Ishimaru Naoki, Tabuchi Akiko, Hara Daichi, Hayashi Hiroyuki, Sugimoto Takayuki, Yasuhara Masahiro, Shiota Jun, Tsuda Masaaki
Department of Biological Chemistry, Graduate School of Medicine, University of Toyama, Sugitani, Japan.
J Neurochem. 2007 Jan;100(2):520-31. doi: 10.1111/j.1471-4159.2006.04216.x. Epub 2006 Oct 24.
Neurotrophin-3 (NT-3), a neurotrophin member, plays crucial roles in neuronal development, function and plasticity. Previous studies have demonstrated that NT-3 gene transcription is driven by alternative promoters A and B, located upstream of exons 1A (EIA) and 1B (EIB), respectively. However, the transcription factors and DNA elements that drive NT-3 gene transcription remain to be identified. Here, we analysed the promoter region of the NT-3 gene and found that an NT-3 transcript containing EIB is predominantly expressed in cortical neurons which preferentially utilize promoter B, and two tandemly repeated GC-boxes, located between -100 and -60 base pairs within promoter B, are required for the transcription. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that both specificity protein (Sp)3 and Sp4 were able to bind to the Sp1 binding sequences within the GC boxes. Expression of dominant-negative Sp3 and Sp4 small interfering RNA in cortical neurons reduced the activity of the NT-3 gene promoter. Over-expression of Sp1 family members, especially Sp4, resulted in an increase of the NT-3 gene promoter. These findings indicate that the NT-3 gene is a target gene for Sp4 that is abundantly expressed in the brain.
神经营养因子-3（NT-3）作为神经营养因子家族的一员，在神经元发育、功能及可塑性方面发挥着关键作用。先前的研究表明，NT-3基因转录由分别位于外显子1A（EIA）和1B（EIB）上游的A和B两个可变启动子驱动。然而，驱动NT-3基因转录的转录因子和DNA元件仍有待确定。在此，我们分析了NT-3基因的启动子区域，发现包含EIB的NT-3转录本主要在优先利用启动子B的皮质神经元中表达，且启动子B内-100至-60碱基对之间的两个串联重复GC盒是转录所必需的。电泳迁移率变动分析和染色质免疫沉淀分析表明，特异性蛋白（Sp）3和Sp4均能与GC盒内的Sp1结合序列结合。在皮质神经元中表达显性负性Sp3和Sp4小干扰RNA可降低NT-3基因启动子的活性。Sp1家族成员，尤其是Sp4的过表达导致NT-3基因启动子活性增加。这些发现表明，NT-3基因是在脑中大量表达的Sp4的靶基因。
