RE-1 silencing transcription factor-4 (REST4) is neither a transcriptional repressor nor a de-repressor

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Abstract

The zinc finger protein RE-1 silencing transcription factor (REST) is a transcriptional repressor that represses neuronal genes in non-neuronal tissues. A neuronal splice form of REST, termed REST4, has been described in the rat. It encompasses the N-terminus of REST, including the N-terminal repressor domain and five of the eight zinc fingers of the DNA-binding domain. The biological function of REST4 is controversial. Transcriptional repression as well as transcriptional de-repression activity has been attributed to the REST4 protein of rat. Here, we have expressed a ‘humanized’ version of REST4 (hREST4) to facilitate a comparison of the biological functions of hREST4 and REST. The biological activity the human REST protein has been extensively studied in the past. Additionally, hREST4 has a high degree of homology with the REST4 protein of rat. An immunofluorescence analysis showed that hREST4 is expressed in the nucleus, indicating that the protein may have a potential impact on gene regulation. We analyzed the biological function of hREST4 in NS20Y neuroblastoma cells using human synapsin I promoter/reporter gene constructs. The human synapsin I gene is negatively regulated by REST. The results show that hREST4, in contrast to the full-length human REST protein, does not impair human synapsin I promoter activity. Moreover, co-transfection experiments with expression vectors encoding REST and hREST4 did not reveal any evidence that REST4 blocks the transcriptional repression activity of REST.

Introduction

RE-1 silencing transcription factor (REST), also known as neuron-restrictive silencer factor (NRSF), functions as a transcriptional repressor of neuronal genes in non-neuronal tissues (Chong et al., 1995, Schoenherr and Anderson, 1995). Target genes of REST are the genes encoding synapsin I, brain-derived neurotrophic factor (BDNF), choline acetyltransferase, type II sodium channel, SCG10, m4 muscarinic acetylcholine receptor, N-methyl-d-aspartate receptor subunit NR2C, glutamate receptor 2, adhesion proteins L1 and NgCAM and others (reviewed in Thiel et al., 1999).

While the widespread expression of REST in non-neuronal tissues (Chong et al., 1995, Schoenherr and Anderson, 1995) is in good agreement with the role of REST as a negative regulator of neuron-specific gene transcription, the expression of REST in neuronal progenitor cells, neurons and neuronal cell lines has been a matter of controversy. While some studies described the absence of REST or REST repressor activity in neurons (Schoenherr and Anderson, 1995), others detected REST mRNA in low concentrations in neuronal cell lines (Chong et al., 1995, Lönnerberg et al., 1996, Bessis et al., 1997). We showed that the expression pattern of REST and that of a REST target gene, synapsin I, is in a direct inverse relationship in human neuroblastoma cells. Increased levels of REST mRNA resulted in reduced synapsin I mRNA levels and vice versa (Lietz et al., 1998). However, both REST and synapsin I were expressed in neuroblastoma cells, although at different concentrations. This observation indicates that low levels of REST are tolerable for allowing neuron-specific gene transcription. We concluded that the concentration of REST is of major importance for REST function and for the expression of neuronal genes (Lietz et al., 1998). A detailed analysis using in-situ hybridization and RNase protection mapping techniques revealed that REST is expressed in the adult rat nervous system, although at lower amounts than in undifferentiated neuronal progenitor cells (Palm et al., 1998).

In addition, several splice variants of REST were discovered (Palm et al., 1998) encoding proteins with five or four zinc finger motifs. Two variants of REST termed REST4 and REST5 were only detected in neuronal tissues. These transcripts are generated by alternative splicing of a neuron-specific exon (exon N) located between exons V and VI. REST4 retains the N-terminal repression domain and five of the eight zinc fingers that function as a DNA-tethering domain. The neuron-specific exon adds the amino acid sequence CDLVG, followed by an in-frame stop codon, to the REST4 protein C-terminal of the fifth zinc finger. (Palm et al., 1998). The neuron-specific splicing of REST is conserved in human, mouse and rat (Palm et al., 1999). The biological function of REST4 is controversial. While transcriptional repression activity was attributed to the REST isoforms by Palm et al. (1998), Shimojo et al. (1999) described REST4 as a de-repressor. They suggested that REST4 binds to the full-length REST protein and silences the silencing activity of REST. We have extensively studied the biological activity of REST and REST mutants in the past. We showed that REST fullfils the criterion of a transcriptional silencer binding protein that blocks transcription regardless of its location with respect to the enhancer and the promoter (Thiel et al., 1998). Here, we have compared the biological activities of REST and the ‘humanized’ REST4 protein (hREST4), to impair transcription of the human synapsin I promoter. Furthermore, we have studied the ability of hREST4 to block REST-mediated transcriptional repression.

Section snippets

Reporter constructs

The reporter constructs are derivatives of pGL3-Basic (Promega). The human synapsin I promoter/luciferase gene encoding plasmid pSyIluc contains the human synapsin I promoter sequences from −422 to +47. In plasmid pSyIlucΔNRSE, a deletion within the human synapsin I promoter removed the neural-restrictive silencer element (NRSE), the DNA-binding site for REST. The β-galactosidase expression vector pRSVβ has been described (Jüngling et al., 1994).

Expression constructs

The REST expression vector pCMVmycREST, encoding

Modular structure and expression of a ‘humanized’ REST4 protein

The REST protein displays a modular structure (Fig. 1A). The DNA-binding domain has been localized within the cluster of eight zinc fingers at the N-terminus. Two repressor domains were mapped at the N- and the C-termini of the protein (Tapia-Ramı́rez et al., 1997, Thiel et al., 1998). Recently, several splice forms of REST have been discovered derived from cDNA libraries made from human, mouse and rat mRNAs. One of these splice forms, REST4, was first obtained from a rat cDNA library,

Discussion

Binding sites for the transcriptional repressor protein REST were detected in many neuronal genes (Schoenherr et al., 1996, Thiel et al., 1999) that encode proteins with fundamental importance in brain function, i.e. neuronal receptors and synaptic vesicle proteins, adhesion molecules and signaling proteins, transcription factors and channel proteins. Thus, REST control of neuronal genes touches many biological functions in neurons. REST has two distinct transcriptional repression domains

Acknowledgements

We thank Libby Guethlein for critical reading of the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (TH 377/6-2).

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