Abstract
A xenobiotic-responsive element (XRE)-binding factor(s) other than the AhR.Arnt complex was found to inhibit the transcription of CYP1A1 gene in the liver from adult rabbits, known to be nonresponsive to CYP1A1 inducers. The constitutive factor(s) in liver nuclear extracts bound to the core sequence of XRE. The binding was eliminated by the presence of an excess amount of the AhR.Arnt complex synthesized in vitro. To identify the constitutive factor(s), a sequence similar to rabbit XRE was sought. It was found that the sequence of rabbit XRE overlapped with that of the upstream stimulatory factor 1 (USF1)-binding site in the mouse metallothionein I promoter. In fact, a super shift assay using a specific antibody against human USF1 indicated that USF1 was capable of binding to rabbit XRE. Additionally, the AhR.Arnt-mediated activation of XRE-TK/Luc reporter gene in RK13 cells was blocked by the transfection with a USF1 expression vector with the amounts of the expression vector transfected. These results indicate that the XRE of the rabbit CYP1A1 gene is recognized by the basic helix-loop-helix proteins to regulate the expression of CYP1A1 in both an agonistic (AhR.Arnt) and an antagonistic (USF1) manner.
Highlights
A xenobiotic-responsive element (XRE)-binding factor(s) other than the AhR1⁄7Arnt complex was found to inhibit the transcription of CYP1A1 gene in the liver from adult rabbits, known to be nonresponsive to CYP1A1 inducers
The luciferase activity was significantly decreased by the deletion of the sequences between nucleotides Ϫ1.06 and Ϫ0.95 kb, Ϫ0.95 and Ϫ0.83 kb, and Ϫ0.68 and Ϫ0.45 kb from the transcriptional start sites. These results indicate that at least three regions containing essential XREs are responsible for the induction of rabbit CYP1A1 gene
Competition between the Constitutive Factor(s) and the AhR1⁄7Arnt Complex to Bind to the XRE—As a first step to prove our idea that the unknown XRE-binding factor(s) in the nuclear extracts from adult rabbit livers bound to XRE to compete with the AhR1⁄7Arnt complex, we examined the possibility that the constitutive factor(s) recognized the core sequence of XRE
Summary
A xenobiotic-responsive element (XRE)-binding factor(s) other than the AhR1⁄7Arnt complex was found to inhibit the transcription of CYP1A1 gene in the liver from adult rabbits, known to be nonresponsive to CYP1A1 inducers. The AhR1⁄7Arnt-mediated activation of XRE-TK/ Luc reporter gene in RK13 cells was blocked by the transfection with a USF1 expression vector with the amounts of the expression vector transfected. One of the two domains is the Per-Arnt-Sim region composed of approximately 300 amino acids that mediates the ligand binding and interaction with Hsp90 This region is found in the Drosophila regulatory proteins Per and Sim [21] and the mammalian hypoxia-inducible factor 1␣ [22]. We found USF1 to be an additional factor capable of binding to the rabbit XRE to inhibit the interaction of the AhR1⁄7Arnt complex with XRE in rabbits
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