The role of CREM2 gene in the expression of CYP1A1 gene

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Our group identified CERM gene as a one of the components in a new signal transduction pathway that mediates CYP1A1 induction. In order to clarify the function of CREM gene product, we analyzed the cis-element in the regulatory sequence of Cyp1a1 gene using reporter gene assay. We successfully narrowed down the responsive sequence of CREM and found BTE (basic transcription element) in this sequence. Then, several mutations in the consensus sequence were introduced to test the initiation ability of transcription by the method of reporter gene assay. The mutated cis-element of BTE decreased the transcription of Cyp1a1.Furthermore, a possible protein factor(s) that can bind to this sequence was investigated by EMSA (electromobility shift assay) method. The nuclear extract was prepared from HepG2 cells and was mixed with 32P-labeled cis-element. After 20 min incubation at 25℃, the nuclear protein and oligonucleotide mixture was separated on non-denaturation acrylamide gel and the dried gel was exposed to imaging plate to visualize the retarded band that was a complex of proteins with DNA. There were several specific bands that were abolished by the addition of vast excess of non-labeled oligonucleotide. Although these bands were observed in the nuclear extract from non-treated sample, the band intensity increased remarkably by the treatment of omeprazole. Therefore, it was suggested that CREM might make a complex with Sp1 and might stimulate the transcription of Cyp1a1. To verify the possible involvement of CREM in the process of Cyp1a1 induction, gene-silencing experiment of CREM in HepG2 cells is performing with siRNA of CREM.