Liu Anguo, Hoffman Peter W, Lu Weiwei, Bai Guang
Department of Biomedical Sciences, Dental School, Program in Neuroscience, and Program in Cellular and Molecular Biology, University of Maryland, Baltimore, Maryland 21201, USA.
J Biol Chem. 2004 Apr 23;279(17):17449-58. doi: 10.1074/jbc.M311267200. Epub 2004 Feb 16.
The NR1 gene undergoes induction in neurogenesis mainly via promoter de-repression, and up-regulation during neuronal differentiation by undefined mechanism(s). Here, we show that in the distal region the NR1 promoter has an active NF-kappaB site sharing the consensus with the immunoglobulin (Ig)/human immunodeficiency virus NF-kappaB site. Mutation of this site significantly reduced NR1 promoter up-regulation during neuronal differentiation of P19 cells. Electrophoretic mobility shift assays revealed that P19 nuclei constitutively contained p50 and that neuronal differentiation not only increased nuclear p50 but also induced p65 nuclear translocation. Responding to this change was an up-regulation of NF-kappaB-dependent promoter activity. However, inhibition of NF-kappaB nuclear translocation by an IkappaBalpha super-repressor or decoy DNA only moderately inhibited NR1 promoter up-regulation. Interestingly, the NR1 NF-kappaB site strongly interacted with Sp3/Sp1, instead of NF-kappaB factors, in P19 nuclear extracts. This interaction was reduced for Sp3 following neuronal differentiation, accompanied by dynamic expression of Sp factors. Cotransfection of Sp factors (Sp1, 3, or 4) upregulated the NR1 NF-kappaB site dramatically in differentiated neurons, but only moderately in undifferentiated P19 cells. This up-regulation was strong for Sp1 in differentiated cells and for Sp3 in undifferentiated cells. Chromatin-immunoprecipitation assays further demonstrated that Sp1 and Sp3 interacted with the NR1 NF-kappaB site in situ, and Sp3 lost its interaction after neuronal differentiation. We conclude that the NF-kappaB site positively regulates the NR1 promoter during neuronal differentiation via interacting mainly with Sp factors and neuronal differentiation reduces the effect of Sp3 factor on this site.
NR1基因在神经发生过程中主要通过启动子去抑制作用被诱导，并且在神经元分化过程中通过未知机制上调。在此，我们表明在远端区域，NR1启动子有一个活跃的NF-κB位点，与免疫球蛋白（Ig）/人类免疫缺陷病毒NF-κB位点具有相同的共有序列。该位点的突变显著降低了P19细胞神经元分化过程中NR1启动子的上调。电泳迁移率变动分析表明，P19细胞核中组成性地含有p50，并且神经元分化不仅增加了细胞核中的p50，还诱导了p65的核转位。响应这种变化的是NF-κB依赖性启动子活性的上调。然而，用IkappaBalpha超级阻遏物或诱饵DNA抑制NF-κB核转位仅适度抑制了NR1启动子的上调。有趣的是，在P19核提取物中，NR1的NF-κB位点与Sp3/Sp1强烈相互作用，而不是与NF-κB因子相互作用。神经元分化后，Sp3的这种相互作用减弱，同时伴随着Sp因子的动态表达。在分化的神经元中，共转染Sp因子（Sp1、3或4）可显著上调NR1的NF-κB位点，但在未分化的P19细胞中仅适度上调。这种上调在分化细胞中对Sp1很强，在未分化细胞中对Sp3很强。染色质免疫沉淀分析进一步证明，Sp1和Sp3在原位与NR1的NF-κB位点相互作用，并且神经元分化后Sp3失去了这种相互作用。我们得出结论，NF-κB位点在神经元分化过程中通过主要与Sp因子相互作用来正向调节NR1启动子，并且神经元分化降低了Sp3因子对该位点的作用。
