Research report
Role of NRSF/REST in the molecular mechanisms regulating neural-specific expression of trkC/neurotrophin-3 receptor gene

https://doi.org/10.1016/j.molbrainres.2004.12.019Get rights and content

Abstract

The processes of differentiation and development of neurons involve the induction of neuron-specific genes by instructive signals with subsequent neurotrophic factor-driven survival and functional maturation. We have previously shown that bone morphogenetic protein-2 (BMP2) and retinoic acid synergistically induce the responsiveness of developing sympathetic neurons to neurotrophic factors, neurotrophin 3 (NT-3), and GDNF by upregulating corresponding receptors concomitantly with the induction of other neuron-specific genes including BRINP1, a neuron-specific cell-cycle regulatory protein. In the present study, we analyzed transcriptional mechanisms regulating the neuron-specific expression of TrkC/NT-3 receptor gene. TrkC gene contains at least four NRSE/RE-1 (neuron-restrictive silencing element/repressor element 1)-like elements (TrkC-NRSE A–D). Consequently, we found that in non-neuronal cells, neuron-restrictive silencing factor (NRSF) acts on TrkC-NRSE D located at the downstream of exon 3 to suppress the promoter activity of TrkC gene in a manner similar to the mechanism of NRSF suppressing BRINP1 transcription. In contrast, in neuronal cells, the biological activity of NRSF on TrkC was suppressed. From these observations, molecular mechanisms regulating the expression of neuron-specific genes via NRSE during neuronal differentiation are discussed.

Introduction

The mechanisms underlying the generation of multiple neuronal cell types during the course of stem cell differentiation include the actions of extracellular instructive signals as well as the transcriptional activation of a set of neuronal genes. A range of neurotrophic factors also act on postmitotic neurons to support their survival and promote their phenotypic specification. In particular mature sympathetic neurons of rat superior cervical ganglia (SCG) depend for their survival on the target-derived NGF that interacts with the TrkA receptor. However, preceding the onset of TrkA expression, developing SCG neurons show significant responsiveness to other neurotrophic factors, neurotrophin 3 (NT-3), and glial cell line-derived neurotrophic factor (GDNF). Gene disruption analysis has also indicated important roles of both NT-3 and GDNF in the development of SCG neurons [5], [25]. Recently, it was found that bone morphogenetic protein-2 (BMP2) plays major roles in the determination of autonomic identity of peripheral neurons [6], [8], [33], [36]. We also reported that BMP2 in combination with RA induces the initial responsiveness of the developing SCG neurons to NT-3 and GDNF by upregulating expression of the relevant receptors, TrkC and GFRá-1, that bind to NT3 and GDNF, respectively [19], [41]. In the screen of genes regulated by BMP2/RA in developing SCG neurons, we then identified a group of genes that are induced synergistically by BMP2 and RA. These included BRINP1, which is also known as DBCCR1 (DBC1), a putative tumor suppresser gene for bladder cancer on chromosome 9q 32-33 [11], [12], [16], [27]. We have found that the expression of BRINP1 gene is tightly coupled to neural development [16]. BRINP1 expression in the brain is further increased by neural activity (unpublished data). Furthermore, when over-expressed in mammalian cells, BRINP1 suppresses cell cycle progression at G1/S transition [16], [29]. Hence, it is suggested that BRINP1 plays important physiological roles in the development of the nervous system such as cessation of stem cell proliferation and the suppression of unwanted cell cycle progression in the postmitotic neurons. Clarification of the mechanism regulating the expression of BRINP1 and TrkC genes should thus shed light on the elucidation of mechanisms regulating neuronal gene expression induction and on the action of BMP2/RA in the differentiation of sympathetic neurons.

The expression of cell-type-specific genes is regulated by both positive and negative mechanisms acting on promoter elements to regulate transcription. Increasing numbers of reports have indicated negative regulatory mechanisms play important roles in the tissue-specific gene expression. One such mechanism involved in the neural-specific gene expression is mediated by neuron restrictive silencing factor/RE-1 silencing transcription factor (NRSF/REST) whose activity is mainly detected in non-neural tissues [3], [26], [30], [34]. NRSF blocks gene transcription in non-neural tissues by binding to NRSE/RE-1 located in the proximity of core promoter region of the target genes [3], [26], [34]. In the previous study, we showed that NRSE located on the proximal upstream of promoter region of BRINP1 gene determines the neural-specific expression of BRINP1. In the present study, to further confirm the roles of NRSF in the development of sympathetic neurons, we investigated the mechanisms regulating the neuron-specific transcription of TrkC gene. The results indicated that NRSF also regulates the neural-specific expression of TrkC gene via TrkC-NRSE located at the third intron of the TrkC gene. Molecular mechanisms regulating concomitant induction of BRINP1 and TrkC genes via NRSE during neuronal differentiation are discussed.

Section snippets

Determination of the transcription start site

The transcription start site of human TrkC gene was determined by the cap site hunting method using human brain cap site cDNA-dT (Nippon gene) as the templates for the first PCR, according to the manufacturer's instructions. The capped ends of mRNAs in human brain cap site cDNA-dT were specifically labeled by synthetic r-oligo. Then, the cap site region of the mRNA was identified by sequencing of the cDNA clones obtained from nested PCR amplifications. The first PCR was performed with 1RDT

Determination of human TrkC transcription start sites

In the present study, we first determined the transcription start site on the exon 1 of TrkC gene by using the cap site hunting method on human brain cap site cDNA. The cDNA containing cap site was amplified by nested PCR using 2RDT primer designed for r-oligo and hTrkC-32 primer or hTrkC-33 primers designed for human TrkC gene. The amplified products were electrophoresed on 2% agarose gels and stained with ethidium bromide (Fig. 1a). PCR products with lengths of ∼180 bp and ∼130 bp

Discussion

Neurotrophic factors play important roles in survival and functional differentiation of different types of neurons. Therefore, different types of neurons respond to different types of neurotrophic factors [18], [20]. Neurotrophins act on specific receptor tyrosine kinases, Trks, on neuronal cells. Thus, TrkA serves as a high affinity NGF receptor, while, TrkB and TrkC serve as BDNF/NT4 receptor and NT-3 receptor, respectively [20]. It is therefore assumed that individual neurotrophic factor

Acknowledgments

This work was supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sport, Science and Technology of Japan. Authors are grateful to Dr. K. Matsuomoto and T. Taira (Hokkaido University) for technical guidance on some molecular biological assays. Authors are indebted to Mr. Simon Bayley (Asahikawa Medical College, Asahikawa) for correcting our use of language.

References (41)

  • L.M. McGregor et al.

    Molecular cloning of the cDNA for human TrkC (NTRK3), chromosomal assignment, and evidence for a splice variant

    Genomics

    (1994)
  • N. Mori et al.

    A common silencer element in the SCG10 and type II Na+ channel genes binds a factor present in nonneuronal cells but not in neuronal cells

    Neuron

    (1992)
  • K. Palm et al.

    Neuron-specific splicing of zinc finger transcription factor REST/NRSF/XBR is frequent in neuroblastomas and conserved in human, mouse and rat

    Mol. Brain Res.

    (1999)
  • C. Schneider et al.

    Bone morphogenetic proteins are required in vivo for the generation of sympathetic neurons

    Neuron

    (1999)
  • N.M. Shah et al.

    Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members

    Cell

    (1996)
  • S.T. Smale

    Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes

    Biochim. Biophys. Acta

    (1997)
  • A. Tabuchi et al.

    REST4-mediated modulation of REST/NRSF-silencing function during BDNF gene promoter activation

    Biochem. Biophys. Res. Commun.

    (2002)
  • J.D. Dignam et al.

    Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei

    Nucleic Acids Res.

    (1983)
  • M.J. Fann et al.

    Depolarization differentially regulates the effects of bone morphogenetic protein (BMP)-2, BMP-6, and activin A on sympathetic neuronal phenotype

    J. Neurochem.

    (1994)
  • M.M. Garner et al.

    A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system

    Nucleic Acids Res.

    (1981)
  • Cited by (9)

    View all citing articles on Scopus
    View full text