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 promoter activity was found to reside within the first 285 bp, ∼75% of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (∼55%) on reporter gene expression independent of orientation and position, consistent with properties expected for a silencer. The sequence upstream of -665 bp to -5.5 kb contains mainly repressive elements which further reduce the promoter activity by 30%. The 65 bp PPRS worked in an orientation-independent, but position-dependent, manner and could be further divided into two independent elements, PPRS₁ (-90 to -50 bp) and PPRS₂ (-50 to -25 bp). While similar nuclear factor(s) from different cell types interact with PPRS₂, those interacting with PPRS₁ exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS₁ (-60 tgctgattcat -50) was important for enhancing TS promoter activity in HL-60 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 TS promoter/luc construct. Despite the fact that NF-E2 and the cis-elements could alter the efficiency of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status at the TS promoter in several human cell lines reveals 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-elements, trans-activator(s), and possibly genomic methylation.