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, approximately 75% of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (approximately 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.
Lee
K D
KD
Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore, MD USA.
Baek
S J
SJ
Shen
R F
RF
D000818:Animals; D016466:CHO Cells; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D006224:Cricetinae; D019175:DNA Methylation; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D018922:HL-60 Cells; D006801:Humans; D008113:Liver Neoplasms; D008156:Luciferases / Q000096:biosynthesis; D051379:Mice; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D013930:Thromboxane-A Synthase / Q000096:biosynthesis* / Q000235:genetics*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D008156:Luciferases; D013930:Thromboxane-A Synthase