MULTIPLE FACTORS REGULATING THE EXPRESSION OF HUMAN THROMBOXANE SYNTHASE GENE

Characterization of the 5.5 kb promoter of human thromboxane synthase (TS) gene revealed a proximal positive regulatory sequence (PPRS, -90 to -25 bp) and several distal repressive elements. The maximal promote r activity was found to reside within the first 285 bp, similar to 75 % of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (similar to 55 %) on report er gene expression independent of orientation and position, consistent with properties expected for a silencer. The sequence upstream of - 6 65 bp to -5.5 kb contains mainly repressive elements which further red uce the promoter activity by 30 %. The 65 bp PPRS worked in an orienta tion-independent, but position-dependent, manner and could be further divided into two independent elements, PPRS, (-90 to -50 bp) and PPRS, (-50 to -25 bp), While similar nuclear factor(s) from different cell types interact with PPRS(2), those interacting with PPRS(2) exhibit ce ll specificity. Internal sequence deletion and oligonucleotide competi tion established that a binding sequence for NF-E2 in PPRS(1) (-60 tgc tgattcat -50) was important for enhancing TS promoter activity in HL-6 0 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by reverse-transcription PCR amplification of the cDNA and Northern blot analysis, A 9-fold transactivation of luciferase (luc) reporter gene expression had been detected when NF-E2 cDNA was co-expressed with a T S promoter/luc construct. Despite the fact that NF-E2 and the cis-elem ents could alter the efficiency of TS transcription, they were not suf ficient for restricting cell-specific TS expression. Analysis of the m ethylation status at the TS promoter in several human cell lines revea ls cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elemen ts, trans-activator(s), and possibly genomic methylation.