Cheng Chi Keung, Yeung Chung Man, Hoo Ruby L C, Chow Billy K C, Leung Peter C K
Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada V6H 3V5.
Endocrinology. 2002 Dec;143(12):4693-701. doi: 10.1210/en.2002-220576.
Previous deletion analysis of the 5'-flanking region of human GnRH receptor (GnRHR) gene has revealed a powerful negative regulatory element (NRE) located between nucleotide -1017 and -771. In the present study, we demonstrated that this NRE could repress the homologous promoter, irrespective of its position and completely abolish the activity of a heterologous thymidine kinase promoter in an orientation-dependent manner. Progressive 3'-deletion analysis revealed that most of the silencing activity of the NRE resided in a putative octamer regulatory sequence (5'AAGCAAACT3'), which alone could repress the promoter activities by 69-90% in ovarian OVCAR-3, placental JEG-3, and gonadotrope-derived alphaT3-1 cells. Mutation of the AAAC residues of the octamer sequence completely removed its silencing activity. Interestingly, conversion of the octamer sequence into that of the rodent GnRHR promoter (5'AAGCAAAGT3') did not attenuate its silencing effect, indicating that the repressive role of the octamer sequence is evolutionarily conserved. EMSAs showed that common DNA-protein complexes of the same mobility were formed with nuclear extracts from the reproductive cells and gonadotropes, and a consensus octamer transcription factor-1 (Oct-1) binding sequence could dose dependently inhibit the complex formation. Antibody supershift and Southwestern blot assays confirmed that the protein binding to the octamer sequence was the ubiquitously expressed transcription factor Oct-1. Overexpression of Oct-1 augmented the silencing activity of the octamer sequence in alphaT3-1 cells. Taken together, our results clearly indicate a role of Oct-1 in the transcriptional repression of the human GnRHR gene.
先前对人促性腺激素释放激素受体（GnRHR）基因5'侧翼区的缺失分析显示，在核苷酸-1017至-771之间存在一个强大的负调控元件（NRE）。在本研究中，我们证明该NRE可抑制同源启动子，无论其位置如何，并以方向依赖的方式完全消除异源胸苷激酶启动子的活性。渐进性3'缺失分析表明，NRE的大多数沉默活性存在于一个假定的八聚体调控序列（5'AAGCAAACT3'）中，该序列单独可使卵巢OVCAR-3、胎盘JEG-3和促性腺激素细胞来源的αT3-1细胞中的启动子活性降低69-90%。八聚体序列中AAAC残基的突变完全消除了其沉默活性。有趣的是，将八聚体序列转换为啮齿动物GnRHR启动子的序列（5'AAGCAAAGT3'）并没有减弱其沉默效果，表明八聚体序列的抑制作用在进化上是保守的。电泳迁移率变动分析（EMSA）显示，生殖细胞和促性腺激素细胞的核提取物形成了迁移率相同的常见DNA-蛋白质复合物，并且一个共有八聚体转录因子-1（Oct-1）结合序列可剂量依赖性地抑制复合物的形成。抗体超迁移和蛋白质印迹分析证实，与八聚体序列结合的蛋白质是普遍表达的转录因子Oct-1。Oct-1的过表达增强了αT3-1细胞中八聚体序列的沉默活性。综上所述，我们的结果清楚地表明Oct-1在人GnRHR基因的转录抑制中起作用。
