Cooperative regulation of CYP3A5 gene transcription by NF-Y and Sp family members

The purpose of this study was to clarify the mechanism(s) responsible for t he transcriptional regulation of the human CYP3A5 gene. It was found that a region from nucleotides -90 to -40 was involved in the transcriptional act ivity of the CYP3A5 gene by transfection of a series of 5 ' -truncated prom oter-luciferase chimeric genes into human hepatoma HepG2 cells. A gel shift assay using nuclear extracts prepared from HepG2 cells showed that nuclear factor-Y (NF-Y) and specificity protein (Sp) 1 and Sp3 bound to CCAAT box (-78/-68) and a basic transcription element (BTE) (-67/-46) in the CYP3A5 g ene. Furthermore, introduction of mutations in the CCAAT box, the BTE, or b oth elements decreased the transcriptional activity to 10, 21, or 4% of tha t seen with the intact gene. Thus, we conclude that the transcription of th e CYP3A5 gene is cooperatively regulated by NF-Y, Sp1, and Sp3 in HepG2 cel ls. (C) 2001 Academic Press.