Repressor Element-1 Silencing Transcription Research Articles

Lithium beyond psychiatric indications: the reincarnation of a new old drug.

Lithium has been used in the treatment of bipolar disorders for decades, but the exact mechanisms of action remain elusive to this day. Recent evidence suggests that lithium is critically involved in a variety of signaling pathways affecting apoptosis, inflammation, and neurogenesis, all of which contributing to the complex pathophysiology of various neurological diseases. As a matter of fact, preclinical work reports both acute and long-term neuroprotection in distinct neurological disease models such as Parkinson’s disease, traumatic brain injury, Alzheimer’s disease, and ischemic stroke. Lithium treatment reduces cell injury, decreases α-synuclein aggregation and Tau protein phosphorylation, modulates inflammation and even stimulates neuroregeneration under experimental conditions of Parkinson’s disease, traumatic brain injury, and Alzheimer’s disease. The therapeutic impact of lithium under conditions of ischemic stroke was also studied in numerous preclinical in vitro and in vivo studies, giving rise to a randomized double-blind clinical stroke trial. The preclinic data revealed a lithium-induced upregulation of anti-apoptotic proteins such as B-cell lymphoma 2, heat shock protein 70, and activated protein 1, resulting in decreased neuronal cell loss. Lithium, however, does not only yield postischemic neuroprotection but also enhances endogenous neuroregeneration by stimulating neural stem cell proliferation and by regulating distinct signaling pathways such as the RE1-silencing transcription factor. In line with this, lithium treatment has been shown to modulate postischemic cytokine secretion patterns, diminishing microglial activation and stabilizing blood-brain barrier integrity yielding reduced levels of neuroinflammation. The aforementioned observations culminated in a first clinical trial, which revealed an improved motor recovery in patients with cortical stroke after lithium treatment. Beside its well-known psychiatric indications, lithium is thus a promising neuroprotective candidate for the aforementioned neurological diseases. A detailed understanding of the lithium-induced mechanisms, however, is important for prospective clinical trials which may pave the way for a successful bench-to-bedside translation in the future. In this review, we will give an overview of lithium-induced neuroprotective mechanisms under various pathological conditions, with special emphasis on ischemic stroke.

MBRS-53. CONTROL OF MEDULLOBLASTOMA VASCULATURE BY A REGULATOR OF NEUROGENESIS

Medulloblastomas are characterized by poor neuronal lineage specification. Expression of the RE1 Silencing Transcription Factor (REST), a regulator of neurogenesis, is aberrantly elevated in human sonic hedgehog (SHH) medulloblastomas. Using a novel transgenic mouse (RESTTG) model, we demonstrated that REST is a driver of medulloblastoma genesis and promotes tumor progression in mice with loss of an allele of Ptch1 (Ptch+/−). Tumor formation in Ptch+/−/RESTTG mice occurred with 100% penetrance and a latency of 10–90 days in contrast to Ptch+/− mice, which developed tumors at a frequency of 15–20% at 6–9 months of age. Histopathological analyses showed leptomeningeal dissemination of tumors in Ptch+/−/RESTTG mice, in addition to a significant increase in tumor vasculature compared to tumors in Ptch+/− mice. These findings were recapitulated in xenografted tumors of isogenic low and high-REST medulloblastomas in mice. Proteome profiler human angiogenesis array analyses revealed a REST-dependent increase in vascular endothelial growth factor (VEGF) and placental growth factor (PLGF). Surprisingly, REST elevation also caused co-localization of tumor cells with tumor vasculature, specifically endothelial cells, and was associated with upregulated expression of a number of pro-angiogenic genes, including receptor VEGFR1 and the positive regulator of endothelial differentiation, E26 transformation specific-1 (ETS1), in tumor cells. In addition, expression of several anti-angiogenic molecules was downregulated. Knockdown of ETS1 reversed the above findings. Thus, our data demonstrate that REST elevation not only blocks neurogenesis in medulloblastoma cells, but also modulates the tumor microenvironment by mechanisms that likely involve vascular mimicry.

MBRS-64. STUDY OF ARGININE METHYL TRANSFERASES IN MEDULLOBLASTOMA

Medulloblastoma is the most common malignant pediatric cancer and a leading cause of childhood cancer-related mortality. There is dire need for new therapies. Molecular sub-classification of medulloblastomas has identified chromatin modifiers as potential drivers of tumorigenesis. Expression of the RE1 Silencing Transcription Factor (REST), a hub for assembly of repressive chromatin remodelers and a known regulator of neurogenesis, is elevated in a subset of human sonic hedgehog (SHH) subgroup medulloblastomas, and is associated with poor prognosis. Using a novel transgenic mouse model, we showed REST to be a driver of medulloblastoma development. Surprisingly, our studies also revealed a role for REST in promoting proliferation of granule cell progenitors (GCPs), the cells of origin of SHH-driven medulloblastoma, and a concomitant loss of telomeres and increased genomic instability. We performed a gain of function screen using a library of chromatin modifiers to understand the mechanism by which proliferative potential was maintained, despite the loss of telomeres. This screen identified the Protein Arginine Methyltransferase 6 (PRMT6) as a high confidence hit. PRMT6 upregulation caused a reduction in CDKN2A, an important regulator of replicative senescence. Evasion of senescence is frequently implicated in tumor progression. Using a chemical screen, we also identified a novel, selective, reversible and competitive inhibitor of PRMT6. The consequence of genetic and pharmacological inhibition of PRMT6 on cell proliferation and senescence will be reported. Thus, our studies are the first to demonstrate a role for arginine methyl transferase family of chromatin modifiers in medulloblastoma genesis.

MBRS-70. FUNCTIONAL DEPENDENCY BETWEEN REST AND DNMT1 IN MEDULLOBLASTOMA

Medulloblastomas exhibit poor neuronal lineage specification. Expression of RE1 Silencing Transcription Factor (REST), a repressor of neurogenesis, is aberrantly elevated in human sonic hedgehog (SHH) medulloblastomas. Constitutive REST expression in mice (RESTTG) drives medulloblastoma genesis and promotes tumor progression in the context of Ptch1 haploinsufficiency (Ptch+/−), implicating it as a driver of tumorigenesis. Tumor formation in Ptch+/−/RESTTG mice showed significantly decreased latency and increased penetrance compared to that in Ptch+/− mice. Since REST silences gene expression by chromatin remodeling, we sought to identify cooperating epigenetic events that contributed to its oncogenic activity. To this end, we performed a loss of function screen employing a bar-coded library of short hairpin RNAs against epigenes, to identify candidates whose loss could create a proliferative vulnerability in the context of REST-elevation. This screen identified DNA methyltransferase 1 (DNMT1) as a high-priority epigenetic modifier. DNMT1 and the Ubiquitin like with PHD and Ring finger domain 1 (UHRF1) proteins are essential for methylation of hemi-methylated DNA at the replication fork during S-phase. Their expression is downregulated during neuronal differentiation. In human SHH-medulloblastoma tumors, REST and UHRF1 expression are positively correlated with higher levels of both genes noted specifically in the SHH-beta subtype, and is associated with poor prognosis. The requirement for DNMT1/UHRF1 in the context of REST elevation, was established by RNA-Seq and Reduced Representation Bisulfite Sequencing (RRBS), which revealed hypermethylation and downregulated expression of REST-target genes needed for neurogenesis. Thus, DNMT1/UHRF1 is a functional and potential therapeutic vulnerability in REST-elevated SHH medulloblastomas.

MIR448 antagomir reduces arrhythmic risk after myocardial infarction by upregulating the cardiac sodium channel.

Cardiac ischemia is associated with arrhythmias; however, effective therapies are currently limited. The cardiac voltage-gated sodium channel α subunit (SCN5A), encoding the Nav1.5 current, plays a key role in the cardiac electrical conduction and arrhythmic risk. Here, we show that hypoxia reduces Nav1.5 through effects on a miR, miR-448. miR-448 expression is increased in ischemic cardiomyopathy. miR-448 has a conserved binding site in 3′-UTR of SCN5A. miR-448 binding to this site suppressed SCN5A expression and sodium currents. Hypoxia-induced HIF-1α and NF-κB were major transcriptional regulators for MIR448. Moreover, hypoxia relieved MIR448 transcriptional suppression by RE1 silencing transcription factor. Therefore, miR-448 inhibition reduced arrhythmic risk after myocardial infarction. Here, we show that ischemia drove miR-448 expression, reduced Nav1.5 current, and increased arrhythmic risk. Arrhythmic risk was improved by preventing Nav1.5 downregulation, suggesting a new approach to antiarrhythmic therapy.

Spatiotemporal immunolocalisation of REST in the brain of healthy ageing and Alzheimer's disease rats.

In the brain, REST (Repressor Element-1 Silencing Transcription factor) is a key regulator of neuron cell-specific gene expression. Nuclear translocation of neuronal REST has been shown to be neuroprotective in a healthy ageing context. In contrast, inability to upregulate nuclear REST is thought to leave ageing neurons vulnerable to neurodegenerative stimuli, such as Alzheimer's disease (AD) pathology. Hippocampal and cortical neurons are known to be particularly susceptible to AD-associated neurodegeneration. However, REST expression has not been extensively characterised in the healthy ageing brain. Here, we examined the spatiotemporal immunolocalisation of REST in the brains of healthy ageing wild-type Fischer-344 and transgenic Alzheimer's disease rats (TgF344-AD). Nuclear expression of REST increased from 6months to 18months of age in the hippocampus, frontal cortex and subiculum of wild-type rats, but not in TgF344-AD rats. No changes in REST were measured in more posterior cortical regions or in the thalamus. Interestingly, levels of the presynaptic marker synaptophysin, a known gene target of REST, were lower in CA1 hippocampal neurons of 18-month TgF344-AD rats compared to 18-month wild-types, suggesting that elevated nuclear REST may protect against synapse loss in the CA1 of 18-month wild-type rats. High REST expression in ageing wild-type rats did not, however, protect against axonal loss nor against astroglial reactivity in the hippocampus. Taken together, our data confirm that changes in nuclear REST expression are context-, age- and brain region-specific. Moreover, key brain structures involved in learning and memory display elevated REST expression in healthy ageing wild-type rats but not TgF344-AD rats.

Repressor Element 1 Silencing Transcription Factor (REST) Governs Microglia-Like BV2 Cell Migration via Progranulin (PGRN)

Microglia activation contributes to Alzheimer's disease (AD) etiology, and microglia migration is a fundamental function during microglia activation. The repressor element-1 silencing transcription factor (REST), a powerful transcriptional factor, was found to play a neuroprotective role in AD. Despite its possible role in disease progression, little is known about whether REST participates in microglia migration. In this study, we aimed to explore the function of REST and its molecular basis during microglia migration under Aβ1-42-treated pathological conditions. When treated by Aβ1-42 REST was upregulated through JAK2/STAT3 signal pathway in BV2 cells. And transwell coculture system was used to evaluate cell migration function of microglia-like BV2. Small interfering RNA (siRNA) targeting progranulin (PGRN) were delivered into BV2 cells, and results showed that PGRN functions to promote BV2 migration. REST expression was inhibited by sh-RNA, which induced BV2 cell migration obviously. On the contrary, REST was overexpressed by REST recombinant plasmid transfection, which repressed BV2 cell migration, indicating that REST may act as a repressor of cell migration. To more comprehensively examine the molecular basis, we analyzed the promoter sequence of PGRN and found that it has the potential binding site of REST. Moreover, knocking-down of REST can increase the expression of PGRN, which confirms the inhibiting effect of REST on PGRN expression. Further detection of double luciferase reporter gene also confirmed the inhibition of REST on the activity of PGRN promoter, indicating that REST may be an inhibitory transcription factor of PGRN which governs microglia-like BV2 cell migration. In conclusion, the present study demonstrates that transcription factor REST may act as a repressor of microglia migration through PGRN.

Regulating the Regulators: The Role of Histone Deacetylase 1 (HDAC1) in Erythropoiesis.

Histone deacetylases (HDACs) play important roles in transcriptional regulation in eukaryotic cells. Class I deacetylase HDAC1/2 often associates with repressor complexes, such as Sin3 (Switch Independent 3), NuRD (Nucleosome remodeling and deacetylase) and CoREST (Corepressor of RE1 silencing transcription factor) complexes. It has been shown that HDAC1 interacts with and modulates all essential transcription factors for erythropoiesis. During erythropoiesis, histone deacetylase activity is dramatically reduced. Consistently, inhibition of HDAC activity promotes erythroid differentiation. The reduction of HDAC activity not only results in the activation of transcription activators such as GATA-1 (GATA-binding factor 1), TAL1 (TAL BHLH Transcription Factor 1) and KLF1 (Krüpple-like factor 1), but also represses transcription repressors such as PU.1 (Putative oncogene Spi-1). The reduction of histone deacetylase activity is mainly through HDAC1 acetylation that attenuates HDAC1 activity and trans-repress HDAC2 activity through dimerization with HDAC1. Therefore, the acetylation of HDAC1 can convert the corepressor complex to an activator complex for gene activation. HDAC1 also can deacetylate non-histone proteins that play a role on erythropoiesis, therefore adds another layer of gene regulation through HDAC1. Clinically, it has been shown HDACi can reactivate fetal globin in adult erythroid cells. This review will cover the up to date research on the role of HDAC1 in modulating key transcription factors for erythropoiesis and its clinical relevance.

Environmental enrichment enhances sociability by regulating glutamate signaling pathway through GR by epigenetic mechanisms in amygdala of Indian field mice Mus booduga

Environmental enrichment (EE) dynamically regulates gene expression and synaptic plasticity with positive consequences on behavior. The present study was performed on field-mice to explore the effects of EE on both captive-condition inducing social stress and epigenetic changes of molecules resilience stress. For this purpose, field-mice were caught and allowed to habituate in standard laboratory conditions for 7 days. The next day animals were randomly assigned to three groups: i) mice at short-term standard condition (STSC); which were subjected to social interaction test (SIT) on day 9, ii) mice continuously maintainedfor additional 30 days, with these long-term standard conditions (LTSC), and iii) mice maintained in an EE cage for additional 30 days. After achieving SIT, we examined epigenetic changes of a repertory of molecules associated with resilience stress, by determining their levels by Western blot. Thus, the main findings were that during SIT, EE exerted more social interaction of field-mice with the strangers compared with STSC and LTSC mice. Related with social behavior results, we found that in mice subjected to EE the levels of histone 3 lysine 9 di-methylation (H3K9me2), glucocorticoid receptor (GR), N-methyl-D asparate (NMDA) receptor subunits NR2A and NR2B, postsynaptic density protein-95 (PSD-95), and mature brain-derived neurotrophic factor (mBDNF) were significantly elevated; whereas the levels of DNA methyltransferase-3A (DNMT3A), methyl-CpG-binding protein-2 (MeCP2), repressor element-1 silencing transcription factor (REST), H3K4me2 and lysine demethylase-1A (KDM1A) decreased. These results suggest that enhanced sociability of EE mice could be mediated, in part, by altered expression of molecules regulating glutamate signaling pathway through GR by epigenetic mechanisms.

REST-repressed lncRNA NPPA-AS1 regulates cervical cancer progression by modulating miR-302e/DKK1/Wnt/β-catenin signaling pathway.

Long noncoding RNAs (lncRNAs), a group of transcripts, have been revealed to be critical participants in regulating multiple biological processes of malignant tumors. The knowledge of NPPA-AS1 (a new lncRNA) in cancer research is hardly known. Thus, it is of urgent need to study the underlying role of NPPA antisense RNA 1 (NPPA-AS1) in cervical cancer (CC). In this study, NPPA-AS1 was discovered to be lowly expressed and upregulation of it impaired cell proliferation and migration in CC. Besides, downregulation of it led to opposite results. Molecular mechanism assays uncovered that increased expression of NPPA-AS1 could inactivate Wnt/β-catenin signaling pathway in CC. In addition, NPPA-AS1 was found to negatively interact with miR-302e whereas positively correlate with dickkopf-1 (DKK1, an inhibitor of Wnt pathway) in CC. Besides, loss of function assay illuminated that miR-302e inhibition restrained cell proliferation and migration in CC. Subsequent rescue assays confirmed that NPPA-AS1 acted as a competing endogenous RNA in CC by sponging miR-302e to upregulate DKK1 expression. Finally, the RE-1 silencing transcription factor (REST) was testified to function as a transcription suppressor of NPPA-AS1 in CC. In brief, REST-repressed NPPA-AS1 regulates CC progression by modulating miR-302e/DKK1/Wnt/β-catenin signaling pathway.

Aβ-Induced Repressor Element 1-Silencing Transcription Factor (REST) Gene Delivery Suppresses Activation of Microglia-Like BV-2 Cells.

Compelling evidence from basic molecular biology has demonstrated the crucial role of microglia in the pathogenesis of Alzheimer's disease (AD). Microglia were believed to play a dual role in both promoting and inhibiting Alzheimer's disease progression. It is of great significance to regulate the function of microglia and make them develop in a favorable way. In the present study, we investigated the function of repressor element 1-silencing transcription factor (REST) in Aβ1-42-induced BV-2 cell dysfunction. We concluded that Aβ1-42 could promote type I activation of BV-2 cells and induce cell proliferation, migration, and proinflammation cytokine TNF-α, IL-1β, and IL-6 expression. Meanwhile, REST was upregulated, and nuclear translocalization took place due to Aβ1-42 stimulation. When REST was knocked down by a specific short hairpin RNA (sh-RNA), BV-2 cell proliferation, migration, and proinflammation cytokine expression and secretion induced by Aβ1-42 were increased, demonstrating that REST may act as a repressor of microglia-like BV-2 cell activation.

P-Cresol Sulfate Caused Behavior Disorders and Neurodegeneration in Mice with Unilateral Nephrectomy Involving Oxidative Stress and Neuroinflammation.

Protein-bound uremic toxins, such as p-cresol sulfate (PCS), can be accumulated with declined renal function and aging and is closely linked with central nervous system (CNS) diseases. In the periphery, PCS has effects on oxidative stress and inflammation. Since oxidative stress and inflammation have substantial roles in the pathogenesis of neurological disorders, the CNS effects of PCS were investigated in unilateral nephrectomized C57/BL/6 mice. Unlike intact mice, unilateral nephrectomized mice showed increased circulating levels of PCS after exogenous administration. Upon PCS exposure, the unilateral nephrectomized mice developed depression-like, anxiety-like, and cognitive impairment behaviors with brain PCS accumulation in comparison with the nephrectomy-only group. In the prefrontal cortical tissues, neuronal cell survival and neurogenesis were impaired along with increased apoptosis, oxidative stress, and neuroinflammation. Circulating brain-derived neurotrophic factors (BDNF) and serotonin were decreased in association with increased corticosterone and repressor element-1 silencing transcription factor (REST), regulators involved in neurological disorders. On the contrary, these PCS-induced changes were alleviated by uremic toxin absorbent AST-120. Taken together, PCS administration in mice with nephrectomy contributed to neurological disorders with increased oxidative stress and neuroinflammation, which were alleviated by PCS chelation. It is suggested that PCS may be a therapeutic target for chronic kidney disease-associated CNS diseases.

Mutual regulation between β-TRCP mediated REST protein degradation and Kv1.3 expression controls vascular smooth muscle cell phenotype switch

Background and aimsPhenotypic switch of vascular smooth muscle cells (VSMC) plays a key role in the pathogenesis of atherosclerosis and restenosis after artery intervention. Transcription repressor element 1-silencing transcription factor (REST) has been identified as key regulator of VSMC proliferation. In the present study, we sought to investigate the potential association of E3-ubiquitin ligase β-TRCP mediated REST protein degradation with Kv1.3 expression during VSMC phenotypic switch. MethodsProtein and mRNA expression was measured in ex vivo and in vitro models. Protein interaction and ubiquitination were analyzed by immunoprecipitation assays. ChIP assays were performed to assess the relationship between REST and targeted DNA binding site. ResultsWe found that the expression level of E3-ubiquitin ligase β-TRCP is significantly increased during VSMC phenotypic switch. REST protein ubiquitination mediated by β-TRCP is critical for VSMC proliferation and migration. We also found that the gene KCNA3 encoding potassium channel protein Kv1.3 contains a functional REST binding site and is repressed by REST. Downregulation of REST by β-TRCP and consequently upregulation of Kv1.3 are important events during VSMC phenotypic switch. Furthermore, upregulated Kv1.3 accelerates β-TRCP modulated REST degradation through Erk1/2 signaling. ConclusionsOur results reveal a fundamental role for regulatory interactions between β-TRCP modulated REST degradation and Kv1.3 in the control of the multilayered regulatory programs required for VSMC phenotype switch.

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.

Significant decrease of a master regulator of genes (REST/NRSF) in high-grade squamous intraepithelial lesion and cervical cancer

BackgroundThe repressor element 1-silencing transcription factor (REST) is a regulator of gene expression, and the Ras association domain family member 1 A (RASSF1A) is an important tumor suppressor gene involved in cancer development. Although extensive characterization of the roles of REST and RASSF1A in cancer development have been reported in cellular models, the link between them and their possible role in the development of squamous intraepithelial lesions (SIL) and squamous cell carcinoma (SCC) of the cervix have not been explored. The aim of this study was to evaluate the expression of REST and RASSF1A in cervical cytological and histological samples from patients diagnosed with SIL or SCC and in CC-derived cell lines. MethodsWe analyzed the expression of REST and RASSF1A by immunocyto/histochemistry in cervical samples from patients (n = 271) and in cancer cell lines. Data analyses were performed using the Kruskal–Wallis test and generalized linear models. ResultsWe identified binding sites for REST in RASSF1A and observed a significant reduction in REST and RASSF1A nuclear expression in samples from patients with high-grade SIL (HSIL) and SCC. For REST, we observed an average decrease of 334 and 423 r.u.d. for HSIL (n = 21) and SCC (n = 18) compared with non-LSIL (n = 72), whereas for RASSF1A, this decrease was 126 and 217 r.u.d., respectively (p < 0.001). ConclusionOur results provide evidence of the altered expression of REST and RASSF1A in SIL and SCC, with a significant decrease in HSIL, SCC, and SCC-derived cell lines; findings that can contribute to the diagnosis, prognosis, and post-treatment follow-up of patients diagnosed with SIL or SCC.

Abstract 1295: Myc family members differentially regulate lineage plasticity in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer with a dismal prognosis. The standard-of-care remains to be uniform treatment with chemotherapy and radiotherapy, while emerging evidence suggests its molecular heterogeneity that has been previously under-appreciated. In primary SCLC, the gene loci for Myc family members are amplified mutually exclusively, their expression is correlated with unique neuroendocrine markers and distinct histopathology of xenografts from SCLC cell lines and murine SCLC. In this study, we use integrative genomic and epigenomic analyses to explore a novel role for c-Myc and L-Myc as lineage determining factors to bridge the gap between SCLC molecular subtypes and histological classification. First, we built a novel network using the Bayesian estimation from combined mRNA expression profile datasets for a total of 135 primary SCLC tumors. This revealed distinct transcriptional networks for c-Myc and L-Myc; wherein L-Myc was enriched for neuronal pathways and c-Myc for Notch signaling and epithelial-to-mesenchymal transition. The assay for transposase accessible-chromatin profiling of 3 representative cell lines for each c-Myc and L-Myc, revealed enrichment of biological processes involved in neuronal development for L-Myc expressing cell lines and active Notch signaling in c-Myc expressing cell lines. Together, these analyses implied that c-Myc and L-Myc govern distinct transcriptional programs to impart respective transcriptional networks associated with features unique to SCLC molecular subtypes. Next, we genetically engineered c-Myc amplified SCLC to exchange c-Myc with L-Myc and found L-Myc regulates neuronal associated pathways but was insufficient to induce lineage switch, however; c-Myc was required for the maintenance of NeuroD1-driven lineage state. In contrast, exogenous expression of c-Myc in classical-ASCL1-positive SCLC revealed incompatibility of c-Myc expression in this subtype, and led to suppression of neuronal associated pathways, trans-differentiation to NeuroD1-SCLC accompanied by variant histopathological features. Pharmacological inhibition of neuroendocrine-low associated Notch signaling and its target RE-1 silencing transcription factor (REST), revealed c-Myc-induced suppression of ASCL1 is not mediated by Notch signaling but rather by direct activation of REST expression. Collectively, our findings reveal a previously undescribed role for historically defined general oncogenes, c-Myc and L-Myc, in regulating lineage plasticity across SCLC molecular subtypes as well as histological classes. Citation Format: Ayushi S. Patel, Seungyeul Yoo, Ranran Kong, Takashi Sato, Maya Fridrikh, German Nudelman, Charles A. Powell, Jun Zhu, Hideo Watanabe. Myc family members differentially regulate lineage plasticity in small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1295.

The microRNA/TET3/REST axis is required for olfactory globose basal cell proliferation and male behavior.

In the main olfactory epithelium (MOE), new olfactory sensory neurons (OSNs) are persistently generated to replace lost neurons throughout an organism's lifespan. This process predominantly depends on the proliferation of globose basal cells (GBCs), the actively dividing stem cells in the MOE. Here, by using CRISPR/Cas9 and RNAi coupled with adeno-associated virus (AAV) nose delivery approaches, we demonstrated that knockdown of miR-200b/a in the MOE resulted in supernumerary Mash1-marked GBCs and decreased numbers of differentiated OSNs, accompanied by abrogation of male behaviors. We further showed that in the MOE, miR-200b/a targets the ten-eleven translocation methylcytosine dioxygenase TET3, which cooperates with RE1-silencing transcription factor (REST) to exert their functions. Deficiencies including proliferation, differentiation, and behaviors illustrated in miR-200b/a knockdown mice were rescued by suppressing either TET3 or REST. Our work describes a mechanism of coordination of GBC proliferation and differentiation in the MOE and olfactory male behaviors through miR-200/TET3/REST signaling.

Transcriptional Dysregulation in Huntington's Disease: The Role in Pathogenesis and Potency for Pharmacological Targeting.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a mutation in the gene that encodes a critical cell regulatory protein, huntingtin (Htt). The expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats causes improper folding of functional proteins and is an initial trigger of pathological changes in the brain. Recent research has indicated that the functional dysregulation of many transcription factors underlies the neurodegenerative processes that accompany HD. These disturbances are caused not only by the loss of wild-type Htt (WT Htt) function but also by the occurrence of abnormalities that result from the action of mutant Htt (mHtt). In this review, we aim to describe the role of transcription factors that are currently thought to be strongly associated with HD pathogenesis, namely, RE1-silencing transcription factor, also known as neuron-restrictive silencer factor (REST/NRSF), forkhead box proteins (FOXPs), peroxisome proliferator-activated receptor gamma coactivator-1a (PGC1α), heat shock transcription factor 1 (HSF1), and nuclear factor κ light-chain-enhancer of activated B cells (NF- κB). We also take into account the role of these factors in the phenotype of HD as well as potential pharmacological interventions targeting the analyzed proteins. Furthermore, we considered whether molecular manipulation resulting in changes in transcription factor function may have clinical potency for treating HD.

Molecular mechanism of long-term neuroprotective effects of gradual flow restoration on cerebral ischemia reperfusion injury in MCAO rats

Background and purposeIschemia-reperfusion injuries (IRIs) can aggravate the condition of some patients with acute occlusion of major intracranial artery (AOMIA) who received endovascular thrombectomy. Here, we provided data confirming the association of Repressor Element-1 Silencing Transcription factor (REST) with the long-term neuroprotective effect of the middle cerebral artery occlusion (MCAO) rats underwent Gradual Flow Restoration (GFR). MethodsLong term neuroprotective effects of GFR intervention were evaluated on MCAO rats model after 3d and 7d reperfusion. The neurological deficit score and TTC staining were performed to evaluate the degree of brain damage in GFR and other interventions at different time. Differentially expressed genes related to cerebral ischemia reperfusion injury (CIRI) were initially screened and identified using GSE32529 microarray analysis. REST protein expression in rat brain cortex infarction was detected by Western blot analysis. ResultsMCAO rats intervened with GFR exhibited reduced neurological deficit (P < 0.05) and alleviated brain infarction volume (P < 0.01). The REST gene with up-regulated expression and its downstream genes with down-regulated expression were screened by Microarray analysis. The brain cortex infarction in MCAO rats produced high levels of REST expression. The GFR intervention inhibited REST expression, and alleviated brain injury on MCAO rats. ConclusionOur results demonstrated that GFR intervention plays a long-term neuroprotective role and reduces brain edema and damage at reperfusion, possibly by inhibiting REST expression.

1947-P: RE1-Silencing Transcription Factor (REST) Regulates Mitochondrial Activity in White Adipocytes

RE1-silencing transcription factor (REST) is a transcriptional factor which negatively regulates the expression of numerous neuronal genes. In this study, model mice lacking REST in adipocytes were generated to investigate the role of REST in adipocytes. This was achieved by mating Rest2lox/2lox mice with Adipoq-Cre mice, and the resultant Adipoq-Cre/Rest2lox/2lox mice were compared with Adipoq-Cre mice. Adipoq-Cre/Rest2lox/2lox mice exceeding 10 weeks of age showed increased body weight, fat mass, and adipocyte size; however no difference was observed in food intake. Glucose tolerance deteriorated under a high fat diet, but not under normal diets in Adipoq-Cre/Rest2lox/2lox mice. Oxygen consumption decreased in Adipoq-Cre/Rest2lox/2lox mice. The characteristics of gene expression in epididymal fat, i.e., white adipose tissue (WAT) and brown adipose tissue (BAT), isolated from Adipoq-Cre/Rest2lox/2lox mice, were compared by microarray with those from Adipoq-Cre mice. Although expression of numerous neuronal genes increased in Adipoq-Cre/Rest2lox/2lox mice, there were no differences in adipocyte-specific genes. Gene analyses utilizing a cluster one program detected a significant decrease in the mitochondrial translation gene group in WAT, but not BAT. There was no difference in the amount of mitochondrial DNA, whereas the expression of cytochrome C protein was reduced in WAT but not BAT, which was isolated from Adipoq-Cre/Rest2lox/2lox mice. These results indicate that obesity in Adipoq-Cre/Rest2lox/2lox mice is due to a mitochondrial dysfunction in WAT. It is also suggested that REST might regulate the mitochondrial function in WAT. Disclosure M. Fuwa: None. K. Kajita: None. K. Taguchi: None. T. Ikeda: None. T. Ishizuka: None. H. Morita: None.