CHARACTERIZATION OF THE PROXIMAL PROMOTER AND 2 SILENCER ELEMENTS IN THE CYP2C11 GENE EXPRESSED IN RAT-LIVER

The cytochrome P450 gene CYP2C11, expressed in the liver of male rats, is transcriptionally regulated in a dual fashion by the sexually dimo rphic secretion pattern of growth hormone. To enable analysis of trans criptional regulatory DNA elements, Fat genomic sequences were cloned. DNase I hypersensitivity analysis of rat liver nuclei revealed the ex istence of two hypersensitive sites whose presence in the vicinity of the transcription start site correlates to high transcriptional activi ty of the gene. Deletion mutants of the 5' flank were fused to reporte r genes and transiently transfected into HepG2 cells or into primary a dult rat hypatocytes. Transfection experiments in combination with DNa se I footprinting analysis in vitro led to the identification of two n egative regulatory regions spanning nucleotides -1,230 to -1,188 and - 409 to -368 and designated (SIL1200) and (SIL400), respectively. When placed in front of the heterologous thymidine kinase promoter, SIL1200 and SIL400 reduced the activity of the chloramphenicol acetyl transfe rase reporter gene to 13% and 23% of the control value, respectively. No sex-dependent binding of liver nuclear extracts to the two silencer s could be detected by in vitro footprinting or gel retardation assays . However, a sex-dependent footprint consistently stronger with male l iver nuclear extracts than with female extracts was observed in the -3 20 to -294 region. A significant level of identity was found between t he DNA sequence corresponding to this footprint and that of orphan ste roid receptor elements as well as with that of a basal transcription e lement common to several CYP2C genes. However, the identity of a poten tial trans-acting factor binding between -320 and -294 or response of this element to growth hormone is as yet unknown. A sex- and GH secret ory profile-dependent protein-DNA interaction in vitro was observed in the -107 to -95 region. In spite of the sequence similarity that exis ts between this region and the consensus binding site for HNF-1, this region does not bind HNF-1 alpha. This element acted as a repressor on the heterologous thymidine kinase promoter. To date, the two silencer elements and possibly also the HNF-1-like element are the only functi onal elements defined in the CYP2C11 gene, and it is conceivable that induction of the gene involves derepression of the silencer elements.