Elsevier

Molecular Brain Research

Volume 90, Issue 2, 20 June 2001, Pages 174-186
Molecular Brain Research

Research report
Cell-type non-selective transcription of mouse and human genes encoding neural-restrictive silencer factor

https://doi.org/10.1016/S0169-328X(01)00107-3Get rights and content

Abstract

Neural-restrictive silencer (NRS) has been identified in at least twenty neuron-specific genes, and its nuclear DNA-binding factor, NRSF (also known as RE1-silencing transcription factor (REST)), has been cloned from human and rat, and was shown to repress transcription by recruiting corepressors mSin3 and/or CoREST via its N- and C-terminal domains, leading to chromatin reorganization by mSin3-associated histone deacetylase, HDAC. However, it is largely unknown how NRSF gene expression is regulated. To elucidate the mechanisms for gene expression of NRSF, we isolated the transcriptional unit of the NRSF gene from mouse and human, identified three 5′-non-coding exons in addition to three coding exons, determined transcription start sites, and identified two basal promoter activities in the upstream of the first two non-coding exons. Both promoters functioned equally in neuronal and non-neuronal cells, suggesting that levels of initial transcripts of NRSF gene are similar in neuronal and non-neuronal cells. These results suggest that the level of NRSF gene expression is not determined by transcription per se, and rather is modulated at the post-transcriptional level, e.g. splicing, mRNA stability, and/or post-translational modifications, in a cell-specific manner. Consistent with this idea, NRSF protein was apparently present even in neuronal cells and tissues, but was unable to bind to the NRS element, suggesting that NRSF is regulated at least in part post-translationally.

Introduction

The roles of cell-type-specific transcription factors in lineage specification and terminal differentiation during development are beginning to be established in both vertebrates and invertebrates [2], [35]. In the mammalian nervous system, cell-specific expression of transcription factors containing either POU, helix–loop–helix (HLH), or zinc-coordinated finger motifs as a sequence-specific DNA-binding domain have been shown to play crucial roles at various stages of neuronal differentiation [8], [16], [22], [48], [49]. These transcription factors are either activators or repressors (or could be both depending upon configuration) for other transcription factors, thus forming a transcription factor cascade of positive and negative regulators; however, only a few factors have been shown to be directly involved in establishing the terminally differentiated neuronal phenotype.

Neural restrictive silencer factor (NRSF) [39], also known as RE1-silencing transcription factor (REST) [7], functions as a transcriptional repressor of multiple neuron-specific genes in neuronal precursor cells during development and in non-neuronal cells and tissues in adulthood [6], [40]. Recent results from our own laboratory and others revealed that NRSF/REST functions as a repressor by binding to corepressors mSin3 and CoREST, thereby, at least in part, via recruiting histone deacetylase (HDAC) [3], [10], [11], [31], [37]. Target genes of NRSF [41] relates directly to neuronal function, and thus include ion channel [21], neurotransmitter synthetase [13], [14], [19], [25], [26], receptors [4], [5], [27], [50], synaptosomal proteins [24], [38], neuronal cell adhesion molecules [17], [18], neuronal cytoskeleton [41], neurotrophic factors [43], [47] and neuronal growth-associated proteins [29], [30]. In these neuron-specific target genes of NRSF, direct target DNA-sequences, which are collectively known as the neural-restrictive silencer element (NRSE) [28], [30], [40], also called repressing element-1 (RE-1) [21], appear either in the promoter-proximal, distal, or intragenic regions including introns. Although NRSF is considered to function primarily in non-neuronal cells and neuronal progenitors, recent evidence indicated that NRSF is expressed in mature neuron [32], [33] [N. Mori, Y. Naruse, T. Kojima, Soc. Neurosci. Abstr. 23 (1997) 144.4], suggesting more complex but interesting possibilities of NRSF’s roles in neuronal differentiation and function in the nervous system.

A considerable level of NRSF gene expression occurs even in neuron; however the level of NRSF mRNA is far more abundant in non-neuronal cells and tissues [7] [N. Mori, T. Kojima, Y. Naruse, S. Muraoka, ASBMB Abstract, #1258 in FASEB J. 11(9) (1997) A1072], and the NRSE-binding activity is only detectable in non-neuronal cells [21], [30]. These results suggest that NRSF gene expression is likely to be restricted to non-neuronal cells and undifferentiated neuronal progenitors. A simple explanation for this regulation would be that the gene encoding NRSF is repressed selectively in differentiating neuronal precursors and mature neurons. If this were the case, neurons should induce and maintain a specific repressor that may act on the NRSF gene transcription. Thus, the presence of a neuron-specific silencer, i.e. silencer of a silencer, becomes an intriguing possibility for the gene regulation of NRSF during neuronal differentiation. Alternatively, such a non-neuronal cell preferred expression of NRSF might be determined at the post-transcriptional level. Even if the latter is the case, it is still of importance to approach the transcriptional regulatory mechanisms of the NRSF gene in order to link, if possible, further upstream transcriptional regulators (such as neurogenin, MASH, NeuroD, and HES) [12], [15], [23] for better understanding of the mechanisms of neural development [1].

To approach these issues, we sought to isolate and characterize the promoter function of the NRSF gene. As described herein, we established the overall genomic organization of the mouse and human NRSF genes, and showed that the NRSF gene has multiple promoters associated with distinct exons in both species. We also provided evidence of cell-type non-selective promoters in the mouse and human NRSF gene, and further showed that NESF/REST is indeed expressed equally in neuronal cells.

Section snippets

Genomic cloning

A human PAC library (Genome Systems) was screened with an HZ4-derived fragment [39] as a probe. Two positive candidates were isolated (#7097, #7098), and restriction maps of those clones were determined by standard molecular biological techniques. Exon-containing regions were subcloned by shotgun methods, and a detail map and sequences of the inserts were determined. A murine ES cell-derived P1 genomic library (Genome Systems) was screened by PCR using a primer pair of MNR13 and MNR14, i.e.

Genomic organization of the mouse and human NRSF genes

Screening of a human PAC library with the HZ4 cDNA fragment as a probe identified two positive clones, i.e. HNR-PAC 1 and 2 (original clone numbers were 7097 and 7098, respectively) (Fig. 1A). Restriction and sequence analyses revealed that the human NRSF coding exons were divided into three exons, which spanned about 15 kb in length (Fig. 1B). We also screened a mouse P1 library and obtained three positive clones, i.e. MNR-P11, 12, and 13 (7326, 7327 and 7328, respectively) (Fig. 1C).

Discussion

We described here the genomic organization of mouse and human NRSF genes, determined the promoter characteristics at the first two exons, showed evidence of alternate use of these promoters, which functioned equally in neuronal and non-neuronal cells. NRSF protein was indeed expressed efficiently even in neuronal cells, suggesting that NRSE-binding activity of this protein is regulated mostly post-translationally.

Despite an extensive effort, we were unable to identify any cell-type specific

Acknowledgements

We thank D.J. Anderson for providing us with HZ4 and M5 plasmids, and encouragement. The early phase of this study was initiated in the laboratory of Inheritance and Variation at Keihanna Plaza; we therefore thank S. Muraoka for excellent technical assistance and laboratory maintenance. This work was supported by grants from PRESTO and CREST, JST and also, in part, by Strategic Promotion System for Brain Science from STA and Funds for Comprehensive Research on Aging and Health from the Ministry

References (50)

  • S.D. Kraner et al.

    Silencing the type II sodium channel gene: a model for neural-specific gene regulation

    Neuron

    (1992)
  • P. Lonnerberg et al.

    Cell type-specific regulation of choline acetyltransferase gene expression

    Role of the neuron-restrictive silencer element and cholinergic-specific enhancer sequences. J. Biol. Chem.

    (1996)
  • M. Mieda et al.

    Expression of the rat m4 muscarinic acetylcholine receptor gene is regulated by the neuron-restrictive silencer element/repressor element 1

    J. Biol. Chem.

    (1997)
  • N. Mori et al.

    A cell type-preferred silencer element that controls the neural-specific expression of the SCG10 gene

    Neuron

    (1990)
  • 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

    Brain Res. Mol. Brain Res.

    (1999)
  • J.P. Quinn

    Neuronal-specific gene expression — the interaction of both positive and negative transcriptional regulators

    Prog. Neurobiol.

    (1996)
  • S. Schoch et al.

    Major role of a negative regulatory mechanism

    J. Biol. Chem.

    (1996)
  • C.J. Schoenherr et al.

    Silencing is golden: negative regulation in the control of neuronal gene transcription

    Curr. Opin. Neurobiol.

    (1995)
  • A. Tabuchi et al.

    Silencer-mediated repression and non-mediated activation of BDNF and c-fos gene promoters in primary glial or neuronal cells

    Biochem. Biophys. Res. Commun.

    (1999)
  • A.H. Tang et al.

    PHYL acts to down-regulate TTK88, a transcriptional repressor of neuronal cell fates, by a SINA-dependent mechanism

    Cell

    (1997)
  • G. Thiel et al.

    Biological activity and modular structure of RE-1-silencing transcription factor (REST), a repressor of neuronal genes

    J. Biol. Chem.

    (1998)
  • T. Timmusk et al.

    Brain-derived neurotrophic factor expression in vivo is under the control of neuron-restrictive silencer element

    J. Biol. Chem.

    (1999)
  • I.C. Wood et al.

    Neural specific expression of the m4 muscarinic acetylcholine receptor gene is mediated by a RE1/NRSE-type silencing element

    J. Biol. Chem.

    (1996)
  • D.J. Anderson et al.

    Cell lineage determination and the control of neuronal identity in the neural crest

    Cold Spring Harbor Symp. Quant. Biol.

    (1997)
  • Cited by (18)

    • Regulated clearance of histone deacetylase 3 protects independent formation of nuclear receptor corepressor complexes

      2012, Journal of Biological Chemistry
      Citation Excerpt :

      HDACs can interact with different partners to assemble into distinct multisubunit complexes. These complexes often have different functions, as best exemplified by HDAC1, which is a shared subunit of the functionally distinct NuRD (nucleosome remodeling and deacetylase complex) (5), Sin3A (SIN3 homolog A) (6–8), and CoREST (9–12) corepressor complexes. HDAC3 also interacts with different proteins, including the two homologous nuclear receptor corepressors N-CoR and SMRT (13–17).

    View all citing articles on Scopus

    The nucleotide sequences reported in this paper have been submitted to the GenBank and related Data Banks with the accession numbers AB024497 and AB024498.

    1

    Present address: Kyoto Prefectural University of Medicine, Kyoto, Japan.

    View full text