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.
USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts.
R A
RA
Pimental
B
B
Liang
G K
GK
Yee
A
A
Wilhelmsson
L
L
Poellinger
K E
KE
Paulson
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