TRANSCRIPTIONAL REGULATION OF CYTOCHROME P-450 GENES

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A number of P450 genes are constitutively expressed in rodent liver. Some, such as the rat CYP2EI and CYP2AI genes, are absent until birth at which time they become transcriptionally activated. The CYP2El remains active in both male and female throughout adult life, whereas the CYP2Al gene is repressed in males at puberty but remains active in females. A related gene, CYP2A2, is transcriptionally inactive throughout the life of females but becomes active in males at puberty concomitant with suppression of the CYP2AI gene. The rat CYP2D genes are also activated at certain stages of development. CYP2D3 is activated right after birth, while CYP2D5 is activated at puberty in both sexes. To investigate the mechanism of regulation of the genes, we are using procedures that can identify cis-acting DNA elements and transacting factors responsible for transcriptional activation. In vitro cell free transcription systems are prepared from livers of rats of various ages and used to transcribe cloned P450 gene template. Deletion analyses are undertaken to identify important regulatory regions upstream of the RNA polymerase II promoter. DNA binding analyses are used to identify cis-acting elements capable of binding nuclear transcription factors. Using these procedures we have identified the elements and factors controlling developmental activation of the CYP2EI gene. The mechanism of regulation of the CYP2A genes remains elusive. Studies are also underway to determine the mechanism of induction of P450 genes by foreign compounds. The CYP4AI and CYP4A2 genes were cloned and completely sequenced. These genes can be transcriptionally activated by the hypolipidemic drug clofibrate. The promoter regions of CYP4A genes were fused to the report gene chloramphenicol acetyltransferase. Transfection of these constructs into rat hepatoma cells or rat hepatocytes yielded no activity after treatment with clofibrate. Further studies are being carried out using fragments of DNA up to 20 kilobase pairs upstream of the gene's start site to search for sequences that can activate heterologous promoters in the presence of clofibrate.