in response to TCDD. This result was verified by mutation analysis, suggesting that the conserved DRE at site -981 could represent a significant and universal AhR regulatory element for CYP1A1. The reversed substituted intolerant core sequence (5'-GCGTG-3' or 5'-CACGC-3') of seven DREs reduced the transcriptional efficiency, which illustrated that the adjacent sequences of DRE played a vital role in activating transcription. The core DRE sequence (5'-TNGCGTG-3') tends to show a higher transcriptional level than that of the core DRE sequence (5'-CACGCNA-3') triggered by TCDD. Furthermore, in the core DRE (5'-TNGCGTG-3') sequence, when "N" is thymine or cytosine (T or C), the transcription efficiency was stronger compared with that of the other nucleotides. The effects of DRE orientation, DRE adjacent sequences and the nucleotide "N" in the core DRE (5'-TNGCGTG-3') sequence on the AhR-regulated CYP1A1 transcription in response to TCDD were studied systematically, and our study laid a good foundation for further investigation into the AhR-dependent transcriptional regulation triggered by dioxin and dioxin-like compounds.

Figure 1.

Sequence of the putative dioxin responsive element (DRE) sites on the promoter region of mouse CYP1A1, and the alignments with the corresponding human and rat CYP1A1 promoter sequences are shown. The asterisk “^*” indicates positions which have a single, fully conserved residue.

Figure 2.

The putative DRE at position −488 is enough to activate AhR-dependent transcription. The mouse CYP1A1 promoter-reporter constructs, pCYP1A1W-Luc, which contains 1.4 kb of the promoter sequence, pCYP1A1-T1-Luc, which contains only one DRE at position −488, and pCYP1A1-T2-Luc, which contains no DREs, were transiently transfected into cultured Hepa wide-type (WT) cells one day before treatment. The transfected cells were incubated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10⁻⁹ M) or with solvent alone at 0.1% (control). After 24 h, luciferase assays were performed to determine the promoter activity of mouse CYP1A1. ^* p < 0.05, the difference from solvent-treated cells (control) by one-way ANOVA with Tukey’s test.

Figure 3.

The AhR-dependent transcriptional efficiency of the seven putative DREs are different. The following constructs were transiently transfected into cultured Hepa WT cells one day before drug treatment: seven mouse CYP1A1 promoter-reporter constructs, each of which contains one DRE site, the control vector pGL3-promoter (A) or (B) pCYP1A1W-Luc, which contains the 1.4-kb CYP1A1 promoter sequences, pCYP1A1-M1-Luc and pCYP1A1-M2-Luc, which contain mutations of the DRE at position −981 (DRE4), and the control vector pGL3-basic. After one day of treatment with TCDD (10⁻⁹ M) or with solvent alone at 0.1% (control), luciferase assays were performed to determine the promoter activity of mouse CYP1A1. ^* p < 0.05, the difference from solvent-treated cells (control) by one-way ANOVA with Tukey’s test.

Figure 4.

The DRE orientation and its adjacent sequences can affect the efficiency of transcription in response to TCDD. The mouse CYP1A1 promoter-reporter constructs consisting of a 25-bp region surrounding the core DREs in the original orientation, reverse orientation or the reversed core sequence only with surrounding sequences in the original orientation, were transiently transfected into cultured Hepa WT cells. The pGL3-promoter vector is used as the control. One day later, the transfected cells were incubated with TCDD (10⁻⁹ M) or with solvent alone at 0.1% (control) for one day, followed by luciferase assays to determine the promoter activity. ^* p < 0.05, the difference from solvent-treated cells (control) by one-way ANOVA with Tukey’s test.

Figure 5.

The nucleotide “N” in the core DRE (5′-TNGCGTG-3′) sequence contributed to the AhR-dependent transcriptional differences. The mouse CYP1A1 promoter-reporter constructs, which contain “N” mutations of the DRE sequence at position −488 (5′-CACGCGA-3′) (A) or which contain “N” mutations of the reversed 25-bp DRE sequence at position, −488 (5′-TCGCGTG-3′) (B) were transiently transfected into cultured Hepa WT cells one day before drug treatment. The pGL3-basic vector is used as the control. After one day of treatment, luciferase assays were performed to determine the promoter activity of mouse CYP1A1. The transfected cells were incubated with TCDD (10⁻⁹ M) or with solvent alone at 0.1% (control). ^* p < 0.05, the difference from solvent-treated cells (control) by one-way ANOVA with Tukey’s test.