Synergistic Activation of theN-Methyl-d-aspartate Receptor Subunit 1 Promoter by Myocyte Enhancer Factor 2C and Sp1

doi:10.1074/jbc.273.40.26218

Synergistic Activation of theN-Methyl-d-aspartate Receptor Subunit 1 Promoter by Myocyte Enhancer Factor 2C and Sp1*

From the ^‡CNS Research Institute, Brigham and Women’s Hospital, and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115, the ^§Molecular Neurobiology Unit, Gerontology Research Center, NIA, National Institutes of Health, Baltimore, Maryland 21224, and the ^¶Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02114

Abstract

The N-methyl-d-aspartate (NMDA) subtype of glutamate receptor plays important roles in neuronal development, plasticity, and cell death. NMDA receptor subunit 1 (NR1) is an essential subunit of the NMDA receptor and is developmentally expressed in postnatal neurons of the central nervous system. Here we identify on the NR1 promoter a binding site for myocyte enhancer factor 2C (MEF2C), a developmentally expressed neuron/muscle transcription factor found in cerebrocortical neurons, and study its regulation of the NR1 gene. Co-expression of MEF2C and Sp1 cDNAs in primary neurons or cell lines synergistically activates the NR1 promoter. Disruption of the MEF2 site or the MEF2C DNA binding domain moderately reduces this synergism. Mutation of the Sp1 sites or the activation domains of Sp1 protein strongly reduces the synergism. Results of yeast two-hybrid and co-immunoprecipitation experiments reveal a physical interaction between MEF2C and Sp1 proteins. The MEF2C DNA binding domain is sufficient for this interaction. Dominant-negative MEF2C interferes with expression of NR1 mRNA in neuronally differentiated P19 cells. Growth factors, including epidermal growth factor and basic fibroblast growth factor, can up-regulate NR1 promoter activity in stably transfected PC12 cells, even in the absence of the MEF2 site, but the Sp1 sites are necessary for this growth factor regulation, suggesting that Sp1 sites may mediate these effects.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants R01 EY06087, R01 EY05477, P01 HD29587 (to S. A. L.), Hoechst AG, the Pew Scholar’s Program, an American Chemical Society Faculty Research Award (to R. B), and National Institutes of Health Intramural Research Program (to G. B. and J. W. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ To whom correspondence should be addressed: CNS Research Institute, Brigham and Women’s Hospital and Harvard Medical School, 221 Longwood Ave., LMRC First Floor, Boston, MA 02115. Tel.: 617-278-0363; Fax: 617-264-5277; E-mail:slipton{at}rics.bwh.harvard.edu.
↵2 S. Okamoto, K. Sherman, and S. A. Lipton, manuscript submitted for publication.
↵3 D. Krainc and S. A. Lipton, unpublished observations.
Abbreviations:

NMDA

N-methyl-D-aspartate

NR1

N-methyl-D-aspartate receptor subunit 1

MEF2C

myocyte enhancer factor 2C

EMSA

electrophoretic mobility shift assay

mt

mutation

PCR

polymerase chain reaction.
- Received April 7, 1998.
- Revision received July 15, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.