Identification and Characterization of Transcriptional Regulatory Elements for Liver-Specific Expression of Rabbit CYP2C Genes

108 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.Heme-containing monooxygenases cytochrome P450 are involved in the oxidative metabolism of many xenobiotic substances and endogenous compounds. These enzymes are encoded by a superfamily of CYP genes, among which mammalian CYP2 is the largest family with a predominant liver-specific expression pattern. We have developed a functional analysis system using rabbit CYP2C 5$\sp\prime$ flanking region-luciferase chimeric constructs to transiently transfect hepatoma cells (HepG2) and demonstrated the presence of multiple cis-acting regulatory elements in three closely related CYP2C genes (2C1, 2C2 and 2C3). The most active promoter motifs which confer the hepatic specific activation of these genes are located about 100 bp upstream of their mRNA initiation sites. Both DNA binding and functional analysis strongly suggest that this hepatic specific element is a binding motif of a liver-enriched transcriptional factor hepatocyte nuclear factor 4 (HNF-4), since it was recognized by a nuclear protein with the same tissue distribution, DNA binding specificity and immunochemical reactivity as those of HNF-4. The CYP2C2 promoter-luciferase constructs containing either a native or an authentic HNF-4 site showed comparable promoter activities in HepG2 cells and both could be potently trans-activated by coexpressed HNF-4 in nonhepatic COS-1 cells. The HNF-4 sites in the highly related CYP2C genes bound to HNF-4 with significantly different affinities, with the order of 2C2 $>$ 2C1 $> >$ 2C3. The binding affinity differences correlated well with the transcription activity of each HNF-4 element present in its minimal hepatic promoter or imbedded in the same flanking sequence context. A single nucleotide change in the CYP2C3 HNF-4 element could convert it to a motif of significantly higher binding affinity and hepatic promoter activity. Sequence comparison revealed that at least one copy of an HNF-4 like motif is present within the first 200 bp immediate upstream regions of more than 20 CYP2A, 2C and 2D genes at a conserved position in each subfamily. Taken together, these results clearly indicate that HNF-4 is the crucial transactivator for hepatic cell specificity of these CYP2C promoters, and suggest that HNF-4 is very likely to be a common regulator for the liver-specific expression of the majority of CYP2 genes.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD