Gene Expression and DevelopmentPositive regulation of the rat CYP2B2 phenobarbital response unit by the nuclear receptor hexamer half-site·nuclear factor 1 complex
Section snippets
Materials and methods
Rat hepatocytes were isolated, cultured, transfected, and treated with PB as previously described [6], except that Matrigel was omitted. CAT activity was assayed by the method of Gorman et al.[11]. The following oligos were synthesized by Life Technologies: NF1dm3, 5′-ACTTTCCTGACCTTTTCACAGTGCTTCCAT-3′; and HXm2, 5′-ACTCTGTACTTTCCTCTGGTTGGCACAGTGC CACCA-3′. Substitutions in the wild-type sequence are indicated by bold characters. To generate the construct HXm2, the pSa-Sa163 plasmid [6] was
Results and discussion
As previously mentioned, based on mutational analyses, the HX half-site seems to act positively in rat CYP2B2 and negatively in mouse Cyp2b10. To determine whether this apparent difference might be due to the different mutations introduced into the rat and mouse sequences, we generated an HXm2 mutant that carries the exact 4-bp substitution (AGGTCA → ACCAGA) introduced into the mouse half-site [5]. As we found previously with the HXm and HXm-NF1dm2 mutations [6], the HXm2 mutation reduced but
Acknowledgements
This work was supported by the Medical Research Council. We thank Eric Trottier for careful reading of the manuscript, Pierre Paquin and Guy Langlois for photographic work, Frances Sladek for the HNF-4 cDNA, and Luc Bélanger for the FTF expression vector.
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Mutational analysis of the CYP2B2 phenobarbital response unit and inhibitory effect of the constitutive androstane receptor on phenobarbital responsiveness
2000, Journal of Biological ChemistryCitation Excerpt :Thus, it becomes important to verify the effects of NR1- or NR2-inactivating mutations of the PBREM in the natural sequence context. Mutation of any one of the four NR1 or NR2 half-sites by a 4-bp central CCAG substitution reduced but did not abolish PB responsiveness conferred by the rat PBRU in the natural sequence context (Fig. 3, A and D), in accord with results obtained previously for the identical mutations in the mouse PBREM in the tk sequence context (7) and for the identical NR1B mutation (24) and another NR1B mutation (4) of the rat PBRU in the Ev context. Furthermore, double half-site mutations of both NR1 and NR2 (NR1A-NR2A and NR1A-NR2B) abolished PB responsiveness conferred by the PBRU in the natural sequence context (Fig. 3 B), as do double half-site mutations of the mouse PBREM in the tk sequence context (7).
Identification and functional characterization of a conserved, nuclear factor 1-like element in the proximal promoter region of CYP1A2 gene specifically expressed in the liver and olfactory mucosa
2000, Journal of Biological ChemistryCitation Excerpt :NF-1-like elements containing the highly conserved TGG motif have been found in the promoters and enhancers of many genes. Of particular interest, an NF-1 site is present in the phenobarbital-responsive enhancer module in mouse Cyp2b10 and rat CYP2B2 genes and is important for the magnitude of the induction response (51-53). An NF-1 element was also found to be a functional component of the mouseCyp1a1 promoter in a study with mouse hepatoma cells (54); a similar site is present in rat and human CYP1A1 promoter and was found to be involved in the down-regulation of CYP1A1gene expression by oxidative stress (55).
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