Repressor Element-1 Silencing Research Articles

Functional Analysis of RE1 Silencing Transcription Factor as a Putative Tumor Suppressor in Human Endometrial Cancer.

Uterine cancer is the most common gynecologic malignancy in the United States, with endometrioid endometrial adenocarcinoma (EC) being the most common histologic sub-type. Considering the molecular classifications of EC, efforts have been made to identify additional biomarkers that can assist in diagnosis, prognosis, and individualized therapy. We sought to explore the relationship of Repressor Element 1 (RE1) silencing transcription factor (REST), which downregulates neuronal genes in non-neuronal tissue, along with matrix metalloproteinase-24 (MMP24) and EC. We analyzed the expression of REST and MMP24 in 31 cases of endometrial cancer and 16 controls. We then explored the baseline expression of REST and MMP24 in two EC cell lines (Ishikawa and HEC-1-A) compared to a benign cell line (t-HESC) and subsequently evaluated proliferation, migration, and invasion in the setting of loss of REST gene expression. REST and MMP24 expression were significantly lower in human EC samples compared to control samples. REST was highly expressed in EC cell lines, but decreasing REST gene expression increased proliferation (FC: 1.13X, p < 0.0001), migration (1.72X, p < 0.0001), and invasion (FC: 7.77X, p < 0.05) in Ishikawa cells, which are hallmarks of cancer progression and metastasis. These findings elicit a potential role for REST as a putative tumor suppressor in EC.

Establishment of Novel Protein Interaction Assays between Sin3 and REST Using Surface Plasmon Resonance and Time-Resolved Fluorescence Energy Transfer.

Repressor element-1 (RE-1) or neural restrictive silencer element (NRSE) bound with a zinc finger transcription repressor, RE-1 silencing transcription factor (REST, also known as neural restrictive silencer factor, NRSF) has been identified as a fundamental repressor element in many genes, including neuronal genes. Genes regulated by REST/NRSF regulate multifaceted neuronal phenotypes, and their defects in the machinery cause neuropathies, disorders of neuron activity), autism and so on. In REST repressions, the N-terminal repressor domain recruits Sin3B via its paired amphipathic helix 1 (PAH1) domain, which plays an important role as a scaffold for histone deacetylase 1 and 2. This machinery has a critical role in maintaining neuronal robustness. In this study, in order to establish protein–protein interaction assays mimicking a binding surface between Sin3B and REST, we selected important amino acids from structural information of the PAH1/REST complex and then tried to reconstitute it using recombinant short peptides derived from PAH1/REST. Initially, we validated whether biotinylated REST interacts with glutathione S-transferase (GST)-tagged PAH1 and whether another PAH1 peptide (PAH1-FLAG) competitively binds with biotinylated REST using surface plasmon resonance (SPR). We observed a direct interaction and competitive binding of two PAH1 peptides. Secondly, in order to establish a high-throughput and high-dynamic-range assay, we utilized an easily performed novel time-resolved fluorescence energy transfer (TR-FRET) assay, and closely monitored this interaction. Finally, we succeeded in establishing a novel high-quality TR-FRET assay and a novel interaction assay based on SPR.

Abstract B5: Identification of a role for REST in promoting preneoplastic events in a novel genetically engineered mouse model

Abstract Medulloblastoma is a pediatric cerebellar tumor that arises from undifferentiated neuronal stem/progenitor cells (NSCs). The Repressor Element-1 (RE1) Silencing Transcription Factor (REST) negatively regulates neurogenesis by repressing numerous genes involved in terminal neuronal differentiation. We previously showed that REST levels were abnormally elevated in medulloblastoma samples and that this was associated with poor prognosis for patients. Previous studies from two groups including ours showed that REST knockdown in human tumor cells blocked their tumorigenic potential in mouse orthotopic models, suggesting that REST was required for tumor maintenance. In addition, constitutively elevated expression of REST in neural progenitors in the context of Myc overexpression promoted tumorigenesis in the murine brain, implicating REST in tumor progression. However, a role for REST in tumor initiation has not been demonstrated. To examine if elevated REST expression in neuronal progenitors was sufficient to promote pre-neoplastic events such as proliferation, delayed differentiation and genetic instability, we generated a novel transgenic (Tg) mouse model in which human REST Tg expression could be conditionally elevated in the post-natal cerebellar progenitor cells. These studies revealed that cerebellar progenitors harvested from REST Tg mice, had sustained neurosphere formation and proliferation potential compared to progenitors from age-matched wild type littermates. Progenitors from REST Tg mice also exhibited substantially decreased expression of REST target neuronal differentiation genes such as Syn1 and SCG10 and displayed delayed onset of neurogenesis compared to wild type controls. Interestingly, progenitors with elevated REST expression had significantly perturbed telomere homeostasis associated with altered telomerase levels and increased genomic instability compared to wild type controls. The above findings were recapitulated in vivo where cerebella from REST Tg mice exhibited prolonged Ki-67 staining accompanied by lack of differentiation and accumulation of genetic instability. Furthermore, REST mediated genetic instability was associated with increased sensitivity to DNA damaging reagents such as etoposide compared to normal wild type progenitors. Thus, we provide the first demonstration of a role for elevated REST expression in neural progenitors in promoting tumor initiating events such as hyperproliferation, blockade of differentiation and telomere defects. We also show that the genetic instability contributes to the accelerated response of these cells to clinically relevant DNA damage-inducing agents. These observations may have therapeutic implications for high REST medulloblastomas. Citation Format: Tara Dobson, Ellen Busschers, Pete Taylor, Julianne Humphries, Sadhan Majumder, Vidya Gopalakrishnan. Identification of a role for REST in promoting preneoplastic events in a novel genetically engineered mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B5.

The role of the RE1 element in activation of the NR1 promoter during neuronal differentiation.

To understand the genetic mechanism controlling the expression of the NMDA subtype of glutamate receptors during neuronal differentiation, we studied activation of the N-methyl-D-aspartate receptor subunit 1 (NR1) gene and the role of the repressor element-1 (RE1) element in NR1 promoter activation. Following neuronal differentiation of P19 embryonic carcinoma cells, the NR1 transcription rate and mRNA level were significantly increased, while the nuclear level of the repressor RE1 silencing transcription factor (REST)/neuron-restriction silencer factor (NRSF) was reduced. Nuclear REST/NRSF from undifferentiated cells formed a large complex with the NR1 RE1 element. While this complex was significantly reduced after the differentiation, REST/NRSF from differentiated cells formed a new, faster migrating complex. In transient transfections, deletion of the RE1 element increased activity of the 5.4-kb NR1 promoter sixfold in undifferentiated cells, but only induced approximately 1.4-fold increase in differentiated cells. Forced expression of REST/NRSF in differentiated cells suppressed the promoter, while forced expression of a dominant-negative REST/NRSF induced promoter activity as well as the mRNA of the NR1 gene in undifferentiated cells. In stable transfectants, the wild-type promoter showed a robust increase in activity following differentiation in a pattern similar to the NR1 mRNA increase. Conversely, the promoter lacking the RE1 element showed only a moderate increase. Our data suggest that the NR1 gene up-regulation during neuronal differentiation is controlled by its promoter activation, which is largely determined by the interaction between the RE1 element and the repressor REST/NRSF.

Repressor Element-1 Silencing Transcription/Neuron-Restrictive Silencer Factor Is Required for Neural Sodium Channel Expression during Development ofXenopus

The ability of neurons to fire rapid action potential relies on the expression of voltage-gated sodium channels; the onset of the transcription of genes that encode these channels occurs during early neuronal development. The factors that direct and regulate the specific expression of ion channels are not well understood. Repressor element-1 silencing transcription/neuron-restrictive silencer factor (REST/NRSF) is a transcriptional regulator characterized as a repressor of the expression of NaV1.2, the gene encoding the voltage-gated sodium channel most abundantly expressed in the CNS, as well as of the expression of numerous other neuronal genes. In mammals, REST/NRSF is expressed mostly in non-neural cell types and immature neurons, and it is downregulated on neural maturation. To understand the mechanisms that govern sodium channel gene transcription and to explore the role of REST/NRSF in vivo, we inhibited REST/NRSF action in developing Xenopus laevis embryos by means of a dominant negative protein or antisense oligonucleotides. Contrary to what was expected, these maneuvers result in the decrease of the expression of the NaV1.2 gene, as well as of other neuronal genes in the primary spinal neurons and cranial ganglia, without overt perturbation of neurogenesis. These results, together with the demonstration of robust REST/NRSF expression in primary spinal neurons, suggest that REST/NRSF is required for the acquisition of the differentiated functional neuronal phenotype during early development. Furthermore, they suggest that REST/NRSF may be used to activate or repress transcription of neuronal genes in distinct cellular and developmental contexts.