Functional analysis of the proximal 5'-flanking region of the N-methyl-D-aspartate receptor subunit gene, NMDAR1.
The NMDAR1 receptor subunit is a common subunit of N-methyl-D-aspartate receptors. We have previously characterized 3 kilobases (kb) of 5'-flanking sequence of the NMDAR1 gene and now report on the ability of this region to direct transcription of a reporter gene and on its interaction with nuclear proteins. The sequence 356 base pairs (bp) 5' of the first nucleotide of codon 1 was sufficient to express a luciferase reporter gene in rat PC12 pheochromocytoma cells. Additional sequences upstream of nucleotide -356 influenced the activity approximately 2-fold. A labeled 112-bp fragment (position -356 to -245) formed six complexes (C1A and -B, C2A and -B, and C3A and -B), grouped as three double bands, with nuclear extracts from PC12 cells. Competition with Sp1 oligonucleotides abolished formation of C2A and -B and C3A and -B complexes. Sp1 antibody recognized the C3A complex in supershift experiments. Prior immunoprecipitation of nuclear extracts with Sp1 antibody abolished formation CA2 and -B and C3A and -B complexes. Purified Sp1 protein alone did not form a C3A complex but potentiated its formation when PC12 nuclear extract was added. A GC-rich sequence in this fragment was protected from DNase I digestion by nuclear extract. These results suggest that a 356-bp sequence comprises the NMDAR1 basal promoter, and that NMDAR1 gene expression may be regulated by Sp1-like nuclear factors.
Bai
G
G
Molecular Neurobiology Unit, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
Kusiak
J W
JW
D000310:Adrenal Gland Neoplasms; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D016384:Consensus Sequence; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D008156:Luciferases / Q000096:biosynthesis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D016716:PC12 Cells; D010673:Pheochromocytoma; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014158:Transcription, Genetic
D015342:DNA Probes; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D008156:Luciferases