Neuron-restrictive Silencer Element Research Articles

Cysteine 397 plays important roles in the folding of the neuron-restricted silencer factor/RE1-silencing transcription factor

The neuron-restrictive silencer factor/RE1-silencing transcription factor (NRSF/REST) is regarded as not only a key transcriptional repressor but also an activator in neuron gene expression by specifically interacting with neuron-restrictive silencer element (NRSE/RE1) dsDNA and small NRSE/RE1 dsRNA, respectively. But its exact mechanism remains unclear. One major problem is that it is hard to obtain its functional multiple zinc finger (ZnF) domains in a large quantity for further structural studies. To address this issue, in this study, we for the first time attained soluble NRSF/REST functional domains named as ZnF5–8, ZnF4–8, ZnF3–8 and ZnF2–8 containing four, five, six and seven ZnF motifs in tandem, respectively, by using Circular Dichroism (CD) spectrum and two-dimensional (2D) nucleic magnetic resonance (NMR) 1H–1H NOESY spectrum to monitor the folding of each single ZnF peptide. The data indicated that the residue cysteine 397 (Cys397) plays important roles in the global folding of NRSF/REST multiple ZnFs domain.

Plasmids containing NRSE/RE1 sites enhance neurite outgrowth of retinal ganglion cells via sequestration of REST independent of NRSE dsRNA expression

Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor of neuron-specific genes that binds to a conserved DNA element, the neuron restrictive silencer element (NRSE/RE1). Interestingly, increased REST activity is found in several neurological diseases like Huntington's disease and cerebral ischemia. Recently, it was shown that NRSE dsRNA, a double-stranded non-coding RNA can bind to REST during a defined period of neuronal differentiation, and thereby changes REST from a transcriptional repressor to an activator of neuron-specific genes. Here, we analyzed the effects of NRSE dsRNA expression in primary retinal ganglion cells. We found that NRSE dsRNA expression vectors significantly enhance neurite outgrowth even when axonal degeneration is induced by neurotrophin deprivation. Transfection of HEK cells with NRSE dsRNA-expressing vectors altered their morphology leading to the formation of thin processes and induced the expression of neurofilament-68. Surprisingly, control vectors containing REST-binding sites, but not expressing NRSE dsRNA, resulted in the same effects, also in the retinal ganglion cell model. Reporter assays and retention of REST in the cytoplasm with a labeled NRSE/RE1-containing plasmid incapable of entering the nucleus suggest that sequestration of REST in the cytoplasm is the reason for the observed effects. No evidence for a biological function of NRSE dsRNA could be found in these models. We conclude that sequestration of REST leads to enhanced neurite outgrowth in retinal ganglion cells and that an increased activity of REST, as it is found in several neurodegenerative diseases, can be effectively modulated by sequestration of REST with plasmids containing NRSE/RE1 sites.

Neuron‐restrictive silencer factor‐mediated hyperpolarization‐activated cyclic nucleotide gated channelopathy in experimental temporal lobe epilepsy

Enduring, abnormal expression and function of the ion channel hyperpolarization-activated cyclic adenosine monophosphate gated channel type 1 (HCN1) occurs in temporal lobe epilepsy (TLE). We examined the underlying mechanisms, and investigated whether interfering with these mechanisms could modify disease course. Experimental TLE was provoked by kainic acid-induced status epilepticus (SE). HCN1 channel repression was examined at mRNA, protein, and functional levels. Chromatin immunoprecipitation was employed to identify the transcriptional mechanism of repressed HCN1 expression, and the basis for their endurance. Physical interaction of the repressor, NRSF, was abolished using decoy oligodeoxynucleotides (ODNs). Video/electroencephalographic recordings were performed to assess the onset and initial pattern of spontaneous seizures. Levels of NRSF and its physical binding to the Hcn1 gene were augmented after SE, resulting in repression of HCN1 expression and HCN1-mediated currents (I(h) ), and reduced I(h) -dependent resonance in hippocampal CA1 pyramidal cell dendrites. Chromatin changes typical of enduring, epigenetic gene repression were apparent at the Hcn1 gene within a week after SE. Administration of decoy ODNs comprising the NRSF DNA-binding sequence (neuron restrictive silencer element [NRSE]), in vitro and in vivo, reduced NRSF binding to Hcn1, prevented its repression, and restored I(h) function. In vivo, decoy NRSE ODN treatment restored theta rhythm and altered the initial pattern of spontaneous seizures. Acquired HCN1 channelopathy derives from NRSF-mediated transcriptional repression that endures via chromatin modification and may provide insight into the mechanisms of a number of channelopathies that coexist with, and may contribute to, the conversion of a normal brain into an epileptic one.

The regulation and role of neuronal gap junctions during development.

Coupling of neurons by electrical synapses (gap junctions) transiently increases in the mammalian CNS during development and plays a role in a number of developmental events, including neuronal death. The coupling subsequently decreases and remains low in the adult, confined to specific subsets of neurons. In a recent study we have demonstrated that the developmental increase in neuronal gap junction coupling is regulated by the balance between the activity of two neurotransmitter receptors, group II metabotropic glutamate receptors (mGluR) and GABA(A) receptors. Specifically, we found that activation of group II mGluRs induces the developmental increases in neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein) and activation of GABA(A) receptors counteracts to these increases. We also established that the regulation by both neurotransmitter receptors is via a neuron-restrictive silencer element in the Cx36 gene promoter and the 3'-untranslated region of the Cx36 mRNA. Importantly, we demonstrated that mechanisms for the developmental increase in neuronal gap junction coupling directly control the death/survival mechanisms in developing neurons.

The regulation and role of neuronal gap junctions during development

Coupling of neurons by electrical synapses (gap junctions) transiently increases in the mammalian CNS during development and plays a role in a number of developmental events, including neuronal death. The coupling subsequently decreases and remains low in the adult, confined to specific subsets of neurons. In a recent study we have demonstrated that the developmental increase in neuronal gap junction coupling is regulated by the balance between the activity of two neurotransmitter receptors, group II metabotropic glutamate receptors (mGluR) and GABAA receptors. Specifically, we found that activation of group II mGluRs induces the developmental increases in neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein) and activation of GABAA receptors counteracts to these increases. We also established that the regulation by both neurotransmitter receptors is via a neuron-restrictive silencer element in the Cx36 gene promoter and the 3'-untranslated region of the Cx36 mRNA. Importantly, we demonstrated that mechanisms for the developmental increase in neuronal gap junction coupling directly control the death/survival mechanisms in developing neurons.

Interplay of Chemical Neurotransmitters Regulates Developmental Increase in Electrical Synapses

Coupling of neurons by electrical synapses (gap junctions) transiently increases in the mammalian CNS during development. We report here that the developmental increase in neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein) are regulated by an interplay between the activity of group II metabotropic glutamate receptors (mGluRs) and GABA(A) receptors. Specifically, using dye coupling, electrotonic coupling, Western blots and small interfering RNA in the rat and mouse hypothalamus and cortex in vivo and in vitro, we demonstrate that activation of group II mGluRs augments, and inactivation prevents, the developmental increase in neuronal gap junction coupling and Cx36 expression. However, changes in GABA(A) receptor activity have the opposite effects. The regulation by group II mGluRs is via cAMP/PKA-dependent signaling, and regulation by GABA(A) receptors is via Ca(2+)/PKC-dependent signaling. Furthermore, the receptor-mediated upregulation of Cx36 requires a neuron-restrictive silencer element in the Cx36 gene promoter, and the downregulation involves the 3'-untranslated region of the Cx36 mRNA, as shown using reverse-transcription quantitative real-time PCR and luciferase reporter activity analysis. In addition, the methyl thiazolyl tetrazolium analysis indicates that mechanisms for the developmental increase in neuronal gap junction coupling directly control the death/survival mechanisms in developing neurons. Together, the results suggest a multitiered strategy for chemical synapses in developmental regulation of electrical synapses.

REST Regulates DYRK1A Transcription in a Negative Feedback Loop

DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1A) has been shown to be involved in learning and memory impairments in Alzheimer disease and Down syndrome. As a homolog of Drosophila minibrain gene, DYRK1A also plays important roles in neurodevelopment; however, the function and regulatory mechanism of DYRK1A in neurodevelopment remain elusive. REST (RE1 silencing transcription factor) plays vital roles in neuronal differentiation. Here, we found that REST can activate DYRK1A transcription via a neuron-restrictive silencer element at bp -833 to -815 of human DYRK1A promoter. The coordinated expression of DYRK1A and REST in mouse brain further supports the cross-interaction of DYRK1A and REST during neurodevelopment. Moreover, we showed that DYRK1A dosage imbalance reduced REST protein stability and transcriptional activity through facilitating ubiquitination and subsequent degradation of REST protein. Therefore, the regulation of DYRK1A by REST in a negative feedback loop suggests that DYRK1A and REST are closely related in neurodevelopment.

Current biochemistry, molecular biology, and clinical relevance of natriuretic peptides

The mammalian natriuretic peptide family consists of atrial (ANP), brain [B-type; BNP] and C-type natriuretic peptide (CNP) and three receptors, natriuretic receptors-A (NPR-A), -B (NPR-B) and -C (NPR-C). Both ANP and BNP are abundantly expressed in the heart and are secreted mainly from the atria and ventricles, respectively. By contrast, CNP is mainly expressed in the central nervous system, bone and vasculature. Plasma concentrations of both ANP and BNP are elevated in patients with cardiovascular disease, though the magnitude of the increase in BNP is usually greater than the increase in ANP. This makes BNP is a clinically useful diagnostic marker for several pathophysiological conditions, including heart failure, ventricular remodeling and pulmonary hypertension, among others. Recent studies have shown that in addition to BNP-32, proBNP-108 also circulates in human plasma and that levels of both forms are increased in heart failure. Furthermore, proBNP-108 is O-glycosylated and circulates at higher levels in patients with severe heart failure. In this review we discuss recent progress in our understanding of the biochemistry, molecular biology and clinical relevance of the natriuretic peptide system.

Non-Coding RNAs in Neural Networks, REST-Assured

In the nervous system, several key steps in cellular complexity and development are regulated by non-coding RNAs (ncRNAs) and the repressor element-1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF). REST recruits gene regulatory complexes to regulatory sequences, among them the repressor element-1/neuron-restrictive silencer element, and mediates developmental stage-specific gene expression or repression, chromatin (re-)organization or silencing for protein-coding genes as well as for several ncRNAs like microRNAs, short interfering RNAs or long ncRNAs. NcRNAs are far from being just transcriptional noise and are involved in chromatin accessibility, transcription and post-transcriptional processing, trafficking, or RNA editing. REST and its cofactor CoREST are both highly regulated through various ncRNAs. The importance of the correct regulation within the ncRNA network, the ncRNAome, is demonstrated when it comes to a deregulation of REST and/or ncRNAs associated with molecular pathophysiology underlying diverse disorders including neurodegenerative diseases or brain tumors.

Epigenetic Gene Silencing Underlies C-Fiber Dysfunctions in Neuropathic Pain

Peripheral nerve injury causes neuropathic pain, which is characterized by the paradoxical sensations of positive and negative symptoms. Clinically, negative signs are frequently observed; however, their underlying molecular mechanisms are largely unknown. Dysfunction of C-fibers is assumed to underlie negative symptoms and is accompanied by long-lasting downregulation of Na(v)1.8 sodium channel and mu-opioid receptor (MOP) in the dorsal root ganglion (DRG). In the present study, we found that nerve injury upregulates neuron-restrictive silencer factor (NRSF) expression in the DRG neurons mediated through epigenetic mechanisms. In addition, chromatin immunoprecipitation analysis revealed that nerve injury promotes NRSF binding to the neuron-restrictive silencer element within MOP and Na(v)1.8 genes, thereby causing epigenetic silencing. Furthermore, NRSF knockdown significantly blocked nerve injury-induced downregulations of MOP and Na(v)1.8 gene expressions, C-fiber hypoesthesia, and the losses of peripheral morphine analgesia and Na(v)1.8-selective blocker-induced hypoesthesia. Together, these data suggest that NRSF causes pathological and pharmacological dysfunction of C-fibers, which underlies the negative symptoms in neuropathic pain.

Early-Life Experience Reduces Excitation to Stress-Responsive Hypothalamic Neurons and Reprograms the Expression of Corticotropin-Releasing Hormone

Increased sensory input from maternal care attenuates neuroendocrine and behavioral responses to stress long term and results in a lifelong phenotype of resilience to depression and improved cognitive function. Whereas the mechanisms of this clinically important effect remain unclear, the early, persistent suppression of the expression of the stress neurohormone corticotropin-releasing hormone (CRH) in hypothalamic neurons has been implicated as a key aspect of this experience-induced neuroplasticity. Here, we tested whether the innervation of hypothalamic CRH neurons of rat pups that received augmented maternal care was altered in a manner that might promote the suppression of CRH expression and studied the cellular mechanisms underlying this suppression. We found that the number of excitatory synapses and the frequency of miniature excitatory synaptic currents onto CRH neurons were reduced in "care-augmented" rats compared with controls, as were the levels of the glutamate vesicular transporter vGlut2. In contrast, analogous parameters of inhibitory synapses were unchanged. Levels of the transcriptional repressor neuron-restrictive silencer factor (NRSF), which negatively regulates Crh gene transcription, were markedly elevated in care-augmented rats, and chromatin immunoprecipitation demonstrated that this repressor was bound to a cognate element (neuron-restrictive silencing element) on the Crh gene. Whereas the reduced excitatory innervation of CRH-expressing neurons dissipated by adulthood, increased NRSF levels and repression of CRH expression persisted, suggesting that augmented early-life experience reprograms Crh gene expression via mechanisms involving transcriptional repression by NRSF.

Neuron-restrictive silencer factor causes epigenetic silencing of K v4.3 gene after peripheral nerve injury

Peripheral nerve injury causes a variety of alterations in pain-related gene expression in primary afferent, which underlie the neuronal plasticity in neuropathic pain. One of the characteristic alterations is a long-lasting downregulation of voltage-gated potassium (K v) channel, including K v4.3, in the dorsal root ganglion (DRG). The present study showed that nerve injury reduces the messenger RNA (mRNA) expression level of K v4.3 gene, which contains a conserved neuron-restrictive silencer element (NRSE), a binding site for neuron-restrictive silencer factor (NRSF). Moreover, we found that injury causes an increase in direct NRSF binding to K v4.3-NRSE in the DRG, using chromatin immunoprecipitation (ChIP) assay. ChIP assay further revealed that acetylation of histone H4, but not H3, at K v4.3-NRSE is markedly reduced at day 7 post-injury. Finally, the injury-induced K v4.3 downregulation was significantly blocked by antisense-knockdown of NRSF. Taken together, these data suggest that nerve injury causes an epigenetic silencing of K v4.3 gene mediated through transcriptional suppressor NRSF in the DRG.

Analysis of the Repressor Element‐1 Silencing Transcription Factor/Neuron‐Restrictive Silencer Factor Occupancy of Non‐Neuronal Genes in Peripheral Lymphocytes from Patients with Huntington's Disease

We have previously demonstrated that the transcription of neuronal repressor element-1/neuron-restrictive silencer element (RE1/NRSE)-regulated genes is reduced in the brain of subjects with Huntington's disease (HD) as a result of increased binding of the repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) to its RE1/NRSE targets. As specific non-neuronal REST/NRSF-regulated genes have been identified in the human genome, we exploited the possibility that the binding of REST/NRSF to its target RE1/NRSE sites may also be altered in the peripheral tissues of HD patients. Our results show that REST/NRSF occupancy is increased in lymphocytes from HD subjects, thus indicating for the first time that the activity of the RE1/NRSE sites is dysfunctional in vivo. Chromatin immunoprecipitation (ChIP) of the RE1/NRSE sites in lymphocytes may therefore be a reproducible, sensitive and specific means of searching for candidate markers of HD onset and progression.

Genome-wide identification of target genes repressed by the zinc finger transcription factor REST/NRSF in the HEK 293 cell line

Transcriptional repression is as important as transcriptional activation in establishing cell-type specific patterns of gene expression. RE1-silencing transcription factor (REST), also known as neuronal restrictive silencing factor (NRSF), is a transcriptional regulator that represses a battery of neuronal differentiation genes in non-neuronal cells or in neural progenitor cells by binding to a specific DNA sequence (repressor element-1/neuron-restrictive silencer element, RE1/NRSE). REST/NRSF functions in the neuronal development are widely studied, however, little is known about target genes in various non-neuronal lineages that may result in cell differentiation. Here, we use RNA interference (RNAi) technology combined with the microarray strategy to identify potential REST/NRSF targets and RE1/NRSEs in human non-neuronal cell line HEK 293. Expression of 54 genes was up-regulated by inhibition of REST/NRSF in the HEK 293 cells according to the microarray experiment and 13 of those were further confirmed by quantitative RT-PCR. Our results confirmed the good confidence and reliability of current research data based on in silico, chromatin immunoprecipitation in combination with microarrays (ChIP-chip), and high-throughput sequencing (ChIP-seq). However, in view of the fact that thousands of genes have been testified or predicted to be recognized by REST/NRSF, our data show that only a few genes among those are directly up-regulated by the interaction of REST/NRSF with RE1/NRSEs sites in gene sequences.

Construction and identification of the lentiviral vector of repressor element-1/neuron-restrictive silencer element double-stranded RNA

To construct a lentiviral vector of repressor element-1/neuron-restrictive silencer element (RE-1/NRSE) double-stranded RNA (dsRNA). The RE-1/NRSE cDNA containing both sense and antisense oligo DNA fragments of the targeting sequence was synthesized and cloned into the pGC-LV vector. The obtained lentiviral vector containing RE-1/NRSE dsRNA was confirmed by PCR and sequencing. A total of 293T cells were cotransfected with lentiviral vector of L-smNRSE/RE-1, pHelper 1.0, and pHelper 2.0. The titer of virus was measured based on the expression level of green fluorescent protein. The transfection efficiency of green fluorescent protein into rat mesenchymal stem cells was calculated. PCR and DNA sequencing demonstrated that the constructed lentivirus vector of L-smNRSE/RE-1 produced RE-1/NRSE dsRNA.The titer of the concentrated virus was 4x108 TU/m1. The virus was stably transfected into rat mesenchymal stem cells, and the infection efficiency reached 100% when the multiplicity of infection was 80. The lentivirus vector of RE-1/NRSE dsRNA is successfully constructed.

Regulation of Aldosterone and Cortisol Production by the Transcriptional Repressor Neuron Restrictive Silencer Factor

Aldosterone synthase (CYP11B2) and 11 beta-hydroxylase (CYP11B1) regulate aldosterone and cortisol production, respectively. The expression of these enzymes is promoted by calcium influx through Cav3.2, a T-type calcium channel. Neuron-restrictive silencer factor (NRSF) binds to neuron-restrictive silencer element (NRSE) to suppress the transcription of NRSE-containing genes. We found a NRSE-like sequence in human CYP11B2 and CYP11B1 genes as well as the CACNA1H gene of many mammalian species. The CACNA1H gene encodes the alpha-subunit of Cav3.2. Here we investigated how NRSF/NRSE regulates aldosterone and cortisol synthesis. Inhibition of endogenous NRSF by an adenovirus-expressing dominant-negative NRSF (AD/dnNRSF) increased human CYP11B2 and CYP11B1 mRNA expression, leading to aldosterone and cortisol secretion in human adrenocortical (H295R) cells. In reporter gene experiments, NRSE suppressed luciferase reporters driven by CYP11B2 and CYP11B1 promoters and dnNRSF enhanced them. Moreover, cotransfection of dnNRSF increased luciferase activity of reporter genes after deletion or mutation of NRSE, suggesting that NRSF/NRSE regulates transcription of CYP11B2 and CYP11B1 genes indirectly. AD/dnNRSF augmented mRNA expression of rat CYP11B2 and CYP11B1 genes, neither of which contains a NRSE-like sequence in rat adrenal cells. AD/dnNRSE also significantly increased CACNA1H mRNA in H295R and rat adrenal cells. Efonidipine, a T/L-type calcium channel blocker, significantly suppressed dnNRSF-mediated up-regulation of CYP11B2 and CYP11B1 expression. Moreover, NRSF/NRSE is also involved in angiotensin II- and K(+)-stimulated augmentation of CYP11B2 and CYP11B1 gene transcription. In conclusion, NRSF/NRSE controls aldosterone and cortisol synthesis by regulating CYP11B2 and CYP11B1 gene transcription mainly through NRSF/NRSE-mediated enhancement of the CACNA1H gene.

Transcription of the chicken Grin1 gene is regulated by the activity of SP3 and NRSF in undifferentiated cells and neurons

The NMDA (N-methyl-D-aspartate) receptors are important in the regulation of neuronal development, synaptic plasticity, learning and memory, and are involved in several brain pathologies. The NR1 subunit is essential for the assembly of functional receptors, as it forms the calcium-permeable ion channel and contains the obligatory co-agonist binding site. Previous studies have shown that NR1 gene (Grin1) expression is up-regulated during neuronal differentiation and its expression is widespread in the central nervous system. We have previously cloned the chicken Grin1 gene and 1.9 kb of the 5'-regulatory region. In the present study, we analysed the molecular mechanisms that regulate chicken Grin1 gene transcription in undifferentiated cells and neurons. By functional analysis of chicken Grin1-luciferase gene 5'-regulatory region constructs, we demonstrate that the basal promoter is delimited within 210 bp upstream from the main transcription initiation site. DNA-protein binding and functional assays revealed that the 5'-UTR (untranslated region) has one consensus NRSE (neuron-restrictive silencing element) that binds NRSF (neuron-restrictive silencing factor), and one SP (stimulating protein transcription factor) element that binds SP3, both repressing Grin1 gene transcription in undifferentiated P19 cells (embryonic terato-carcinoma cells) and PC12 cells (phaeochromocytoma cells). The promoter region lacks a consensus TATA box, but contains one GSG/SP (GSG-like box near a SP-consensus site) that binds SP3 and up-regulates gene transcription in embryonic chicken cortical neurons. Taken together, these results demonstrate a dual role of SP3 in regulating the expression of the Grin1 gene, by repressing transcription in the 5'-UTR in undifferentiated cells as well as acting as a transcription factor, increasing Grin1 gene transcription in neurons.

Regulatory role of neuron‐restrictive silencing factor in the specific expression of cocaine‐ and amphetamine‐regulated transcript gene

Cocaine- and amphetamine-regulated transcript (CART) peptide is an endogenous peptide which is widely expressed in the CNS and PNS as well as in endocrine cells. Despite the functional knowledge about CART, the mechanisms that regulate CART gene transcription are poorly characterized. Here, we showed that neuron-restrictive silencer factor (NRSF) functions as a negative regulator of CART gene expression in neuroendocrine cells. A putative neuron-restrictive silencer element (NRSE) conserved between the rodent and human CART promoter was identified and demonstrated to bind to NRSF in sequence-specific manner by the electrophoretic mobility shift and chromatin immunoprecipitation assays. Ectopic expression of NRSF in pheochromocytoma cells (PC12) and insulin-secreting cells (INS-1) induced a marked reduction in the level of CART mRNA and the activity of CART promoter or NRSE reporter. The CART promoter showed very low activity in endogenous NRSF-expressing HeLa cells. When expression of NRSF was down-regulated in HeLa cells using a RNA interfering technique, the transcriptional activity of the CART promoter or a NRSE reporter was significantly increased. Taken together, our data suggested that CART gene expression in neuroendocrine cells is strictly controlled by NRSF, via a mechanism dependent upon the CART NRSE.

Novel function of neuron-restrictive silencer factor (NRSF) for posttranscriptional regulation

The neuron-restrictive silencer factor (NRSF) functions as a transcriptional repressor of neuronal genes in nonneuronal cells. However, it is expressed in certain mature neurons in adults, suggesting that it might have complex and novel roles depending on its cellular and physiological context. Overexpression of NRSF led to both increased opioid ligand-binding activity of the endogenous MOR and MOR–GFP fusion protein expression. In RNA immunoprecipitation and gel-shift assays, NRSF specifically interacted with the NRSE sequence of MOR mRNA. When MOR and NRSF genes were coexpressed, the specific ligand-binding activity of MOR was increased in neuroblastoma NMB cells, but decreased in PC12 cells result from its localization. Indeed, after overexpressing NRSF in NMB cells, the target RNA moved to the translationally active polysomal fraction. Overexpression of NRSF also led to enhanced phosphorylation of eIF4G. In contrast, knockdown of NRSF by siRNA transfection significantly decreased eIF4G phosphorylation. These findings indicate that NRSF may deliver the target MOR transcripts to the polyribosomal complex and activate eIF4G phosphorylation, resulting in translational activation. We report here a novel function of NRSF that enhance the translation of the mu opioid receptor (MOR) gene through its RNA binding sequence, the neuron-restrictive silencer element (NRSE).

REST is a key regulator in brain‐specific homeobox gene expression during neuronal differentiation

Brain-specific homeobox (Bsx) is specifically expressed at the early embryonic stages during brain development. Several studies show that Bsx plays important roles in brain development; however, the mechanisms of its transcriptional regulation remain to be established. In this study, we show that binding of repressor element silencing transcription factor (REST) to the neuron restrictive silencer element (NRSE) represses Bsx transcription in non-neuronal P19 cells. The Bsx promoter contains several putative binding sites for transcription factors, including NRSE for REST and the GC box for the transcriptional activator, Sp1. Upon neuronal differentiation of P19 cells with retinoic acid, Bsx gene expression increased, whereas that of the REST gene decreased. Electrophoretic mobility shift analyses demonstrated that recombinant REST proteins bound the NRSE region of the Bsx promoter. In neuronal NS20Y cells, transcriptional activity of the Bsx promoter was decreased upon expression of REST. Moreover, dominant-negative REST derepressed Bsx transcription in P19 cells. Sp1-mediated transcriptional activity of the Bsx promoter was attenuated by treatment with mithramycin A, a GC box-binding drug, but was enhanced upon mutation of NRSE. Co-immunoprecipitation and chromatin immunoprecipitation assays showed that the Bsx promoter appeared to be modulated by direct interactions between REST and Sp1. The CpG sites of NRSE and GC box were completely unmethylated, signifying no interference of DNA methylation. Our results suggest that binding of REST to NRSE suppresses the Sp1-mediated activation of Bsx in non-neuronal cells.