Functional Analysis of the Rat N-Methyl-D-aspartate Receptor 2A Promoter

Abstract

N-Methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A) is an important modulatory component of the NMDA subtype of glutamate receptors. To investigate the transcription mechanism of the NR2A gene, we cloned the 5 -flanking sequence from a rat genomic library. RNA mapping with rat brain RNA revealed two sets of major and several minor transcription start points in a single exon of 1140 bp. Reporter gene and mutation studies indicated that core promoter activity resided in exon 1, whereas the 5 -flanking sequence up to 1.5 kb showed no significant impact on promoter activity. Fragments containing minor transcription start points were able to drive a reporter gene in transfected cells and produce nascent RNAs in an in vitro transcription system. All fragments tested showed more promoter activity in dissociated neurons of the rat embryonic cerebrocortex and cell lines expressing NR2A mRNA than that in glial cultures and non-neuronal cells. Within exon 1 there are three GC-box elements that displayed distinct binding affinity to both Sp1-and Sp4-like factors. Overexpression of Sp1 or Sp4, but not Sp3, significantly increased the activity of the promoter containing these elements. Inclusion of exon 2 and 3 sequences, which contain five short open-reading frames, attenuated promoter-driven reporter activity more than 3-fold but attenuated the level of reporter mRNA less than 1.4-fold. Our results suggest that the core promoter of the rat NR2A gene requires exon 1, that Sp factors positively regulate this core promoter, and that a post-transcriptional mechanism may negatively regulate expression of the gene.

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

“…For example, Sp3 represses several promoters that positively respond to Sp1 (23), whereas Sp3 and Sp1 both activate some promoters (21,22,27), including the GC-boxes of the NR1 promoter (26). From data of the current study, we believe that Sp1, 3, and 4 are activators of the NR1 promoter via interaction with the NF-B site.…”

“…Construction of Reporter Genes and Expression Constructs-All plasmids used in this study have been described previously (4,27) with the following exceptions. Mutation of the NF-B site in the rat NR1 promoter was conducted with a Quick Exchange kit (Stratagene, La Jolla, CA) with primers GB402, 5Ј-TTAATGTCCTGGtaccTCCTGTCTAC, and GB403, 5Ј-GTAGACAGGAggtaCCAGGACATTAA (lowercased letters represent replaced bases) to form the construct pNRL5.4mtNFB.…”

“…Cell Culture, Neuronal Differentiation, Transfection, Infection, and Reporter Assay-Previously described methods were used for culture of P19 cells (4), HEK cells (5), and dissociated rat cerebrocortical neurons (27). Differentiation of P19 cells was induced by retinoic acid (RA) as described previously (4).…”

“…Each experiment was repeated at least three times. Immunoblot analysis of Sp1 and Sp3 in nuclear extracts was performed using methods described previously (4,27).…”

“…Nuclei were prepared by incubation of cells in lysis buffer (50 mM Hepes, pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.5 mM phenylmethylsul-fonyl fluoride, 1 g/ml aprotinin, 1 g/ml leupeptin) on ice for 10 min, and then centrifugation at 6,000 ϫ g for 5 min. Nuclei were dissolved in radioimmunoprecipitation assay buffer plus protease inhibitors (27), and DNAs in lysates were sonicated into fragments between 200 and 1000 bp. Cell debris was removed by centrifugation at 16,000 ϫ g for 10 min at 4°C, and supernatants were diluted five times with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Hepes, pH 7.9, 167 mM NaCl).…”