Research reportCell-type non-selective transcription of mouse and human genes encoding neural-restrictive silencer factor☆
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
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The nucleotide sequences reported in this paper have been submitted to the GenBank and related Data Banks with the accession numbers AB024497 and AB024498.
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Present address: Kyoto Prefectural University of Medicine, Kyoto, Japan.