NF-κB Site Interacts with Sp Factors and Up-regulates the NR1 Promoter during Neuronal Differentiation

Abstract

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-B site sharing the consensus with the immunoglobulin (Ig)/human immunodeficiency virus NF-B 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-B-dependent promoter activity. The N-methyl-D-aspartate (NMDA) 1 subtype of glutamate receptors plays an important role in neuronal differentiation and plasticity. The essential component of the NMDA receptor complexes is encoded by the NR1 gene (1, 2). This gene is induced during neurogenesis and up-regulated in neurons undergoing differentiation (3, 4). In studies of NR1 gene upregulation, a negative mechanism has been proposed: repressor element 1 (RE1) silencing transcription factor (REST)/neuronrestriction silencer factor (NRSF) acts on the RE1 site in the NR1 promoter and suppresses transcription in neural progenitors. After neurogenesis, REST/NRSF is down-regulated, and the suppression of the NR1 gene is removed to let de-repression occur (4). In studies of neuronal differentiation of P19 cells, we observed that there was a time gap between the down-regulation of the REST/NRSF protein and the gradually robust upregulation of the NR1 mRNA/promoter activity during neuronal differentiation (4), suggesting that a positive mechanism is required for NR1 gene transcription after de-repression. The NR1 promoter contains in the proximal region GC-boxes and MEF2 sites that interact with transcription activators sensitive to neuronal differentiation and thus could contribute to this positive mechanism (5-7). However, our recent observation suggests that the distal region of this promoter is required for fully up-regulated transcription (4) and very likely contains one or more important enhancers that positively respond to the signals of neuronal differentiation.Sequence analysis of the NR1 promoter uncovered a putative nuclear factor-B (NF-B) site in the region distal to the transcription start points (TSPs) (4). NF-B sites interact with dimerized NF-B factors and positively regulates cis-promoter. Five different NF-B factors have been found in mammalian tissues, p65 (Rel-A), p50, p52, C-Rel, and RelB. Homo-or heterodimers of NF-B factors are associated with IB factor in the cytoplasm and remain inactive. Activating signals triggered by environmental cues induce IB dissociation, thus releasing NF-B dimers that then become activated and nuclearly translocated to regulate transcription of target genes (8 -10). In the nervous system, NF-B activity is differentially distributed in the developing brain at different stages (11). Fo...

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