Research reportRole of NRSF/REST in the molecular mechanisms regulating neural-specific expression of trkC/neurotrophin-3 receptor gene
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 BRINP1 expression in the brain is further increased by neural activity (unpublished data).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.
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