07-579 Sigma-AldrichAnti-REST Antibody

Direct lineage reprogramming induces dramatic shifts in cellular identity, employing poorly understood mechanisms. Recently, we demonstrated that expression of Neurog2 or Ascl1 in postnatal mouse astrocytes generates glutamatergic or GABAergic neurons. Here, we take advantage of this model to study dynamics of neuronal cell fate acquisition at the transcriptional level. We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes. Within this subset, only NeuroD4 could by itself induce neuronal reprogramming in both mouse and human astrocytes, while co-expression with Insm1 was required for glutamatergic maturation. Cultured astrocytes gradually became refractory to reprogramming, in part by the repressor REST preventing Neurog2 from binding to the NeuroD4 promoter. Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4. Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming.

We detected and characterized the binding sites of the representative Rest complex components Rest, Sin3A, and Lsd1. We compared their binding patterns in mouse embryonic stem (ES) cells and epiblast stem (EpiS) cells. We found few Rest sites unique to the EpiS cells. The ES-unique site features were distinct from those of the common sites, namely, the signal intensities were weaker, and the characteristic gene function categories differed. Our analyses showed that the Rest binding sites do not always overlap with the Sin3A and Lsd1 binding sites. The Sin3A binding pattern differed remarkably between the ES and EpiS cells and was accompanied by significant changes in acetylated-histone patterns in the surrounding regions. A series of transcriptome analyses in the same cell types unexpectedly showed that the putative target gene transcript levels were not dramatically different despite dynamic changes in the Rest complex binding patterns and chromatin statuses, which suggests that Rest is not the sole determinant of repression at its targets. Nevertheless, we identified putative Rest targets with explicitly enhanced transcription upon Rest knock-down in 143 and 60 common and ES-unique Rest target genes, respectively. Among such sites, several genes are involved in ES cell proliferation. In addition, we also found that long, intergenic non-coding RNAs were apparent Rest targets and shared similar features with the protein-coding target genes. Interestingly, such non-coding target genes showed less conservation through evolution than protein-coding targets. As a result of differences in the components and targets of the Rest complex, its functional roles may differ in ES and EpiS cells.

Diets high in sugar and saturated fat (Western diet) contribute to obesity and pathophysiology of metabolic syndrome. A common physiological response to obesity is hypertension, which induces cardiac remodeling and hypertrophy. Hypertrophy is regulated at the level of chromatin by repressor element 1-silencing transcription factor (REST), and pathological hypertrophy is associated with reexpression of a fetal cardiac gene program. Reactivation of fetal genes is commonly observed in hypertension-induced hypertrophy; however, this response is blunted in diabetic hearts, partially due to upregulation of the posttranslational modification O-linked-β-N-acetylglucosamine (O-GlcNAc) to proteins by O-GlcNAc transferase (OGT). OGT and O-GlcNAc are found in chromatin-modifying complexes, but it is unknown whether they play a role in Western diet-induced hypertrophic remodeling. Therefore, we investigated the interactions between O-GlcNAc, OGT, and the fetal gene-regulating transcription factor complex REST/mammalian switch-independent 3A/histone deacetylase (HDAC). Five-week-old male C57BL/6 mice were fed a Western (n = 12) or control diet (n = 12) for 2 wk to examine the early hypertrophic response. Western diet-fed mice exhibited fasting hyperglycemia and increased body weight (P less than 0.05). As expected for this short duration of feeding, cardiac hypertrophy was not yet evident. We found that REST is O-GlcNAcylated and physically interacts with OGT in mouse hearts. Western blot analysis showed that HDAC protein levels were not different between groups; however, relative to controls, Western diet hearts showed increased REST and decreased ANP and skeletal α-actin. Transcript levels of HDAC2 and cardiac α-actin were decreased in Western diet hearts. These data suggest that REST coordinates regulation of diet-induced hypertrophy at the level of chromatin.

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase β-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis.

Recent studies have shown that the transcriptional functions of REST are much broader than repressing neuronal genes in non-neuronal systems. Whether REST occupies similar chromatin regions in different cell types and how it interacts with other transcriptional regulators to execute its functions in a context-dependent manner has not been adequately investigated. We have applied ChIP-seq analysis to identify the REST cistrome in human CD4+ T cells and compared it with published data from 15 other cell types. We found that REST cistromes were distinct among cell types, with REST binding to several tumor suppressors specifically in cancer cells, whereas 7% of the REST peaks in non-neuronal cells were ubiquitously called and less than 25% were identified for ≥ 5 cell types. Nevertheless, using a quantitative metric directly comparing raw ChIP-seq signals, we found the majority (∼80%) was shared by ≥ 2 cell types. Integration with RNA-seq data showed that REST binding was generally correlated with low gene expression. Close examination revealed that multiple contexts were correlated with reduced expression of REST targets, e.g., the presence of a cognate RE1 motif and cellular specificity of REST binding. These contexts were shown to play a role in differential corepressor recruitment. Furthermore, transcriptional outcome was highly influenced by REST cofactors, e.g., SIN3 and EZH2 co-occupancy marked higher and lower expression of REST targets, respectively. Unexpectedly, the REST cistrome in differentiated neurons exhibited unique features not observed in non-neuronal cells, e.g., the lack of RE1 motifs and an association with active gene expression. Finally, our analysis demonstrated how REST could differentially regulate a transcription network constituted of miRNAs, REST complex and neuronal factors. Overall, our findings of contexts playing critical roles in REST occupancy and regulatory outcome provide insights into the molecular interactions underlying REST's diverse functions, and point to novel roles of REST in differentiated neurons.

The RASSF1A gene is one of the most frequently inactivated genes in over 30 different types of cancers (H. Donninger, M. D. Vos, and G. J. Clark, J. Cell Sci. 120:3163-3172, 2007, http://dx.doi.org/10.1242/jcs.010389). Despite the prevalence of RASSF1A silencing in human cancer, the mechanism by which RASSF1A functions as a tumor suppressor is not well understood. Characterization of the consequences of RASSF1A loss on epithelial cell proliferation revealed that RASSF1A expression suppresses both microRNA 21 (miR-21) expression and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. The mechanism of the former is through restraint of SCF(βTrCP)-dependent destruction of the repressor element 1 silencing transcription factor (REST) tumor suppressor and consequent inhibition of miR-21 promoter activation. The mechanism of the latter is through physical sequestration of MST2, which results in accumulation of inactivating S259 phosphorylation of RAF1. Whether or not inactivation of these RASSF1A regulatory relationships can unleash enhanced proliferative capacity is dependent upon the coupling of SCF(βTrCP) and miR-21 to suppression of SKP2 protein translation and stability. Airway epithelial cultures retain this coupling and therefore respond to RASSF1A inactivation by p27-dependent cell cycle arrest. In contrast, colonic crypt-derived epithelial cells have uncoupled SCF(βTrCP) from SKP2 and respond to RASSF1A inactivation by enhanced proliferation rates. These observations help account for context-specific molecular etiology of oncogenic transformation and suggest intervention strategies for recently developed SKP2 inhibitors.

Human neurons are functional over an entire lifetime, yet the mechanisms that preserve function and protect against neurodegeneration during ageing are unknown. Here we show that induction of the repressor element 1-silencing transcription factor (REST; also known as neuron-restrictive silencer factor, NRSF) is a universal feature of normal ageing in human cortical and hippocampal neurons. REST is lost, however, in mild cognitive impairment and Alzheimer's disease. Chromatin immunoprecipitation with deep sequencing and expression analysis show that REST represses genes that promote cell death and Alzheimer's disease pathology, and induces the expression of stress response genes. Moreover, REST potently protects neurons from oxidative stress and amyloid β-protein toxicity, and conditional deletion of REST in the mouse brain leads to age-related neurodegeneration. A functional orthologue of REST, Caenorhabditis elegans SPR-4, also protects against oxidative stress and amyloid β-protein toxicity. During normal ageing, REST is induced in part by cell non-autonomous Wnt signalling. However, in Alzheimer's disease, frontotemporal dementia and dementia with Lewy bodies, REST is lost from the nucleus and appears in autophagosomes together with pathological misfolded proteins. Finally, REST levels during ageing are closely correlated with cognitive preservation and longevity. Thus, the activation state of REST may distinguish neuroprotection from neurodegeneration in the ageing brain.

Transient global cerebral ischemia induces profound changes in the transcriptome of brain cells, which is partially associated with the induction or repression of genes that influence the ischemic response. However, the mechanisms responsible for the selective vulnerability of hippocampal neurons to global ischemia remain to be clarified. To identify molecular changes elicited by ischemic insults, we subjected hippocampal primary cultures to oxygen-glucose deprivation (OGD), an in vitro model for global ischemia that resulted in delayed neuronal death with an excitotoxic component. To investigate changes in the transcriptome of hippocampal neurons submitted to OGD, total RNA was extracted at early (7 h) and delayed (24 h) time points after OGD and used in a whole-genome RNA microarray. We observed that at 7 h after OGD there was a general repression of genes, whereas at 24 h there was a general induction of gene expression. Genes related with functions such as transcription and RNA biosynthesis were highly regulated at both periods of incubation after OGD, confirming that the response to ischemia is a dynamic and coordinated process. Our analysis showed that genes for synaptic proteins, such as those encoding for PICK1, GRIP1, TARPγ3, calsyntenin-2/3, SAPAP2 and SNAP-25, were down-regulated after OGD. Additionally, OGD decreased the mRNA and protein expression levels of the GluA1 AMPA receptor subunit as well as the GluN2A and GluN2B subunits of NMDA receptors, but increased the mRNA expression of the GluN3A subunit, thus altering the composition of ionotropic glutamate receptors in hippocampal neurons. Together, our results present the expression profile elicited by in vitro ischemia in hippocampal neurons, and indicate that OGD activates a transcriptional program leading to down-regulation in the expression of genes coding for synaptic proteins, suggesting that the synaptic proteome may change after ischemia.

Steroidogenic Factor-1 (SF-1) is a nuclear receptor that has a pivotal role in the development of adrenal glands and gonads and in the control of steroid hormone production, being also implicated in the pathogenesis of adrenocortical tumors. We have analyzed the mechanisms how SF-1 controls gene expression in adrenocortical cells and showed that it regulates different categories of genes according to its dosage. Significant correlations exist between the localization of SF-1-binding sites in chromatin under different dosage conditions and dosage-dependent regulation of gene expression. Our study revealed unexpected functional interactions between SF-1 and Neuron-Restrictive Silencer Factor/RE1-Silencing Transcription Factor (NRSF/REST), which was first characterized as a repressor of neuronal gene expression in non-neuronal tissues, in the regulation of gene expression in steroidogenic cells. When overexpressed, SF-1 reshapes the repertoire of NRSF/REST-regulated genes, relieving repression of key steroidogenic genes. These data show that NRSF/REST has a novel function in regulating gene expression in steroidogenic cells and suggest that it may have a broad role in regulating tissue-specific gene expression programs.

The development and function of the nervous system are directly dependent on a well defined pattern of gene expression. Indeed, perturbation of transcriptional activity or epigenetic modifications of chromatin can dramatically influence neuronal phenotypes. The phosphoprotein synapsin I (Syn I) plays a crucial role during axonogenesis and synaptogenesis as well as in synaptic transmission and plasticity of mature neurons. Abnormalities in SYN1 gene expression have been linked to important neuropsychiatric disorders, such as epilepsy and autism. SYN1 gene transcription is suppressed in non-neural tissues by the RE1-silencing transcription factor (REST); however, the molecular mechanisms that allow the constitutive expression of this genetic region in neurons have not been clarified yet. Herein we demonstrate that a conserved region of human and mouse SYN1 promoters contains cis-sites for the transcriptional activator Sp1 in close proximity to REST binding motifs. Through a series of functional assays, we demonstrate a physical interaction of Sp1 on the SYN1 promoter and show that REST directly inhibits Sp1-mediated transcription, resulting in SYN1 down-regulation. Upon differentiation of neuroblastoma Neuro2a cells, we observe a decrease in endogenous REST and a higher stability of Sp1 on target GC boxes, resulting in an increase of SYN1 transcription. Moreover, methylation of Sp1 cis-sites in the SYN1 promoter region could provide an additional level of transcriptional regulation. Our results introduce Sp1 as a fundamental activator of basal SYN1 gene expression, whose activity is modulated by the neural master regulator REST and CpG methylation.