Kwak Inseok, Song Sihong, Blum Jason L, Simmen Rosalia C M, Simmen Frank A
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.
DNA Cell Biol. 2006 Jan;25(1):6-18. doi: 10.1089/dna.2006.25.6.
The regulation of insulin-like growth factor-binding protein-2 (IGFBP-2) gene transcription in specific cell and developmental contexts is not well understood. Here, we identified DNA regions that mediate IGFBP-2 gene transcription in two of the three major cell types of the uterine endometrium of the early pregnant pig. Two clusters of transcriptional start sites at nucleotides -109/-105 and -96/-87 (+1, translational initiation site) in the porcine IGFBP-2 gene were localized in uterine endometrium and in primary cultures of endometrial glandular epithelial (GE) and stromal (ST) fibroblastic cells. Upstream regions of this gene (spanning -1,397/+73) were fused to a luciferase reporter gene, and the constructs were transiently transfected into endometrial GE and ST cells representative of pregnancy days 12 and 18 (day 115 = parturition). A short (110 bp) upstream region (-874/-765) stimulated the IGFBP-2 and heterologous SV40 promoters in the two cell types at both pregnancy days. Two noncontiguous copies of the novel sequence motif TCAGGG within the 110-bp fragment were implicated in transcriptional activity, since block mutation of these sequences led to a repression of SV40 basal promoter activity in endometrial cells. Southwestern blotting identified an endometrial nuclear protein of 34-kDa molecular weight that bound an oligonucleotide containing this motif, and EMSA suggested robust expression of this protein in early pregnancy endometrium and in ovary but at much reduced levels in endometrium at later pregnancy. A pair of E-box elements (CANNTG) within the 110 bp region was stimulatory to IGFBP-2 promoter activity; block mutation of these converted the 110-bp region into a potent transcriptional silencer in all but day 18 ST cells. Results identify novel DNA motifs that regulate the IGFBP-2 gene promoter in uterine endometrium in pregnancy-associated fashion.
胰岛素样生长因子结合蛋白2（IGFBP-2）基因转录在特定细胞和发育环境中的调控机制尚不清楚。在此，我们在妊娠早期母猪子宫内膜的三种主要细胞类型中的两种中鉴定出介导IGFBP-2基因转录的DNA区域。猪IGFBP-2基因中位于核苷酸-109/-105和-96/-87（+1，翻译起始位点）的两个转录起始位点簇定位于子宫内膜以及子宫内膜腺上皮（GE）和基质（ST）成纤维细胞的原代培养物中。该基因的上游区域（跨度为-1,397/+73）与荧光素酶报告基因融合，并将构建体瞬时转染到代表妊娠第12天和第18天（第115天=分娩）的子宫内膜GE和ST细胞中。一个短的（110 bp）上游区域（-874/-765）在两个妊娠日刺激了两种细胞类型中的IGFBP-2和异源SV40启动子。110 bp片段内新的序列基序TCAGGG的两个不连续拷贝与转录活性有关，因为这些序列的阻断突变导致子宫内膜细胞中SV40基础启动子活性的抑制。蛋白质印迹法鉴定出一种分子量为34 kDa的子宫内膜核蛋白，其与含有该基序的寡核苷酸结合，电泳迁移率变动分析表明该蛋白在妊娠早期子宫内膜和卵巢中大量表达，但在妊娠后期子宫内膜中的表达水平大大降低。110 bp区域内一对E-box元件（CANNTG）对IGFBP-2启动子活性有刺激作用；除第18天的ST细胞外，这些元件的阻断突变将110 bp区域转变为强效转录沉默子。结果鉴定出以妊娠相关方式调节子宫内膜中IGFBP-2基因启动子的新DNA基序。
