Repressor Element-1 Silencing Transcription Factor/neuron-restrictive Silencer Factor Research Articles

Conditional knockout of REST/NRSF in excitatory neurons reduces seizure susceptibility to chemical kindling.

The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is an epigenetic master regulator that plays a crucial role during nervous system development and maturation. REST function was originally described during development, where it determines neuronal phenotype. However, recent studies showed that REST participates in several processes in the adult brain, including neuronal plasticity and epileptogenesis. In this regard, the relationships between REST and epilepsy are still controversial and need further investigation. As forebrain excitatory neurons are the common final pathway of seizure susceptibility, we investigated the role of REST in epilepsy by inducing REST conditional knockout (REST-cKO) specifically in excitatory neurons of the hippocampus. To target the excitatory neuronal population, we cloned the calcium/calmodulin-dependent protein kinase IIα minimal promoter upstream of Cre recombinase. After assessing the specificity of the promoter's expression, the transgenes were packaged in an engineered adeno-associated virus able to cross the blood-brain and blood-cerebrospinal fluid barriers and delivered in the lateral ventricles of 2-month-old RESTflox/flox mice to characterize, after 1 month, the cognitive phenotype and the seizure propensity. We show that REST-cKO mice display lower levels of anxiety in the light-dark test with respect to control mice but have unaltered motor, social, and cognitive profiles. The evaluation of the susceptibility to epileptic seizures showed that REST-cKO mice are more resistant to pentylenetetrazole-induced kindling but not to seizures induced by a single administration of the convulsant and show higher survival rates. Overall, these data suggest that the absence of REST in forebrain excitatory neurons decreases seizure susceptibility, pointing to a pro-epileptogenic role of the transcriptional repressor under conditions of pathological excitation/inhibition imbalance.

Loss of the repressor REST affects progesterone receptor function and promotes uterine leiomyoma pathogenesis

Uterine leiomyomas (UL) are benign tumors that arise in the myometrial layer of the uterus. The standard treatment option for UL is hysterectomy, although hormonal therapies, such as selective progesterone receptor modulators, are often used as temporary treatment options to reduce symptoms or to slow the growth of tumors. However, since the pathogenesis of UL is poorly understood and most hormonal therapies are not based on UL-specific, divergent hormone signaling pathways, hallmarks that predict long-term efficacy and safety of pharmacotherapies remain largely undefined. In a previous study, we reported that aberrant expression of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes activate UL growth due to the near ubiquitous loss of REST. Here, we show that ablation of the Rest gene in mouse uterus leads to UL phenotype and gene-expression patterns analogous to UL, including altered estrogen and progesterone signaling pathways. We demonstrate that many of the genes dysregulated in UL harbor cis-regulatory elements bound by REST and progesterone receptor (PGR) adjacent to each other. Crucially, we identify an interaction between REST and PGR in healthy myometrium and present a putative mechanism for the dysregulation of progesterone-responsive genes in UL ensuing in the loss of REST. Using three Rest conditional knockout mouse lines, we provide a comprehensive picture of the impact loss of REST has in UL pathogenesis and in altering the response of UL to steroid hormones.

Expression of the Neural REST/NRSF-SIN3 Transcriptional Corepressor Complex as a Target for Small-Molecule Inhibitors.

The repressor element 1 (RE1) silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) modulates the expression of genes with RE1/neuron-restrictive silencing element (RE1/NRSE) sites by recruiting the switch independent 3 (SIN3) factorand the REST corepressor (COREST) to its N and C-terminal repressor domain, respectively. Both, SIN3 and COREST assemble into protein complexes that are composed of multiple subunits including a druggable histone deacetylase (HDAC) enzyme. The SIN3 core complex comprises the eponymous proteins SIN3A or SIN3B, the catalytically active proteins HDAC1 or HDAC2, the histone chaperone retinoblastoma-associated protein 46/retinoblastoma-binding protein 7 (RBAP46/RBBP7) or RBAP48/RBBP4, the SIN3-associated protein 30 (SAP30), and the suppressor of defective silencing 3 (SDS3). Here, we overcome a bottleneck limiting the molecular characterization of the REST/NRSF-SIN3 transcriptional corepressor complex. To this end, SIN3 genes were amplified from the complementary DNA of neural stem/progenitor cells, and expressed in a baculovirus/insect cell expression system. We show that the isolates bind to DNA harboring RE1/NRSE sites and demonstrate that the histone deacetylase activity is blocked by small-molecule inhibitors. Thus, our isolates open up for future biomedical research on this critical transcriptional repressor complex and are envisioned as tool for drug testing.

MGluR5 regulates REST/NRSF signaling through N-cadherin/\u03b2-catenin complex in Huntington\u2019s disease

Repressor element 1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a transcription repressor and its expression is regulated by the Wnt pathway through β-catenin. Metabotropic glutamate receptor 5 (mGluR5) signaling plays a key role in controlling neuronal gene expression. Interestingly, REST/NRSF nuclear translocation and signaling, as well as mGluR5 signaling are altered in the presence of mutant huntingtin. It remains unclear whether mGluR5 can modulate Wnt and REST/NRSF signaling under physiological conditions and whether this modulation is altered in Huntington’s disease (HD). Using primary corticostriatal neurons derived from wild type mouse embryos, we find that targeting mGluR5 using the agonist, DHPG, or the negative allosteric modulator, CTEP, modulates REST/NRSF expression by regulating the assembly of N-cadherin/ β-catenin complex in a Src kinase-dependent manner. We have validated our in vitro findings in vivo using two HD mouse models. Specifically, we show that pharmacological inhibition of mGluR5 in zQ175 mice and genetic ablation of mGluR5 in BACHD mice corrected the pathological activation of Src and rescued REST/NRSF-dependent signaling. Together, our data provide evidence that mGluR5 regulates REST/NRSF expression via the Wnt pathway and highlight the contribution of impaired REST/ NRSF signaling to HD pathology.

The role of REST/NRSF, TrkB and BDNF in neurobiological mechanisms of different susceptibility to seizure in a PTZ model of epilepsy

Global transcriptional disturbances are believed to play a major role in the course of epilepsy. Due to the high complexity, the neurobiological mechanisms underlying different susceptibility to seizure and epilepsy are not well known. A transcription factor called REST/NRSF (repressor element 1-silencing transcription factor/neuron-restrictive silencer factor) is believed to contribute to processes associated with seizure development. Its downstream genes, those encoding BDNF (brain-derived neurotrophic factor) and TrkB (BDNF receptor; tropomyosin receptor kinase B), are also thought to play a role. To verify this hypothesis, we used a PTZ kindling model of epilepsy and divided animals into groups according to their different susceptibility to seizure. The concentrations of REST/NRSF, BDNF, and TrkB protein and mRNA were measured in hippocampal homogenates.The level of REST/NRSF protein measured 24 h after the last PTZ injection was increased in animals resistant to kindling and was unchanged in groups of rats kindled after 5, 10 and 20 in.ections of PTZ. In contrast, TrkB protein concentration was enhanced in all kindled rats and was unchanged in the resistant rats. There were no changes in the protein concentration of BDNF in rats with different susceptibility to kindling; however, data from the combined kindled groups vs. the resistant group revealed an increased level of BDNF in resistant animals.In sum, the increased level of protein REST/NRSF in resistant animals may reflect its neuroprotective role against seizure development. The increased concentration of TrkB protein in kindled animals indicates its pivotal role in the process of epileptogenesis. We propose that in resistant rats, REST/NRSF could contribute to the prevention of TrkB activation related to seizures.

The FlpTRAP system for purification of specific, endogenous chromatin regions

The repressor element 1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) binds to repressor element 1/neuron-restrictive silencer element (RE1/NRSE) sites in the genome and recruits effector proteins to repress its target genes. Here, we developed the FlpTRAP system to isolate endogenously assembled DNA-protein complexes such as the REST/NRSF complex. In the FlpTRAP system, we take advantage of the step-arrest variant of the Flp recombinase, FlpH305L, which, in the presence of Flp recognition target (FRT) DNA, accumulates as FRT DNA-protein adduct. The FlpTRAP system consists of three elements: (i) FlpH305L-containing cell extracts or isolates, (ii) a cell line engineered to harbor the DNA motif of interest flanked by FRT sites, and (iii) affinity selection steps to isolate the target chromatin. Specifically, 3×FLAG-tagged FlpH305L was expressed in insect cell cultures infected with baculovirus, and cell lysates were prepared. The lysate was used to capture the FRT-SNAP25 RE1/NRSE-FRT chromatin from a human medulloblastoma cell line, and the target RE1/NRSE chromatin was isolated by anti-FLAG immunoaffinity chromatography. Using electrophoretic mobility shift assays (EMSAs) and chromatin immunopurification (ChIP), we show that FlpH305L recognized and bound to the FRT sites. Overall, we suggest the FlpTRAP system as a tool to purify endogenous, specific chromatin loci from eukaryotic cells.

Loss of nuclear REST/NRSF in aged-dopaminergic neurons in Parkinson’s disease patients

Parkinson’s disease (PD) is the second most common neurodegenerative disease. Lewy bodies and pale bodies in dopaminergic neurons in the substantia nigra are pathological hallmarks of PD. A number of neurodegenerative diseases demonstrate aggregate formation, but how these aggregates are associated with their pathogenesis remains unknown. It has been reported that repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is induced in the nuclei of aged neurons, preserves neuronal function, and protects against neurodegeneration during aging through the repression of cell death-inducing genes. The loss of REST is associated with Alzheimer’s disease pathology. However, its function in dopaminergic neurons remains unknown. Here we demonstrated that REST enters the nucleus of aged dopaminergic neurons. On the other hand, REST is partially sequestrated in Lewy bodies and is mostly absent from the nucleus of neurons in brains with PD and dementia with Lewy bodies (DLB). Dopaminergic neuron-specific autophagy-deficient mice exhibit REST accumulation in aggregates. Defects in the protein quality control system induce REST mRNA expression; its gene product mainly appears in aggregates. Our results suggest that Lewy pathology disturbs normal aging processes in dopaminergic neurons by sequestering REST and the loss of REST may associate with the PD pathology.

Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target.

In human cells, thousands of predominantly neuronal genes are regulated by the repressor element 1 (RE1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). REST/NRSF represses transcription of these genes in stem cells and non-neuronal cells by tethering corepressor complexes. Aberrant REST/NRSF expression and intracellular localization are associated with cancer and neurodegeneration in humans. To date, detailed molecular analyses of REST/NRSF and its C-terminal repressor complex have been hampered largely by the lack of sufficient amounts of purified REST/NRSF and its complexes. Therefore, the aim of this study was to express and purify human REST/NRSF and its C-terminal interactors in a baculovirus multiprotein expression system as individual proteins and coexpressed complexes. All proteins were enriched in the nucleus, and REST/NRSF was isolated as a slower migrating form, characteristic of nuclear REST/NRSF in mammalian cells. Both REST/NRSF alone and its C-terminal repressor complex were functionally active in histone deacetylation and histone demethylation and bound to RE1/neuron-restrictive silencer element (NRSE) sites. Additionally, the mechanisms of inhibition of the small-molecule drugs 4SC-202 and SP2509 were analyzed. These drugs interfered with the viability of medulloblastoma cells, where REST/NRSF has been implicated in cancer pathogenesis. Thus, a resource for molecular REST/NRSF studies and drug development has been established.

Expression of REST4 in human gliomas in vivo and influence of pioglitazone on REST in vitro

The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) has an irreplaceable role during the differentiation of neurons. REST has multiple splice variants which link to various types of cancer. Previous work had highlighted the role of REST in glioma, where the expression of REST is enhanced. But whether alternative splicing of REST is expressed in glioma has not been described. Here, we show that a specific isoform REST4 is expressed in glioma specimens, and will influence the mRNA level of REST in vivo. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have a role of antineoplastic in various tumor cells, which including glioma cells. Moreover, study indicated that PPARγ agonist pioglitazone can promote alternative splicing of REST pre-mRNA. In this study, we selected pioglitazone as a tool drug to explore whether the role of pioglitazone in anti-glioma is mediated by regulating REST expression or promoting alternative splicing of REST in glioma cells. Results show that pioglitazone can inhibit proliferation and induce apoptosis of glioma cell in vitro, which may be mediated by down-regulating REST mRNA level but not by inducing alternative splicing of REST pre-mRNA. Our study firstly reports the expression of REST4 in glioma tissue samples. And we recommend that pioglitazone, which can reduce the expression level of REST, represents a promising drug for therapy of glioma.

Casein kinase 1 suppresses activation of REST in insulted hippocampal neurons and halts ischemia-induced neuronal death.

Repressor Element-1 (RE1) Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF) is a gene-silencing factor that is widely expressed during embryogenesis and plays a strategic role in neuronal differentiation. Recent studies indicate that REST can be activated in differentiated neurons during a critical window of time in postnatal development and in adult neurons in response to neuronal insults such as seizures and ischemia. However, the mechanism by which REST is regulated in neurons is as yet unknown. Here, we show that REST is controlled at the level of protein stability via β-TrCP-dependent, ubiquitin-based proteasomal degradation in differentiated neurons under physiological conditions and identify Casein Kinase 1 (CK1) as an upstream effector that bidirectionally regulates REST cellular abundance. CK1 associates with and phosphorylates REST at two neighboring, but distinct, motifs within the C terminus of REST critical for binding of β-TrCP and targeting of REST for proteasomal degradation. We further show that global ischemia in rats in vivo triggers a decrease in CK1 and an increase in REST in selectively vulnerable hippocampal CA1 neurons. Administration of the CK1 activator pyrvinium pamoate by in vivo injection immediately after ischemia restores CK1 activity, suppresses REST expression, and rescues neurons destined to die. Our results identify a novel and previously unappreciated role for CK1 as a brake on REST stability and abundance in adult neurons and reveal that loss of CK1 is causally related to ischemia-induced neuronal death. These findings point to CK1 as a potential therapeutic target for the amelioration of hippocampal injury and cognitive deficits associated with global ischemia.

Identification of REST-Regulated Genes and Pathways Using a REST-Targeted Antisense Approach

The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is one of the first negative-acting transcriptional regulators implicated in vertebrate development thought to regulate hundreds of neuron-specific genes. However, its function in the adult system remains elusive. Here we employ second-generation antisense oligonucleotides (ASOs) to study the impact of rest-mediated suppression on gene expression. We demonstrate specific reductions in REST levels in vitro, and in vivo in mouse liver following treatment with ASOs, and we show that ASO mediated-REST suppression results in the elevation in expression of many neuronal genes including brain-derived neurotrophic factor, Synapsin1 (syn1) and β3-tubulin in BALB/c liver. Furthermore, we show the elevation of the affected proteins in plasma following ASO treatment. Finally, microarray analysis was applied to identify a broad range of genes modulated by REST suppression in mouse liver. Our findings suggest that REST may be an important target for neurodegenerative diseases like Huntington's disease, is also involved in the regulation of a broad range of additional cellular pathways, and that the antisense approach is a viable strategy for selectively modulating REST activity in vivo.

REST/NRSF-mediated intrinsic homeostasis protects neuronal networks from hyperexcitability

Intrinsic homeostasis enables neuronal circuits to maintain activity levels within an appropriate range by modulating neuronal voltage-gated conductances, but the signalling pathways involved in this process are largely unknown. We characterized the process of intrinsic homeostasis induced by sustained electrical activity in cultured hippocampal neurons based on the activation of the Repressor Element-1 Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF). We showed that 4-aminopyridine-induced hyperactivity enhances the expression of REST/NRSF, which in turn, reduces the expression of voltage-gated Na(+) channels, thereby decreasing the neuronal Na(+) current density. This mechanism plays an important role in the downregulation of the firing activity at the single-cell level, re-establishing a physiological spiking activity in the entire neuronal network. Conversely, interfering with REST/NRSF expression impaired this homeostatic response. Our results identify REST/NRSF as a critical factor linking neuronal activity to the activation of intrinsic homeostasis and restoring a physiological level of activity in the entire neuronal network.

MicroRNA 26b encoded by the intron of small CTD phosphatase (SCP) 1 has an antagonistic effect on its host gene

Tissue-specific patterns of gene expression play an important role in the distinctive features of each organ. Small CTD phosphatases (SCPs) 1-3 are recruited by repressor element 1 (RE-1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) to neuronal genes that contain RE-1 elements, leading to neuronal gene silencing in non-neuronal cells. SCPs are highly expressed in the heart and contain microRNAs (miR)-26b, 26a-2, and 26a-1 with the same seed sequence in their introns. Therefore, we tried to investigate the roles of miR-26b and its host gene in neonatal rat cardiomyocytes. Overexpression of miR-26b suppressed the mRNA expression levels of ANF, βMHC, and ACTA1 and reduced the cell surface area in cardiomyocytes. We confirmed that miR-26b targets the 3' untranslated region (3'UTR) of GATA4 and canonical transient receptor potential channel (TRPC) 3. Conversely, silencing of the endogenous miR-26b family enhanced the expression levels of TRPC3 and GATA4. On the other hand, overexpression of SCP1 induced the mRNA expression of ANF and βMHC and increased the cell surface area in cardiomyocytes. Next, we compared the effect of overexpression of SCP1 with its introns and SCP1 cDNA to observe the net function of SCP1 expression on cardiac hypertrophy. When the expression levels of SCP1 were the same, the overexpression of SCP1 cDNA had a greater effect at inducing cardiac hypertrophy than SCP1 cDNA with its intron. In conclusion, SCP1 itself has the potential to induce cardiac hypertrophy; however, the effect is suppressed by intronic miR-26b in cardiomyocytes. miR-26b has an antagonistic effect on its host gene SCP1.

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.

Corepressor for element-1–silencing transcription factor preferentially mediates gene networks underlying neural stem cell fate decisions

The repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) silences neuronal genes in neural stem cells (NSCs) and nonneuronal cells through its role as a dynamic modular platform for recruitment of transcriptional and epigenetic regulatory cofactors to RE1-containing promoters. In embryonic stem cells, the REST regulatory network is highly integrated with the transcriptional circuitry governing self-renewal and pluripotency, although its exact functional role is unclear. The C-terminal cofactor for REST, CoREST, also acts as a modular scaffold, but its cell type-specific roles have not been elucidated. We used chromatin immunoprecipitation-on-chip to examine CoREST and REST binding sites in NSCs and their proximate progenitor species. In NSCs, we identified a larger number of CoREST (1,820) compared with REST (322) target genes. The majority of these CoREST targets do not contain known RE1 motifs. Notably, these CoREST target genes do play important roles in pluripotency networks, in modulating NSC identity and fate decisions and in epigenetic processes previously associated with both REST and CoREST. Moreover, we found that NSC-mediated developmental transitions were associated primarily with liberation of CoREST from promoters with transcriptional repression favored in less lineage-restricted radial glia and transcriptional activation favored in more lineage-restricted neuronal-oligodendrocyte precursors. Clonal NSC REST and CoREST gene manipulation paradigms further revealed that CoREST has largely independent and previously uncharacterized roles in promoting NSC multilineage potential and modulating early neural fate decisions.

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.

REST and CoREST Modulate Neuronal Subtype Specification, Maturation and Maintenance

BackgroundThe repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a master regulator of neuronal gene expression. REST functions as a modular scaffold for dynamic recruitment of epigenetic regulatory factors including its primary cofactor, the corepressor for element-1-silencing transcription factor (CoREST), to genomic loci that contain the repressor element-1 (RE1) binding motif. While REST was initially believed to silence RE1 containing neuronal genes in neural stem cells (NSCs) and non-neuronal cells, emerging evidence shows an increasingly complex cell type- and developmental stage-specific repertoire of REST target genes and functions that include regulation of neuronal lineage maturation and plasticity.Methodology/Principal FindingsIn this study, we utilized chromatin immunoprecipitation on chip (ChIP-chip) analysis to examine REST and CoREST functions during NSC-mediated specification of cholinergic neurons (CHOLNs), GABAergic neurons (GABANs), glutamatergic neurons (GLUTNs), and medium spiny projection neurons (MSNs). We identified largely distinct but overlapping profiles of REST and CoREST target genes during neuronal subtype specification including a disproportionately high percentage that are exclusive to each neuronal subtype.Conclusions/SignificanceOur findings demonstrate that the differential deployment of REST and CoREST is an important regulatory mechanism that mediates neuronal subtype specification by modulating specific gene networks responsible for inducing and maintaining neuronal subtype identity. Our observations also implicate a broad array of factors in the generation of neuronal diversity including but not limited to those that mediate homeostasis, cell cycle dynamics, cell viability, stress responses and epigenetic regulation.

Differential Deployment of REST and CoREST Promotes Glial Subtype Specification and Oligodendrocyte Lineage Maturation

BackgroundThe repressor element-1 (RE1) silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a master transcriptional regulator that binds to numerous genomic RE1 sites where it acts as a molecular scaffold for dynamic recruitment of modulatory and epigenetic cofactors, including corepressor for element-1-silencing transcription factor (CoREST). CoREST also acts as a hub for various cofactors that play important roles in epigenetic remodeling and transcriptional regulation. While REST can recruit CoREST to its macromolecular complex, CoREST complexes also function at genomic sites independently of REST. REST and CoREST perform a broad array of context-specific functions, which include repression of neuronal differentiation genes in neural stem cells (NSCs) and other non-neuronal cells as well as promotion of neurogenesis. Despite their involvement in multiple aspects of neuronal development, REST and CoREST are not believed to have any direct modulatory roles in glial cell maturation.Methodology/Principal FindingsWe challenged this view by performing the first study of REST and CoREST in NSC-mediated glial lineage specification and differentiation. Utilizing ChIP on chip (ChIP-chip) assays, we identified distinct but overlapping developmental stage-specific profiles for REST and CoREST target genes during astrocyte (AS) and oligodendrocyte (OL) lineage specification and OL lineage maturation and myelination, including many genes not previously implicated in glial cell biology or linked to REST and CoREST regulation. Amongst these factors are those implicated in macroglial (AS and OL) cell identity, maturation, and maintenance, such as members of key developmental signaling pathways and combinatorial transcription factor codes.Conclusions/SignificanceOur results imply that REST and CoREST modulate not only neuronal but also glial lineage elaboration. These factors may therefore mediate critical developmental processes including the coupling of neurogenesis and gliogenesis and neuronal-glial interactions that underlie synaptic and neural network plasticity and homeostasis in health and in specific neurological disease states.

Nontelomeric TRF2-REST Interaction Modulates Neuronal Gene Silencing and Fate of Tumor and Stem Cells

Removal of TRF2, a telomere shelterin protein, recapitulates key aspects of telomere attrition including the DNA-damage response and cell-cycle arrest [1]. Distinct from the response of proliferating cells to loss of TRF2 [2, 3], in rodent noncycling cells, TRF2 inhibition promotes differentiation and growth [4, 5]. However, the mechanism that couples telomere gene-silencing features [6-8] to differentiation programs has yet to be elucidated. Here we describe an extratelomeric function of TRF2 in the regulation of neuronal genes mediated by the interaction of TRF2 with repressor element 1-silencing transcription factor (REST), a master repressor of gene networks devoted to neuronal functions [9-12]. TRF2-REST complexes are readily detected by coimmunoprecipitation assays and are localized to aggregated PML-nuclear bodies in undifferentiated pluripotent human NTera2 stem cells. Inhibition of TRF2, either by a dominant-negative mutant or by RNA interference, dissociates TRF2-REST complexes resulting in ubiquitin-proteasomal degradation of REST. Consequentially, REST-targeted neural genes (L1CAM, beta3-tubulin, synaptophysin, and others) are derepressed, resulting in acquisition of neuronal phenotypes. Notably, selective damage to telomeres without affecting TRF2 levels causes neither REST degradation nor cell differentiation. Thus, in addition to protecting telomeres, TRF2 possesses a novel role in stabilization of REST thereby controlling neural tumor and stem cell fate.

Control of chromosome stability by the β-TrCP–REST–Mad2 axis

REST/NRSF (repressor-element-1-silencing transcription factor/neuron-restrictive silencing factor) negatively regulates the transcription of genes containing RE1 sites. REST is expressed in non-neuronal cells and stem/progenitor neuronal cells, in which it inhibits the expression of neuron-specific genes. Overexpression of REST is frequently found in human medulloblastomas and neuroblastomas, in which it is thought to maintain the stem character of tumour cells. Neural stem cells forced to express REST and c-Myc fail to differentiate and give rise to tumours in the mouse cerebellum. Expression of a splice variant of REST that lacks the carboxy terminus has been associated with neuronal tumours and small-cell lung carcinomas, and a frameshift mutant (REST-FS), which is also truncated at the C terminus, has oncogenic properties. Here we show, by using an unbiased screen, that REST is an interactor of the F-box protein beta-TrCP. REST is degraded by means of the ubiquitin ligase SCF(beta-TrCP) during the G2 phase of the cell cycle to allow transcriptional derepression of Mad2, an essential component of the spindle assembly checkpoint. The expression in cultured cells of a stable REST mutant, which is unable to bind beta-TrCP, inhibited Mad2 expression and resulted in a phenotype analogous to that observed in Mad2(+/-) cells. In particular, we observed defects that were consistent with faulty activation of the spindle checkpoint, such as shortened mitosis, premature sister-chromatid separation, chromosome bridges and mis-segregation in anaphase, tetraploidy, and faster mitotic slippage in the presence of a spindle inhibitor. An indistinguishable phenotype was observed by expressing the oncogenic REST-FS mutant, which does not bind beta-TrCP. Thus, SCF(beta-TrCP)-dependent degradation of REST during G2 permits the optimal activation of the spindle checkpoint, and consequently it is required for the fidelity of mitosis. The high levels of REST or its truncated variants found in certain human tumours may contribute to cellular transformation by promoting genomic instability.