Moon Sung-Min, Kim Jae-Sung, Park Bo Ram, Kim Do Kyung, Kim Su-Gwan, Kim Heung-Joong, Chun Hong Sung, Lee Byung-Kwon, Kim Chun Sung
aSchool of Dentistry, Oral Biology Research Institute bDepartment of Biomedical Science cThe Division of Natural Medical Sciences, College of Health Science, Chosun University, Gwangju, Republic of Korea dDepartment of Microbiology, University of Tennessee, Knoxville, Tennessee, USA.
Neuroreport. 2015 Feb 11;26(3):144-51. doi: 10.1097/WNR.0000000000000316.
The neurotrophin-inducible gene VGF plays an important role in the maintenance of organismal energy balance and in the mediation of hippocampal synaptic activity. The regulatory mechanism of VGF transcription is not fully understood. The neuron-restrictive silencer factor (NRSF) binds with the neuron-restrictive silencer element (NRSE), thereby suppressing the transcription of NRSE-containing genes. In this study, we show that the NRSE sequence of the VGF gene critically regulates the repression of VGF expression in NMB cells. Sequence analysis also establishes the presence of two putative NRSEs (NRSE-1 and NRSE-2) in the promoter region of the VGF gene. In reporter gene experiments, a more than eight-fold increase in the promoter activity was observed when both NRSE-1 and NRSE-2 were deleted. Deletion of NRSE-2 alone did not affect the promoter activity, thus indicating that NRSE-1 could be solely responsible for the repression of VGF gene expression. Mutations in the NRSE-1 sequence increased promoter activity. However, no change in activity was observed when NRSE-1 was coexpressed with dominant-negative NRSF, thereby suggesting that endogenous NRSF interacts with NRSE-1. Binding of NRSF to NRSE in a sequence-specific manner was confirmed with chromatin immunoprecipitation assays, respectively. Furthermore, the overexpressed NRSF in PC12 cells significantly suppressed the VGF gene expression by interacting with the NRSE located in the VGF promoter region. Our results indicate that NRSF plays an important role as a repressor of VGF gene regulation in NMB cells through a mechanism that is dependent on VGF-NRSE.
神经营养因子诱导基因VGF在维持机体能量平衡及介导海马突触活动中发挥重要作用。VGF转录的调控机制尚未完全明确。神经元限制性沉默因子（NRSF）与神经元限制性沉默元件（NRSE）结合，从而抑制含NRSE基因的转录。在本研究中，我们发现VGF基因的NRSE序列对NMB细胞中VGF表达的抑制起着关键调控作用。序列分析还证实VGF基因启动子区域存在两个假定的NRSE（NRSE-1和NRSE-2）。在报告基因实验中，当NRSE-1和NRSE-2均缺失时，启动子活性观察到超过八倍的增加。单独缺失NRSE-2不影响启动子活性，这表明NRSE-1可能单独负责VGF基因表达的抑制。NRSE-1序列中的突变增加了启动子活性。然而，当NRSE-1与显性负性NRSF共表达时，未观察到活性变化，这表明内源性NRSF与NRSE-1相互作用。染色质免疫沉淀试验分别证实了NRSF以序列特异性方式与NRSE结合。此外，PC12细胞中过表达的NRSF通过与VGF启动子区域的NRSE相互作用，显著抑制了VGF基因表达。我们的结果表明，NRSF通过依赖于VGF-NRSE的机制，作为NMB细胞中VGF基因调控的阻遏物发挥重要作用。
