Neuronal Genes In Non-neuronal Cells Research Articles

Abstract 2152: EHMT1 activity to repress neuroendocrine differentiation of prostate cancer is mediated by dual lysine-methylation

Abstract Background: Lysine methylation is an important post-translational regulation on histones, but its molecular functions on non-histone proteins remain largely unknown. In a pilot study, we identified novel lysine methylations at non-histone proteins in prostate cancer (PCa) cells through an affinity pull-down assay. EHMT1/GLP, containing monomethylated K450 and K451, was one of these proteins, and it was known to repress transcription through methylating histone 3 lysine 9 (H3K9). EHMT1 and EHMT2/G9a are members of the REST (NRSF) repressor complex, that includes LSD1/CoREST/HDACs and represses neuronal genes in non-neuronal cells. Loss of function for this complex has been indicated as a mechanism to drive neuroendocrine (NE) transition of PCa. We hypothesize that the K450/451 methylations may inhibit the activity of EHMT1 and that LSD1 and other lysine demethylase(s) may sequentially demethylate these lysines and thus induce EHMT1 activity to silence the expression of NE related genes. Methods: LNCaP stable cell lines overexpressing doxycycline-regulated V5-tagged EHMT1-WT, K450R, K451R, and K450/451R were generated. We performed ChIP-seq analyses of V5 and H3K9me2, and RNA-seq analyses in all four cell lines treated with/out doxycycline. We also conducted in vitro demethylation assays to examine whether LSD1 directly demethylates EHMT1. Moreover, we performed co-immunoprecipitation assays to determine the effect of LSD1 inhibition on the interaction of LSD1 with EHMT1. Neuronal target gene expression was examined through ChIP and mRNA analysis. Results: Using mass-spectrometry analysis, we confirmed K450 and K451 methylations of EHMT1 in LNCaP PCa cell line. The demethylation assays indicated that methylated K450 but not K451 is a potential substrate of LSD1. Moreover, LSD1 interacts with K450/451R mutant but not EHMT1-WT. Importantly, only K450/451R mutant had a significant increase of chromatin binding in V5 and elevated levels of H3K9me2 compared to the weak chromatin binding seen in WT, K450R and K451R. Analyses in RNA-Seq revealed that the K450/K451R mutant repressed genes enriched for neuronal pathways, which was not seen in WT, K450R or K451R. Conclusions: Our studies suggest that both K450 and K451 of EHMT1 are methylated in PCa cells. K450-methylation of EHMT1 can be specifically demethylated by LSD1, but this alone is not sufficient to induce the chromatin recruitment of EHMT1. However, demethylation at both K450/K451 can dramatically increase the chromatin binding of EHMT1, which subsequently represses neuronal pathways. This mechanism may be particularly important for maintaining the cell lineage of adenocarcinoma of PCa and loss of this demethylation might lead to the emergence of NE-like PCa. Citation Format: Anna Besschetnova, Wanting Han, Mingyu Liu, Dong Han, Shuai Gao, Changmeng Cai. EHMT1 activity to repress neuroendocrine differentiation of prostate cancer is mediated by dual lysine-methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2152.

Low REST Expression Indicates a Biomarker of Poor Prognosis in Patients with Renal Cell Carcinoma.

It was initially found that neural-restrictive silencer factor/repressor 1-silencing transcription factor (REST) is a transcriptional repressor of neuronal genes in nonneuronal cells. However, it is reported to be abundantly expressed in various types of aggressive cancer cells. In this study, we evaluated the expression patterns of REST in renal cell carcinoma and found that its expression is lower in tumor tissues compared to normal tissues. The chi-square test showed that the low REST expression was closely related to patients' clinicopathologic parameters, including the pathologic stage and survival status. ROC curve showed that REST had excellent clinical diagnostic prospect. In addition, patients with low REST expression had poor over survival (OS) and relapse-free survival (RFS). Univariate and multivariate Cox regression analysis confirmed that the low REST expression was an independent predictor of poor prognosis in renal cell carcinoma. Gene set enrichment analysis identified P53 pathway, reactive oxygen species pathway, glycolysis, DNA repair, cholesterol homeostasis, and MYC targets V2 enriched with low REST expression phenotype. These results suggested that REST may be a novel biomarker for the diagnosis and prognosis of renal cell carcinoma in clinical applications.

Targeting the C-Terminal Domain Small Phosphatase 1.

The human C-terminal domain small phosphatase 1 (CTDSP1/SCP1) is a protein phosphatase with a conserved catalytic site of DXDXT/V. CTDSP1’s major activity has been identified as dephosphorylation of the 5th Ser residue of the tandem heptad repeat of the RNA polymerase II C-terminal domain (RNAP II CTD). It is also implicated in various pivotal biological activities, such as acting as a driving factor in repressor element 1 (RE-1)-silencing transcription factor (REST) complex, which silences the neuronal genes in non-neuronal cells, G1/S phase transition, and osteoblast differentiation. Recent findings have denoted that negative regulation of CTDSP1 results in suppression of cancer invasion in neuroglioma cells. Several researchers have focused on the development of regulating materials of CTDSP1, due to the significant roles it has in various biological activities. In this review, we focused on this emerging target and explored the biological significance, challenges, and opportunities in targeting CTDSP1 from a drug designing perspective.

Loss of RE-1 silencing transcription factor accelerates exocrine damage from pancreatic injury

Regulation of pancreas plasticity is critical for preventing injury and promoting regeneration upon tissue damage. The intricate process of pancreatic differentiation is governed by an orchestrated network of positive and negative transcription factors for appropriate gene expression. While the transcriptional repressor REST is well characterized as a silencer of neuronal genes in non-neuronal cells, the role of REST in regulating exocrine pancreas cell identity remains largely unexplored. Rest expression is increased upon injury in the mouse pancreas, such as induced acute and chronic pancreatitis and ductal adenocarcinoma. At the cellular level, Rest expression is lower in mature acinar cells compared with pancreas progenitor and ductal cells. To investigate the role of REST activity in pancreatic transdifferentiation and homeostasis, we developed a novel mouse model (Cre/RESTfl/fl) with conditional knockout (KO) of Rest expression within pancreas cells. The high Cre-mediated excision efficiency of Rest exon two KO caused decreased Rest expression and activity within the pancreas. Short-term organoid cultures of pancreatic acini to undergo acinar-to-ductal metaplasia (ADM) showed that loss of REST impedes induced ADM, while overexpression of REST increases ADM. Interestingly, REST ablation accelerated acute pancreatitis in mice treated with the cholecystokinin analog caerulein, as indicated by cellular morphology, elevated serum amylase levels and pancreatic edema. Furthermore, Cre/RESTfl/fl mice were more sensitive to acute pancreatitis injury and displayed augmented tissue damage and cellular lesions. These results suggest REST has a novel protective role against pancreatic tissue damage by acting as a regulator of exocrine cell identity.

Defects in the Alternative Splicing-Dependent Regulation of REST Cause Deafness

The DNA-binding protein REST forms complexes withhistone deacetylases (HDACs) to repress neuronal genes in non-neuronal cells. In differentiating neurons, REST is downregulated predominantly by transcriptional silencing. Here we report that post-transcriptional inactivation of REST by alternative splicing is required for hearing in humans and mice. We show that, in the mechanosensory hair cells of the mouse ear, regulated alternative splicing of a frameshift-causing exon into the Rest mRNA is essential for the derepression of many neuronal genes. Heterozygous deletion of this alternative exon of mouse Rest causes hair cell degeneration and deafness, and the HDAC inhibitor SAHA (Vorinostat) rescues the hearing of these mice. In humans, inhibition of the frameshifting splicing event by a novel REST variant is associated with dominantly inherited deafness. Our data reveal the necessity for alternative splicing-dependent regulation of REST inhair cells, and they identify a potential treatment for a group of hereditary deafness cases.

The RNA helicase DDX17 controls the transcriptional activity of REST and the expression of proneural microRNAs in neuronal differentiation.

The Repressor Element 1-silencing transcription factor (REST) represses a number of neuronal genes in non-neuronal cells or in undifferentiated neural progenitors. Here, we report that the DEAD box RNA helicase DDX17 controls important REST-related processes that are critical during the early phases of neuronal differentiation. First, DDX17 associates with REST, promotes its binding to the promoter of a subset of REST-targeted genes and co-regulates REST transcriptional repression activity. During neuronal differentiation, we observed a downregulation of DDX17 along with that of the REST complex that contributes to the activation of neuronal genes. Second, DDX17 and its paralog DDX5 regulate the expression of several proneural microRNAs that are known to target the REST complex during neurogenesis, including miR-26a/b that are also direct regulators of DDX17 expression. In this context, we propose a new mechanism by which RNA helicases can control the biogenesis of intronic miRNAs. We show that the processing of the miR-26a2 precursor is dependent on RNA helicases, owing to an intronic regulatory region that negatively impacts on both miRNA processing and splicing of its host intron. Our work places DDX17 in the heart of a pathway involving REST and miRNAs that allows neuronal gene repression.

NRSF/REST levels are decreased in cholangiocellular carcinoma but not hepatocellular carcinoma compared with normal liver tissues: A tissue microarray study.

The transcription factor neuron-restrictive silencer factor (NRSF), also termed repressor element 1-silencing transcription factor (REST), has been previously demonstrated to repress the expression of neuronal genes in non-neuronal cells, facilitating the controlled development and organization of nerve tissue. However, previous studies have reported NRSF/REST to be upregulated or downregulated in multiple types of carcinoma. Liver diseases are a major global health concern, with cirrhosis and liver carcinoma among the most common causes of mortality worldwide. A previous study demonstrated that there were >400 NRSF/REST target genes in mouse liver cells; however, the expression profile of NRSF/REST in human liver disease remains unclear. The present study examined NRSF/REST expression in human normal and liver carcinoma samples using tissue microarray immunohistochemistry. The results demonstrated that in normal liver tissues, NRSF/REST can be detected in the cytoplasm and nuclei of the cell; whereas in the liver carcinoma tissue, NRSF/REST is only detected in the cytoplasm. Furthermore, the number of samples with high levels of NRSF/REST was significantly lower in cholangiocellular carcinoma samples compared with normal tissues. Additionally, no detectable sex- or age-associated differences were identified in NRSF/REST expression among all the tissues examined. In conclusion, the results of the present study revealed nuclear loss of NRSF/REST in hepatic carcinomas and decreased expression of NRSF/REST in cholangiocellular carcinoma, indicating that the cytoplasmic translocation of NRSF/REST may be involved in liver tumorigenesis. A low expression level of NRSF/REST may be a novel biomarker for cholangiocellular carcinoma.

Suppression of Sin3A activity promotes differentiation of pluripotent cells into functional neurons

Sin3 is a transcriptional corepressor for REST silencing machinery that represses multiple neuronal genes in non-neuronal cells. However, functions of Sin3 (Sin3A and Sin3B) in suppression of neuronal phenotypes are not well characterized. Herein we show that Sin3A knockdown impedes the repressive activity of REST and enhances differentiation of pluripotent P19 cells into electrophysiologically active neurons without inducing astrogenesis. It is also found that silencing Sin3B induces neurogenesis of P19 cells with a lower efficiency than Sin3A knockdown. The results suggest that Sin3A has a more profound effect on REST repressive machinery for silencing neuronal genes in P19 cells than Sin3B. Furthermore, we show that a peptide inhibitor of Sin3A-REST interactions promotes differentiation of P19 cells into functional neurons. Observations made in studies using genetic deletion and a synthetic inhibitor suggests that Sin3A plays an important role in the repression of neuronal genes by the REST regulatory mechanism.

The developmental and pathogenic roles of BAF57, a special subunit of the BAF chromatin-remodeling complex.

Mammalian SWI/SNF or BAF chromatin-remodeling complexes are polymorphic assemblies of homologous subunit families that remodel nucleosomes. BAF57 is a subunit of the BAF complexes; it is encoded only in higher eukaryotes and is present in all mammalian assemblies. Its main structural feature is a high-mobility group domain, the DNA-binding properties of which suggest that BAF57 may play topological roles as the BAF complex enters or exits the nucleosome. BAF57 displays specific interactions with a number of proteins outside the BAF complex. Through these interactions, it can accomplish specific functions. In the embryo, BAF57 is responsible for the silencing of the CD4 gene during T-cell differentiation, and during the repression of neuronal genes in non-neuronal cells, BAF57 interacts with the transcriptional corepressor, Co-REST, and facilitates repression. Extensive work has demonstrated a specific role of BAF57 in regulating the interactions between BAF and nuclear hormone receptors. Despite its involvement in oncogenic pathways, new generation sequencing studies do not support a prominent role for BAF57 in the initiation of cancer. On the other hand, evidence has emerged to support a role for BAF57 as a metastasis factor, a prognosis marker and a therapeutic target. In humans, BAF57 is associated with disease, as mutations in this gene predispose to important congenital disorders, including menigioma disease or the Coffin-Siris syndrome. In this article, we present an exhaustive analysis of the BAF57 molecular and biochemical properties, cellular functions, loss-of-function phenotypes in living organisms and pathological manifestations in cases of human mutations.

G9a inhibition promotes neuronal differentiation of human bone marrow mesenchymal stem cells through the transcriptional induction of RE-1 containing neuronal specific genes

Recent studies have shown that epigenomic modifications are significantly associated with neuronal differentiation. Many neuronal specific genes contain the repressor element-1 (RE-1), which recruits epigenetic modulators, such as the histone methyltransferase G9a and interrupts the expression of neuronal genes in non-neuronal cells. This study investigated the functional role of G9a during neuronal differentiation of human bone marrow mesenchymal stem cells (BM-MSCs). Human BM-MSCs treated with the G9a inhibitor BIX01294 showed an increased expression of various neuronal-lineage genes. Using genomic sequence analysis, we identified RE-1 consensus sequences in the proximal region of several neuronal-specific genes. Chromatin immunoprecipitation (ChIP) assay results have showed that H3K9me2 (dimethylation of lysine 9 on histone 3) occupancy at RE-1-containing sequences from neuronal-specific genes was significantly decreased in BIX01294-MSCs. When BIX01294-MSCs were differentiated with neuronal induction medium, cells differentiated more effectively into neuron-like cells, complete with a cell body and dendrites. Expression of neuronal-specific genes containing the RE-1 sequences was significantly increased in differentiated BIX01294-MSCs, as confirmed by immunocytochemical staining and immunoblotting. Thus, this study shows that BIX01294 pretreated human BM-MSCs can be effectively differentiated into neuron-like cells by induced expression of neuronal-specific genes containing RE-1 sequences.

Brain REST/NRSF Is Not Only a Silent Repressor but Also an Active Protector.

During neurogenesis, specific transcription factors are needed to repress neuronal genes in nonneuronal cells to ensure precise development. Repressor element-1 binding transcription factor (REST), or neuron-restrictive silencer factor (NRSF), has been shown to be an important regulator for the establishment of neuronal specificity. It restricts the expression of neuronal genes by binding to the neuron-restrictive silencer element (NRSE/RE1) domain in neuron-specific genes. REST/NRSF regulates many target genes in stem cells, nonneural cells, and neurons, which are involved in neuronal differentiation, axonal growth, vesicular transport, and release as well as ionic conductance. However, it is also regulated by some cytokines/regulators such as epigenetic factors (microRNAs) and even its truncated isoform. REST/NRSF is widely detected in brain regions and has been shown to be highly expressed in nonneuronal cells, but current findings also reveal that, at least in the human brain, it is also highly expressed in neurons and increases with ageing. However, its loss in expression and cytoplasmic translocation seems to play a pivotal role in several human dementias. Additionally, REST/NRSF knockdown leads to malformations in nerve and nonneural tissues and embryonic lethality. Altered REST/NRSF expression has been not only related to deficient brain functions such as neurodegenerative diseases, mental disorders, brain tumors, and neurobehavioral disorders but also highly correlated to brain injuries such as alcoholism and stroke. Encouragingly, several compounds such as valproic acid and X5050 that target REST/NRSF have been shown to be clinically effective at rescuing seizures or Niemann-Pick type C disease. Surprisingly, studies have also shown that REST/NRSF can function as an activator to induce neuronal differentiation. These findings strongly indicate that REST/NRSF is not only a classical repressor to maintain normal neurogenesis, but it is also a fine fundamental protector against neurodegeneration and other disorders and may be a novel potent therapeutic target for neural disturbances.

NRSF is an essential mediator for the neuroprotection of trichostatin A in the MPTP mouse model of Parkinson's disease

Neuron-restrictive silencer factor (NRSF) blocks the expression of many neuronal genes in non-neuronal cells and neural stem cells. There is growing body of evidence that NRSF functions in mature neurons and plays critical roles in various neurological disorders. Our previous study demonstrated that the expression of NRSF target genes brain-derived neurotrophic factor (BDNF), and tyrosine hydroxylase (TH) is transiently decreased in 1-methyl-4-phenyl-pyridinium ion (MPP+)-treated SH-SY5Y cells. NRSF neuronal deficient mice are more vulnerable to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Here we investigated the effect of epigenetic modulation on the expression of NRSF target genes in in vitro and in vivo models of Parkinson's disease (PD). Trichostatin A (TSA) was further used to study the effects of histone deacetylase inhibition on NRSF-mediated repression. We found that the repression of NRSF target genes was relieved by TSA in vitro. A single dose TSA pretreatment also upregulated the expression of TH and BDNF and protected the nigrostriatal dopaminergic pathway against MPTP-induced degeneration in wild type mice. However, the protective functions of TSA were fully abolished in NRSF neuronal deficient mice. Our results suggest that NRSF serves as an essential mediator for the neuroprotection of TSA in the MPTP model of PD.

NRSF/REST regulates the mTOR signaling pathway in oral cancer cells.

The neuron-restrictive silencer factor/repressor element 1-silencing transcription factor (NRSF/REST) was originally discovered as a transcriptional repressor of neuronal genes in non-neuronal cells. However, it was recently reported to be abundantly expressed in several types of aggressive cancer cells, as well as in mature neurons. In the present study, the role of NRSF/REST in the human oral squamous cell carcinoma (SCC) KB cell line was evaluated. NRSF/REST was expressed at a higher level in KB cells when compared with that in normal human oral keratinocytes (NHOKs). Knockdown of NRSF/REST by siRNA reduced cell viability only in KB cells in a time-dependent manner, and this effect was due to the activation of apoptosis components and DNA fragmentation. In addition, knockdown of NRSF/REST disrupted the mTOR signaling pathway which is a key survival factor in many types of cancer cells. For example, the phosphorylation of elF4G, elF4E and 4E-BP1 was significantly reduced in the KΒ cells upon NRSF/REST knockdown. These results imply that NRSF/REST plays an important role in the survival of oral cancer cells by regulating the mTOR signaling pathway.

An RNA binding protein promotes axonal integrity in peripheral neurons by destabilizing REST.

The RE1 Silencing Transcription Factor (REST) acts as a governor of the mature neuronal phenotype by repressing a large consortium of neuronal genes in non-neuronal cells. In the developing nervous system, REST is present in progenitors and downregulated at terminal differentiation to promote acquisition of mature neuronal phenotypes. Paradoxically, REST is still detected in some regions of the adult nervous system, but how REST levels are regulated, and whether REST can still repress neuronal genes, is not known. Here, we report that homeostatic levels of REST are maintained in mature peripheral neurons by a constitutive post-transcriptional mechanism. Specifically, using a three-hybrid genetic screen, we identify the RNA binding protein, ZFP36L2, associated previously only with female fertility and hematopoiesis, and show that it regulates REST mRNA stability. Dorsal root ganglia in Zfp36l2 knock-out mice, or wild-type ganglia expressing ZFP36L2 shRNA, show higher steady-state levels of Rest mRNA and protein, and extend thin and disintegrating axons. This phenotype is due, at least in part, to abnormally elevated REST levels in the ganglia because the axonal phenotype is attenuated by acute knockdown of REST in Zfp36l2 KO DRG explants. The higher REST levels result in lower levels of target genes, indicating that REST can still fine-tune gene expression through repression. Thus, REST levels are titrated in mature peripheral neurons, in part through a ZFP36L2-mediated post-transcriptional mechanism, with consequences for axonal integrity.

The Role of the CoREST/REST Repressor Complex in Herpes Simplex Virus 1 Productive Infection and in Latency

REST is a key component of the HDAC1 or 2, CoREST, LSD1, REST (HCLR) repressor complex. The primary function of the HCLR complex is to silence neuronal genes in non-neuronal cells. HCLR plays a role in regulating the expression of viral genes in productive infections as a donor of LDS1 for expression of α genes and as a repressor of genes expressed later in infection. In sensory neurons the HCLR complex is involved in the silencing of viral genome in the course of establishment of latency. The thesis of this article is that (a) sensory neurons evolved a mechanism to respond to the presence and suppress the transmission of infectious agents from the periphery to the CNS and (b) HSV evolved subservience to the HCLR with at least two objectives: to maintain a level of replication consistent with maximal person-to-person spread and to enable it to take advantage of neuronal innate immune responses to survive and be available for reactivation shielded from adaptive immune responses of the host.

REST selectively represses a subset of RE1-containing neuronal genes in mouse embryonic stem cells

REST is a transcriptional repressor that targets a group of neuronal genes in non-neuronal cells. In embryonic stem (ES) cells, REST has been implicated in controlling the expression of transcription factor genes that are crucial for lineage determination and for maintaining ES cell potential. Here, we asked whether REST directly regulates neural-specifying genes in mouse ES cells using siRNA-mediated REST knockdown and ES cells that lack functional REST protein as a result of gene targeting. Loss of REST did not affect the expression of any of ten transcription factor genes known to promote neural commitment and did not affect the expression of several microRNAs, including miR-21, a putative REST target in ES cells. REST-deficient ES cells retained the ability to self-renew and to undergo appropriate differentiation towards mesoderm, endoderm and ectoderm lineages upon LIF withdrawal. Genome-wide expression profiling showed that genes that were deregulated in the absence of REST were preferentially expressed in the brain and highly enriched for the presence of canonical REST binding sites (RE1). Chromatin immunoprecipitation studies confirmed these genes as direct targets of REST in ES cells. Collectively, these data show that REST selectively silences a cohort of neuronal genes in ES cells.

MED19 and MED26 Are Synergistic Functional Targets of the RE1 Silencing Transcription Factor in Epigenetic Silencing of Neuronal Gene Expression

A key hub for the orchestration of epigenetic modifications necessary to restrict neuronal gene expression to the nervous system is the RE1 silencing transcription factor (REST; also known as neuron restrictive silencer factor, NRSF). REST suppresses the nonspecific and premature expression of neuronal genes in non-neuronal and neural progenitor cells, respectively, via recruitment of enzymatically diverse corepressors, including G9a histone methyltransferase (HMTase) that catalyzes di-methylation of histone 3-lysine 9 (H3K9me2). Recently, we identified the RNA polymerase II transcriptional Mediator to be an essential link between RE1-bound REST and G9a in epigenetic suppression of neuronal genes in non-neuronal cells. However, the means by which REST recruits Mediator to facilitate G9a-dependent extra-neuronal gene silencing remains to be elucidated. Here, we identify the MED19 and MED26 subunits in Mediator as direct physical and synergistic functional targets of REST. We show that although REST independently binds to both MED19 and MED26 in isolation, combined depletion of both subunits is required to disrupt the association of REST with Mediator. Furthermore, combined, but not individual, depletion of MED19/MED26 impairs REST-directed recruitment to RE1 elements of Mediator and G9a, leading to a reversal of G9a-dependent H3K9me2 and de-repression of REST-target gene expression. Together, these findings identify MED19/MED26 as a probable composite REST interface in Mediator and further clarify the mechanistic basis by which Mediator facilitates REST-imposed epigenetic restrictions on neuronal gene expression.

Profiling RE1/REST-mediated histone modifications in the human genome

BackgroundThe transcriptional repressor REST (RE1 silencing transcription factor, also called NRSF for neuron-restrictive silencing factor) binds to a conserved RE1 motif and represses many neuronal genes in non-neuronal cells. This transcriptional regulation is transacted by several nucleosome-modifying enzymes recruited by REST to RE1 sites, including histone deacetylases (for example, HDAC1/2), demethylases (for example, LSD1), and methyltransferases (for example, G9a).ResultsWe have investigated a panel of 38 histone modifications by ChIP-Seq analysis for REST-mediated changes. Our study reveals a systematic decline of histone acetylations modulated by the association of RE1 with REST (RE1/REST). By contrast, alteration of histone methylations is more heterogeneous, with some methylations increased (for example, H3K27me3, and H3K9me2/3) and others decreased (for example, H3K4me, and H3K9me1). Furthermore, the observation of such trends of histone modifications in upregulated genes demonstrates convincingly that these changes are not determined by gene expression but are RE1/REST dependent. The outcomes of REST binding to canonical and non-canonical RE1 sites were nearly identical. Our analyses have also provided the first direct evidence that REST induces context-specific nucleosome repositioning, and furthermore demonstrate that REST-mediated histone modifications correlate with the affinity of RE1 motifs and the abundance of RE1-bound REST molecules.ConclusionsOur findings indicate that the landscape of REST-mediated chromatin remodeling is dynamic and complex, with novel histone modifying enzymes and mechanisms yet to be elucidated. Our results should provide valuable insights for selecting the most informative histone marks for investigating the mechanisms and the consequences of REST modulated nucleosome remodeling in both neural and non-neural systems.

Mediator Links Epigenetic Silencing of Neuronal Gene Expression with X-Linked Mental Retardation

Mediator occupies a central role in RNA polymerase II transcription as a sensor, integrator, and processor of regulatory signals that converge on protein-coding gene promoters. Compared to its role in gene activation, little is known regarding the molecular mechanisms and biological implications of Mediator as a transducer of repressive signals. Here we describe a protein interaction network required for extraneuronal gene silencing comprising Mediator, G9a histone methyltransferase, and the RE1 silencing transcription factor (REST; also known as neuron restrictive silencer factor, NRSF). We show that the MED12 interface in Mediator links REST with G9a-dependent histone H3K9 dimethylation to suppress neuronal genes in nonneuronal cells. Notably, missense mutations in MED12 causing the X-linked mental retardation (XLMR) disorders FG syndrome and Lujan syndrome disrupt its REST corepressor function. These findings implicate Mediator in epigenetic restriction of neuronal gene expression to the nervous system and suggest a pathologic basis for MED12-associated XLMR involving impaired REST-dependent neuronal gene regulation.

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).