Transcriptional activation of the human hematopoietic prostaglandin D synthase gene in megakaryoblastic cells. Roles of the oct-1 element in the 5'-flanking region and the AP-2 element in the untranslated exon 1

The human hematopoietic prostaglandin D synthase (H-PGDS) gene is highly expressed in human megakaryoblastic cells, in which phorbol ester induces its expression. We characterized the promoter activity of the 5'-flanking region and the untranslated exon 1 (-1044 to +290) of the human H-PGDS gene in human megakaryoblastic Dami cells. Transient expression analysis using the luciferase reporter gene revealed that the 5'-flanking region and the untranslated exon 1 were sufficient for efficient expression of the H-PGDS gene in Dami cells, but not in monocytic U937 cells. Deletion and site-directed mutagenesis of the Oct-1 element in the 5'-flanking region decreased the promoter activity by approximately 30% compared with that of the entire region from -1044 to +290. An electrophoretic mobility shift assay demonstrated that Oct-1 specifically bound to the promoter region. Interestingly, even only untranslated exon 1 (+1 to +290) showed approximately 60% of the promoter activity of the entire region from -1044 to +290. Site-directed mutagenesis of the AP-2 element within the untranslated exon 1 abolished the basal promoter activity as well as its phorbol ester-mediated up-regulation. In AP-2-deficient HepG2 cells, the H-PGDS promoter activity was enhanced by coexpression with AP-2alpha. These findings indicate that the Oct-1 element in the 5'-flanking region acts as a positive cis-acting element and that the AP-2 element in the untranslated exon 1 is crucial for both basal and phorbol ester-mediated up-regulation of human H-PGDS gene expression in megakaryoblastic Dami cells.     

与人PC-1同源的鼠PC-1基因的启动子的分子克隆和特性分析

为了鉴定鼠mPC-1基因表达的调控元件,克隆并分析了该基因的启动子.构建了一系列mPC-1基因启动子的截短序列.通过荧光素酶报道基因,分析了它们在前列腺癌细胞和其它细胞中的表达.结果表明,在AR阳性细胞系中,mPC-1基因启动子活性远远高于SV40和p61-PSA 启动子,mPC-1基因启动子 599 bp 至449bp 可能含有一个负调控元件; mPC-1 1.1 kb 启动子控制的表达主要在前列腺癌细胞系中; 雄激素可调控mPC-1 1.1kb 启动子表达.mPC-1 1.1kb 序列是一个有前列腺癌细胞特异性和较强的启动子,经过进一步的修饰有可能作为一种有用的前列腺癌基因治疗元件.     

Studies on the promoter region of the c-Ha-ras gene in FRTL5 rat thyroid cells

The functional activity of the promoter region of the rat c-Ha-ras gene was examined in FRTL5 rat thyroid cells, the cell type from which this promoter was cloned. A plasmid (p035-ras-CAT) was constructed containing the untranslated-1 exon as well as 172 base pairs (bp)5' to this exon inserted upstream of the chloramphenicol acetyl transferase (CAT) reporter gene. These 172 bp of 5'-flanking region contain two 10 bp GC box consensus sites and two CAAT boxes. Very weak promoter activity was observed in experiments involving transient transfection of FRTL5 cells with this plasmid, as well as with another plasmid (p5kb-ras-CAT) containing a much more extensive (3.5 kb) 5'-flanking region of the gene. In contrast, strong promoter activity was observed when the same plasmids were transfected into mouse 3T3 fibroblasts. When other promoters (pfos, RSV, and MMTV) were used to drive CAT activity, CAT activity in FRTL5 cells was about 10-fold less than in NIH-3T3 cells and rat embryo fibroblasts. However c-Ha-ras promoter activity was reduced out of proportion in FRTL5 thyroid cells relative to the other cell types (approximately 50-fold less). DNA gel-shift assays performed using crude extracts of FRTL5 and 3T3 nuclear proteins revealed quantitatively similar binding to the same promoter region in the c-Ha-ras 5'-flanking sequence. These data demonstrate that promoter activity of the rat c-Ha-ras gene is contained within the 172 bp 5'-flanking region of the gene. This promoter activity is expressed at a much lower level in slow-growing FRTL5 cells relative to other more rapidly growing cell types.     

The AP-1 sequence is necessary but not sufficient for phorbol induction of collagenase in fibroblasts.

Collagenase, the only enzyme active at neutral pH that initiates collagen degradation, is a major gene product of fibroblasts that have been stimulated with a variety of agents, including phorbol esters. To study mechanisms controlling collagenase gene expression, we transiently transfected rabbit synovial fibroblasts with chimeric constructs containing up to 1.2 kb of the rabbit collagenase 5'-flanking DNA linked to the chloramphenicol acetyltransferase gene (CAT). Our data indicate that the magnitude of the phorbol response is directly linked to the size of the promoter fragment and that the smallest piece of promoter DNA conferring phorbol inducibility is 127 bp. Deletional and mutational analysis of this fragment revealed that the AP-1 sequence alone is insufficient for phorbol inducibility and the presence of at least two additional sequences (a PEA3-like element and a sequence that includes 5'-TTCA-3') is required. In addition, a substantial increase in responsiveness is seen when a fragment containing 182 bp of 5'-flanking DNA is transfected, implicating a 36 bp region located between -182 and -149 as an enhancer. We conclude (1) that the AP-1 sequence is necessary but insufficient for expression of collagenase in adult fibroblasts, (2) that phorbol inducibility depends on cooperation among several sequence elements within the collagenase promoter, and (3) that regulation of this promoter is more complex than previously described.