Dioxin receptor and C/EBP regulate the function of the glutathione S-transferase Ya gene xenobiotic response element.
The rat glutathione S-transferase Ya gene xenobiotic response element (XRE) has both constitutive and xenobiotic-inducible activity. We present evidence that the XRE is regulated by both the constitutive C/EBP transcription factor and the xenobiotic-activated dioxin receptor. A ligand-activated XRE-binding protein was shown to be dioxin receptor by specific antibody immunodepletion and binding of highly purified receptor. Identification of C/EBP alpha as the constitutive binding protein was demonstrated by competition with a C/EBP binding site, protein-DNA cross-linking to determine the molecular weight of the constitutive protein(s), specific antibody immunodepletion, and binding of purified bacterially expressed C/EBP alpha. Mutational analysis of the XRE revealed that the constitutive factor (C/EBP alpha) shares a nearly identical overlapping binding site with the dioxin receptor. In functional testing of the putative C/EBP-XRE interaction, cotransfected C/EBP alpha activated an XRE test promoter in the non-xenobiotic-responsive HeLa cell line. Unexpectedly, cotransfected C/EBP alpha had no effect on basal activity but significantly increased the xenobiotic response of the XRE test promoter in the xenobiotic-responsive, C/EBP-positive HepG2 cell line. Furthermore, inhibition of C/EBP-binding protein(s) in HepG2 cells by transfection of C/EBP oligonucleotides suppressed the xenobiotic response. These results suggest that C/EBP alpha and dioxin receptor recognize the same DNA sequence element and that transcriptional regulation can occur by cooperative interactions between these two transcription factors.
Pimental
R A
RA
USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts.
Liang
B
B
Yee
G K
GK
Wilhelmsson
A
A
Poellinger
L
L
Paulson
K E
KE
D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000378:metabolism*; D015262:Xenobiotics
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; D015262:Xenobiotics; D004247:DNA; D005982:Glutathione Transferase