CHARACTERIZATION OF A REGION UPSTREAM OF EXON I.1 OF THE HUMAN CYP19 (AROMATASE) GENE THAT MEDIATES REGULATION BY RETINOIDS IN HUMAN CHORIOCARCINOMA CELLS

The biosynthesis of estrogens is catalyzed by aromatase P450 (P450(aro m)), the product of the CYP19 gene. The tissue-specific expression of the CYP19 gene is regulated by means of tissue-specific promoters thro ugh the use of alternative splicing mechanisms. Thus, transcripts cont aining various 5'-untranslated termini are present in ovary, brain, ad ipose stromal cells, and placenta. Sequence corresponding to untransla ted exon I.1 is present uniquely in 5'-termini of transcripts expresse d in human placenta and choriocarcinoma cells, as a consequence of exp ression driven by a distal promoter, I.1. The goal of the present stud y was the identification of regulatory elements in this promoter regio n. Various deletion mutations of the upstream nanking region of exon I .1 were constructed using the PCR or restriction enzyme digestion. The genomic fragments were fused up-stream of the luciferase reporter gen e. These constructs were transfected into human choriocarcinoma (JEG3) cells. The longest construct employed, -924/+10 bp, expressed the hig hest luciferase reporter gene activity. The -64/+10 bp and -125/+10 bp constructs showed no reporter gene expression. Transfection of the -2 01/+10 bp construct resulted in reporter gene expression, but at a low er level than that of the -924/+10 bp construct, and this expression w as induced by serum as well as by LG69 and TTNPB, ligands specific for RXR and RAR respectively, as well as by vitamin D. These results para llel the actions of the ligands on aromatase activity. Mutation or del etion of an imperfect palindromic sequence (AGGTCATGCCCC) located at - 183 to -172 bp upstream of the transcriptional start site of exon I.1 resulted in loss of basal-and retinoid-induced reporter gene expressio n. Gel retardation analysis using nuclear extracts of JEG3 cells treat ed with retinoids and the imperfect palindromic sequence as probe, sho wed that proteins present in the nuclear extracts bound to this sequen ce in a specific fashion. The binding activities were elevated by incu bation of the cells with LG69 and TTNPB, ligands specific for RXR and RAR respectively. Binding of nuclear proteins to the palindromic seque nce was displaced either by anti-RXR alpha serum or by anti-VDR serum, suggesting the formation of a heterodimer of RXR alpha and VDR. These results suggest that the imperfect palindromic sequence upstream of e xon I.1 plays an important but novel role in the regulated expression of the CYP19 gene in choriocarcinoma cells.