Single-stranded DNA-binding Proteins and Neuron-restrictive Silencer Factor Participate in Cell-specific Transcriptional Control of the NMDAR1 Gene

Abstract

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 neuronrestrictive 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 dG 4 sequence and the SBPC2 needed a core of TG 3 A plus a 5-flanking sequence. Singlestranded DNA encompassing TG 3 A and/or dG 4 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.The NMDA 1 subtype of glutamate receptor plays important roles in voltage-dependent Ca 2ϩ influx, synaptic plasticity, and excitotoxic neuronal death in the mammalian central nervous system (1-3). Functional NMDA receptors contain a key NMDAR1 subunit in combination with one or more members of the NMDAR2(A-D) subunits (4 -7). The expression pattern of the NMDAR1 gene is widespread in the brain and restricted to neuronal cells (8 -10). A great deal of data suggest that interactions of cis elements in a gene with tissue-specific transacting factors play crucial roles in determining tissue-specific expression of the gene (11-14). We previously isolated a 3-kilobase pair promoter of the rat NMDAR1 gene and observed that a proximal region with 356 bp is sufficient to confer to the promoter a cell-type specificity in neuronal-like PC12 cells compared with C6 glioma and HeLa cervical cancer cells (15)(16)(17). This proximal promoter region includes a 5Ј-untranslated region that contains a neuron-restrictive silencer element (NRSE)/RE1-like element. Several neuronal genes contain this element, and a neuron-restrictive silencer factor/REST, expressed in nonneuronal tissues, may recognize this element and restrict gene expression in nonneuronal cells (11,18).Recently, several factors were identified that preferentially bind transcriptional elements on single-stranded DNA and function as suppressors or activators (19 -31). For example, heterogeneous nuclear ribonucleoprotein K specifically binds to a cytosine-r...

Nerve Growth Factor Uses Ras/ERK and Phosphatidylinositol 3-Kinase Cascades to Up-regulate theN-Methyl-d-aspartate Receptor 1 Promoter

“…5B), and therefore factors binding to double-stranded DNA are unlikely to replace Sp1 from the NR1 promoter. Other potential candidates may be single-stranded DNA-binding proteins that can also be activated by NGF and bind to the same promoter region as revealed by our previous studies (51). In that report, we observed that single-stranded DNA-binding complex 2 interacted with a T(G) 3 A sequence overlapping the GC boxes, but in a single-stranded DNA conformation only, and NGF treatment of PC12 cells for 30 min increased this interaction significantly.…”

“…Therefore, transcription factors interacting with this GSG/GC box region could be the nuclear target(s) of the Ras/ERK cascade. In our previous studies, we showed that Sp1, Egr-1, and single-stranded DNA-binding proteins may interact with this region (22,39,51). ERK regulation of gene transcription can be mediated by two pathways: phosphorylated ERKs enter into the nucleus and modify transcription factors directly (4, 5), or ERKs may modify cytoplasmic ribosomal S6 kinase that translocates to the nucleus and regulates transcription (6).…”

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

“…There is no DNA sequence with more than 65% homology to the initiator (Inr) consensus, YYANWYY, because the initiator may direct transcription initiation for most genes (45). We also did not find an RE1/NRSE site using the same searching criteria to the consensus, TTCAGMACCDYGSASAGHRCC (46), considering that the RE1/NRSE DNA cis-element plays an important role in neuronal expression (41).…”

“…A consensus of GC-Box (Sp1 site) from Promega was used as a competitor. Results of EMSA were analyzed and quantified with a PhosphorImager as described in our previous studies (41).…”

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

“…A 27-bp GCrich region (GC-box) proximal to the transcription start sites has been identified that controls induction of the Grin1 gene upon differentiation of P19 cells, and this site is recognized by Sp1 and myc-associated zinc finger protein transcription factors (Okamoto et al, 2002). These sites previously were known to respond to Sp1, -3, and -4 transcription factors Kusiak, 1995, 1997;Bai et al, 1998;Liu et al, 2001) and interact with an element further 5Ј in the promoter (Ϫ520/Ϫ529) that is recognized by myocyte enhancer factor 2C (Krainc et al, 1998). Studies with Grin1 promoter constructs in PC12 cells suggest that NGF uses both the Ras/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase pathways to up-regulate Grin1 promoter activity through Sp1 (Liu et al, 2001).…”