Effect of the ubiquitous transcription factors, SP1 and MAZ, on NMDA receptor subunit type 1 (NR1) expression during neuronal differentiation

doi:10.1016/S0169-328X(02)00440-0

Molecular Brain Research

Volume 107, Issue 2, 15 November 2002, Pages 89-96

https://doi.org/10.1016/S0169-328X(02)00440-0 Get rights and content

Abstract

The silencer factor NRSF/REST has been reported to restrict expression to neurons of a variety of genes, including that encoding NMDA receptor subunit type 1 (NR1), by suppressing transcription in nonneuronal cells. However, we recently reported that in addition to the absence of NRSF/REST-binding activity, another neuron-specific mechanism is necessary for high level expression of the NR1 gene in neurons. In this study, we explored the mechanism of induction of NR1 promoter activity during neuronal differentiation of the P19 cell line. We identified a 27 base pair GC-rich region in the promoter as an important element responsible for induction of the NR1 gene after neuronal differentiation. We found that the ubiquitous transcription factors SP1 and MAZ bind to this GC-rich region. Surprisingly, the binding activities of SP1 and MAZ are not remarkably changed after neuronal differentiation. Mutations in the SP1 and MAZ sites impair binding of SP1 and MAZ proteins and also decrease NR1 promoter activity. These findings suggest that SP1 and MAZ mediate enhancement of NR1 promoter activity during neuronal differentiation despite the fact that their binding activity does not change.

Introduction

Approximately 30,000 different mRNAs, including splicing variants, have been estimated to be specifically expressed in the brain [26]. Many of these mRNAs have been shown to be exclusively expressed in neurons [33]. The number of cloned cDNAs of neuron-specific genes has been rapidly increasing. However, it is not yet fully understood how the expression of a variety of neuron-specific genes is orchestrated.

N-Methyl-d-aspartate (NMDA) receptors are responsible for a major portion of excitatory synaptic transmission in the central nervous system [39]. NMDA receptors play an important role in synaptic plasticity, neuronal growth, differentiation, migration, and neurodegeneration [39]. Functional analysis of cDNAs encoding NMDA receptors indicates the existence of three families of NMDA receptor subunits, termed NR1, NR2 and NR3 [9], [39]. Among these subunits, only NR1 is indispensable for the formation of functional NMDA receptors [39]. The NR1 gene is widely expressed in the central nervous system [15], [28], [29]. The expression of the NR1 gene is induced during neuronal differentiation in the developing brain [15], [28]. These features of the NR1 gene suggest that it is a good model for the study of transcriptional regulation of neuronal genes in the central nervous system.

One mechanism that has been proposed to regulate transcription of neuronal genes involves a silencer factor. The silencer factor, neuron restrictive silencer factor (NRSF) or repressor element silencing transcription factor (REST), prohibits expression of neuronal genes in nonneuronal cells [8], [36]. Neuron-specific expression of neuronal genes is allowed in the absence of NRSF/REST, as occurs in neurons. In accord with this model, a variety of neuronal genes have been reported to be NRSF/REST-target genes, including type II sodium channels [8], SCG10 [36], m4 muscarinic acetylcholine receptors [25], [40], β2-nicotinic acetylcholine receptors [5], choline acetyltransferase [20], dopamine β-hydroxylase [11], neuron-glia cell adhesion molecule [13], and synapsin I [18], [35]. Recently, we and others reported that the NR1 gene is a target of NRSF/REST and that the absence of NRSF/REST-binding is necessary for expression of the NR1 gene [3], [31]. However, we further demonstrated that the absence of NRSF/REST-binding activity is not sufficient and that additional neuron-specific mechanism(s) are necessary for high level expression of the NR1 gene in neurons [31]. A responsive element for basal and nerve growth factor-induced NR1 gene expression was demonstrated by a series of reports by Bai and co-workers [1], [2], [3]. The proximal GC-rich region was identified as the responsive element. However, the identification of the element was performed using a pheochromocarcinoma PC12 cell line. The molecular mechanism of up-regulation of the NR1 gene has not been previously reported in a model system for the developing central nervous system as we attempt here.

In this study, we investigated the neuron-specific mechanism allowing high expression of the NR1 gene in neurons. As a model system, we utilized the P19 embryonal carcinoma cell line. P19 cells differentiate into neurons when treated with retinoic acid [4], [24]. These retinoic acid-induced neurons have many of the same characteristics as mature neurons in the mammalian central nervous system [4], [24]. Also similar to native neurons in the developing brain, endogenous NR1 mRNA as well as NR1 promoter activity is induced during neuronal differentiation of P19 cells [31], [34]. Notably, NRSF/REST-binding activity is not detectable in either undifferentiated P19 cells or retinoic acid-treated (neuronally differentiated) P19 cells [31]. Thus, during neuronal differentiation, another mechanism likely enhances NR1 promoter activity in an NRSF/REST binding-independent manner. Here we identify a response element mediating induction of NR1 promoter activity during neuronal differentiation of P19 cells. We also characterize binding factors to the response element.

Section snippets

Cell culture, transfection

Mouse embryonal carcinoma P19 cells were purchased from the American Type Culture Collection (CRL 1825). P19 cells were maintained in α-modified minimum essential medium (Sigma) supplemented with 10% fetal bovine serum (Intergen). Neuronal differentiation of P19 cells was induced by adding 300 pM 13-cis retinoic acid (Acros) as described [30], [31]. Proliferative glial cells were killed by 5 μg/ml cytosine arabinoside (Sigma) to enrich for neuronal cells. Under our culture conditions, more than

Deletion of the GC-rich region reduces NR1 promoter activity after neuronal differentiation of P19 cells

Although the NR1 gene is negatively regulated in nonneuronal cells by NRSF/REST, we recently showed that the absence of binding activity of NRSF/REST protein to the NR1 NRSE/RE1 element is not sufficient for high expression of the NR1 gene in neuronal cells [31]. To delineate the mechanism responsible for high level of expression of the NR1 gene in neurons, we first identified the element responsible for induction of NR1 promoter activity during neuronal differentiation. To map the element,

Discussion

The silencer factor NRSF/REST inhibits expression of many neuronal genes, including the NR1 gene, in nonneuronal cells [3], [31], [37]. However, we recently reported that the absence of NRSF/REST-binding activity is necessary, but not sufficient for expression of the NR1 gene, and that an additional neuron-specific mechanism is also necessary for a high level of expression of the NR1 gene in neurons [31]. To investigate the mechanism for this phenomenon, we first determined the element

Acknowledgements

We thank Dr. K.B. Marcu for the MAZ antibody, and Dr. T. Shinohara for critical reading of the manuscript. This work was supported by NIH Grant P01 HD29587 to S.A.L., and by a research fellowship to S.-i.O. from the American Heart Association, Massachusetts Affiliate, Inc., and the American Parkinson Disease Association, Inc.

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Research reportEffect of the ubiquitous transcription factors, SP1 and MAZ, on NMDA receptor subunit type 1 (NR1) expression during neuronal differentiation

Abstract

Introduction

Section snippets

Cell culture, transfection

Deletion of the GC-rich region reduces NR1 promoter activity after neuronal differentiation of P19 cells

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Neuroscience

J. Biol. Chem.

Cell

Brain Res. Mol. Brain Res.

Trends Biochem. Sci.

J. Biol. Chem.

J. Biol. Chem.

Neuroscience

Mol. Brain Res.

J. Biol. Chem.

J. Biol. Chem.

Cell

Neuron

Brain Res. Mol. Brain Res.

Neurosci. Lett.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Research report
Effect of the ubiquitous transcription factors, SP1 and MAZ, on NMDA receptor subunit type 1 (NR1) expression during neuronal differentiation