TY - JOUR
T1 - Transcriptional analysis in vivo of the hepatic genes, Cyp2b9 and Cyp2b10, by intravenous administration of plasmid DNA in mice
AU - Rivera-Rivera, Ilia
AU - Kim, Jongsook
AU - Kemper, Byron
N1 - Funding Information:
This work was supported by grant GM39360 from the National Institutes of Health. We thank Hong Li for isolating the Cyp2b9 PBRU-like region by PCR amplification and cloning.
PY - 2003/2/17
Y1 - 2003/2/17
N2 - Phenobarbital (PB) responsiveness of CYP2B genes has been shown to be mediated by a PB responsive unit (PBRU). The core of the PBRU contains two nuclear receptor sites, NR-1 and NR-2, and a nuclear factor-1 (NF 1) binding site, which are required for PB responsiveness, but the importance of sequences flanking the core is not clear. We have used intravenous administration of plasmid DNA in the tail veins of mice to transfect hepatocytes in vivo and analyze sequence requirements for PB induction. In this assay PB treatment increased transactivation by the Cyp2b10 PBRU about 100-fold, which is similar to the increase in the expression of the endogenous gene while the Cyp2b9 PBRU was unresponsive. Analysis of chimeras of the two PBRUs and deletion mutants of the Cyp2b10 PBRU indicated that the core region containing the NR-1, NR-2 and NF-1 core sites is not sufficient for PB responsiveness. Additional sequence at the 3′ side of the core sequence, which included a previously defined accessory factor-1 (AF-1) site, partially restored responsiveness. This region contained a binding site for NF-1 only in Cyp2b10 and not in Cyp2b9, but the intact site was not required for PB responsiveness. Purified constitutive androstane receptor (CAR)/retinoid X receptor (RXR) bound to the core NR-1 and NR-2 sites and to a third NR-3 site to the 5′ side of the core in Cyp2b10. No binding of CAR/RXR to the Cyp2b9 PBRU was observed. These results indicate that changes in the NR sites which eliminate CAR/RXR binding are sufficient for the non-responsiveness to PB of Cyp2b9, but changes in sequences flanking the core independently eliminate PB responsiveness. The results demonstrate the advantages of transfection of mouse hepatocytes in vivo by tail vein injection of DNA as a method for transcriptional analysis of genes in vivo and show that sequences flanking the core region of the PBRU are required for PB induction in vivo.
AB - Phenobarbital (PB) responsiveness of CYP2B genes has been shown to be mediated by a PB responsive unit (PBRU). The core of the PBRU contains two nuclear receptor sites, NR-1 and NR-2, and a nuclear factor-1 (NF 1) binding site, which are required for PB responsiveness, but the importance of sequences flanking the core is not clear. We have used intravenous administration of plasmid DNA in the tail veins of mice to transfect hepatocytes in vivo and analyze sequence requirements for PB induction. In this assay PB treatment increased transactivation by the Cyp2b10 PBRU about 100-fold, which is similar to the increase in the expression of the endogenous gene while the Cyp2b9 PBRU was unresponsive. Analysis of chimeras of the two PBRUs and deletion mutants of the Cyp2b10 PBRU indicated that the core region containing the NR-1, NR-2 and NF-1 core sites is not sufficient for PB responsiveness. Additional sequence at the 3′ side of the core sequence, which included a previously defined accessory factor-1 (AF-1) site, partially restored responsiveness. This region contained a binding site for NF-1 only in Cyp2b10 and not in Cyp2b9, but the intact site was not required for PB responsiveness. Purified constitutive androstane receptor (CAR)/retinoid X receptor (RXR) bound to the core NR-1 and NR-2 sites and to a third NR-3 site to the 5′ side of the core in Cyp2b10. No binding of CAR/RXR to the Cyp2b9 PBRU was observed. These results indicate that changes in the NR sites which eliminate CAR/RXR binding are sufficient for the non-responsiveness to PB of Cyp2b9, but changes in sequences flanking the core independently eliminate PB responsiveness. The results demonstrate the advantages of transfection of mouse hepatocytes in vivo by tail vein injection of DNA as a method for transcriptional analysis of genes in vivo and show that sequences flanking the core region of the PBRU are required for PB induction in vivo.
KW - Cyp2b gene
KW - Cytochrome P450
KW - In vivo gene delivery
KW - Nuclear receptor
KW - Phenobarbital
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U2 - 10.1016/S0304-4165(02)00484-1
DO - 10.1016/S0304-4165(02)00484-1
M3 - Article
C2 - 12573485
AN - SCOPUS:0037450459
VL - 1619
SP - 254
EP - 262
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
SN - 0304-4165
IS - 3
ER -