Single-stranded DNA-binding proteins and neuron-restrictive silencer factor participate in cell-specific transcriptional control of the NMDAR1 gene.
Our previous studies revealed that a proximal region of the N-methyl-D-aspartate receptor 1 (NMDAR1) promoter is important for cell-type-specific expression. We have now explored the contributions of several regulatory elements to this specificity. Deletion of the neuron-restrictive silencer element partially relieved the suppression of promoter activity in C6 glioma and HeLa cells. An overlapping G(C/G)G/tandem Sp1-containing region crucial for both basal and nerve growth factor (NGF)-regulated promoter activity specifically bound nuclear proteins on its purine-rich sense strand. A faster migrating complex, single-stranded binding protein complex 1 (SBPC1), was highly enriched in HeLa cells, whereas a slower migrating complex, SBPC2, was enriched in PC12 cells. A high ratio of 2/1 complex correlated with a high level of promoter activity. NGF treatment of PC12 cells reduced SBPC1 but increased SBPC2. Competition experiments showed that the SBPC1 binding required a dG4 sequence and the SBPC2 needed a core of TG3A plus a 5'-flanking sequence. Single-stranded DNA encompassing TG3A and/or dG4 specifically suppressed cotransfected NMDAR1 promoter activity. UV cross-linking studies indicated that a 31.5-kDa protein mainly formed SBPC1, whereas SBPC2 contained several larger proteins. Our results suggest that neuron-restrictive silencer factor and single-stranded DNA-binding proteins may both play a role in cell-type specificity of the NMDAR1 gene, and the latter may also be involved in basal and NGF-regulated activity.
Bai
G
G
Molecular Neurobiology Unit, Gerontology Research Center, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA. gbai@rics.bhw.harvard.edu
Norton
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DD
Prenger
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Kusiak
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D000818:Animals; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011687:Purines / Q000378:metabolism; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D000099229:RE1-Silencing Transcription Factor
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011687:Purines; D016194:Receptors, N-Methyl-D-Aspartate; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D000099229:RE1-Silencing Transcription Factor