Miyachi T, Maruyama H, Kitamura T, Nakamura S, Kawakami H
Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Brain Res Mol Brain Res. 1999 Jun 8;69(2):223-31. doi: 10.1016/s0169-328x(99)00112-6.
In this study, we isolated and characterized the human NeuroD (BETA2/BHF1) gene. This gene was found to consist of two exons and one intron. The promoter regions were well-conserved compared with the mouse NeuroD gene. Two transcription start points (TSPs) were determined by the oligo-capping method. One TATA box was located at -31 bp from the lower TSP. The results of a transient transfection assay using the human neuroblastoma cell line IMR-32 and hamster insulin tumor cell line HIT-T15 suggested that there are at least three positive regulatory regions in the promoter. In these regions, four E boxes (CANNTG), named the E1 to E4 boxes, and two GC boxes were present. Cotransfection of the NeuroD expression vector into IMR-32 cells enhanced the NeuroD promoter activity by about 4-fold. A deletion and mutation analysis revealed that the E1 and E4 boxes, especially the E1 box, are associated with autoactivation and that E2 and E3 boxes are not associated with autoactivation. As mutation analysis of E3 box showed a decrease in the enhancer activity to the basal level, it showed that the E3 box is important to activate the NeuroD transcription. These results raised the possibility that the NeuroD gene expression is positively regulated through the E box sequence, not only by NeuroD itself but also by another E box binding protein.
在本研究中，我们分离并鉴定了人类NeuroD（BETA2/BHF1）基因。发现该基因由两个外显子和一个内含子组成。与小鼠NeuroD基因相比，其启动子区域高度保守。通过寡核苷酸帽法确定了两个转录起始点（TSP）。一个TATA盒位于较低TSP下游31 bp处。使用人类神经母细胞瘤细胞系IMR-32和仓鼠胰岛素瘤细胞系HIT-T15进行的瞬时转染分析结果表明，启动子中至少存在三个正调控区域。在这些区域中，存在四个E盒（CANNTG），命名为E1至E4盒，以及两个GC盒。将NeuroD表达载体共转染到IMR-32细胞中可使NeuroD启动子活性增强约4倍。缺失和突变分析表明，E1和E4盒，尤其是E1盒，与自激活相关，而E2和E3盒与自激活无关。由于E盒的突变分析显示增强子活性降至基础水平，表明E3盒对于激活NeuroD转录很重要。这些结果增加了一种可能性，即NeuroD基因表达不仅通过NeuroD自身，还通过另一种E盒结合蛋白，通过E盒序列受到正调控。
