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J. Biol. Chem., Vol. 278, Issue 29, 26423-26434, July 18, 2003

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

MULTIPLE TRANSCRIPTION START POINTS, POSITIVE REGULATION BY Sp FACTORS, AND TRANSLATIONAL REGULATION^*

Anguo Liu , Zhiye Zhuang , Peter W. Hoffman  and Guang Bai  ¶

From the Department of Oral & Craniofacial Biological Sciences, University of Maryland Dental School and Program in Neuroscience, University of Maryland, Baltimore, Maryland 21201 and the Department of Biology, College of Notre Dame of Maryland, Baltimore, Maryland 21210

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.

Received for publication, October 31, 2002 , and in revised form, May 9, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY167029.

^* A part of the preliminary results of this study was reported at the 27th Annual Meeting of the Society for Neuroscience, New Orleans, LA (1997). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by National Institutes of Health Grant NS38077. To whom correspondence should be addressed: The Dept. of Oral & Craniofacial Biological Sciences, University of Maryland Dental School, 666 W. Baltimore St., Baltimore, MD 21201. Tel.: 410-706-2082; Fax: 410-706-0193; E-mail address: GNB001{at}dental.umaryland.edu.

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