Differential regulation by multiple promoters of the gene encoding the neuron-restrictive silencer factor

Abstract

NRSF͞REST is a protein that silences transcription of a number of genes that contain a DNA element called the neuron-restrictive silencer element (NRSE). During embryogenesis, REST is expressed ubiquitously in nonneural cells, but is down-regulated during differentiation of neural progenitors into neurons. REST is also up-regulated in adult neurons by activity, suggesting a possible role for the protein in synaptic plasticity. To understand mechanisms that control expression of REST, we identified and characterized the promoter region of the mouse REST gene (mREST). A 4.5-kb DNA segment containing three exons (A, B, and C) that correspond to alternatively spliced 5 untranslated regions (5 UTRs) was isolated and its DNA sequence was determined. Reverse transcription-PCR analyses of fibroblasts, astrocytes, and neural progenitors identified variants in which these exons were spliced to exon D, suggesting that exons A, B, and C may each have a promoter. Consistent with this hypothesis, primer extension and in vitro transcription experiments revealed clusters of RNA transcription initiation sites upstream of exons A, B, and C. Tests of REST͞luciferase reporter constructs in Neuro2A and NIH 3T3 cells revealed promoters upstream of exons A and B that were active in both cell lines, and a promoter upstream of exon C that was weakly active only in NIH 3T3 cells. Six enhancer and two repressor regions were found to overlap each of the three promoters, and some of these were found to be cell type-specific. Combinatorial arrangements of these promoters with enhancer and repressor regions may allow modulation of REST expression in particular contexts.T he neuron-restrictive silencer factor (NRSF), also known as RE-1 silencing transcription factor (REST) is a 116-kDa C 2 H 2 zinc finger protein that is related to the Gli-Krüppel family of transcriptional repressors. It was originally identified as a factor that binds to a particular 21-bp DNA regulatory element, called the neuron-restrictive silencer element (NRSE; ref. 1), also known as repressor element 1 (RE-1; ref. 2). NRSF is a potent repressor of genes containing the NRSE (3). Several of the genes containing the NRSE are essential for establishment and maintenance of the neuronal phenotype; these include neuron-specific cytoskeletal proteins, neurotransmitters and their biosynthetic enzymes, synaptic vesicle components, neurotrophins, and cell adhesion molecules (3-9).In rodents, NRSF is expressed ubiquitously in developing nonneural tissues between embryonic days 8.5 and 9.5 (E8.5-9.5) (10). At the onset of neuronal differentiation, however, only low levels of NRSF mRNA and protein are found in neural progenitors within the ventricular zone, and NRSF is further down-regulated in neurons (1, 2). Recent phenotypic analyses of REST knockout mice and experiments involving ectopic expression of NRSF in chicken embryos (10) showed that perturbation of REST expression and function results in cellular apoptosis, aberrant differentiation and morphogenesis, and lethality. These...

Hypothermia rescues hippocampal CA1 neurons and attenuates down-regulation of the AMPA receptor GluR2 subunit after forebrain ischemia

“…Recent findings from our laboratory indicate that global ischemia activates a REST-initiated molecular signaling cascade that suppresses GluR2 transcription in CA1 neurons (33). Our findings are consistent with a model whereby the early rise in intracellular free Ca 2ϩ triggered by the ischemic insult activates a Ca 2ϩ -dependent CREB͞CaRE regulatory element in the proximal promoter of the REST gene (34) to induce REST mRNA expression. A likely point of intervention of hypothermia is inhibition of the REST-initiated molecular signaling cascade implicated in down-regulation of the GluR2 gene.…”

Regulation of the NRSF/REST gene by methylation and CREB affects the cellular phenotype of small-cell lung cancer

“…However, responsible regulators still are not yet identified. Although the first promoter region has previously been characterized for human and mouse genes (Koenigsberger et al, 2000;Kojima et al, 2001), it is becoming apparent that they have a role in regulating cell line-specific expression. Indeed, strong promoter activities of pf1 match the abundance of NRSF/REST transcripts initiating with exon 1a (Palm et al, 1998).…”

“…NRSF/REST gene transcription starts with three alternatively used exons, termed 1a, 1b and 1c. Among these, exon 1a is the most abundant transcription start site, accounting for E80% of transcripts (Koenigsberger et al, 2000). Although alternative start exons suggest more than one NRSF/ REST promoter, there was no evidence of a cell line-or tissue-specific regulation, based on luciferase analysis.…”

Epigenetic Gene Silencing Underlies C-Fiber Dysfunctions in Neuropathic Pain

“…The PCR primers used are listed in supplemental Table 1, available at www.jneurosci.org as supplemental material. Some of the primers were published previously (Koenigsberger et al, 2000;Klein et al, 2003;Qiang et al, 2005;Matsumoto et al, 2006;Cheng et al, 2009;Staaf et al, 2009). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for normalization.…”