Repressor Element-1 Silencing Transcription Research Articles

Neuronal differentiation requires BRAT1 complex to remove REST from chromatin

Repressor element-1 silencing transcription factor (REST) is required for the formation of mature neurons. REST dysregulation underlies a key mechanism of neurodegeneration associated with neurological disorders. However, the mechanisms leading to alterations of REST-mediated silencing of key neurogenesis genes are not known. Here, we show that BRCA1 Associated ATM Activator 1 (BRAT1), a gene linked to neurodegenerative diseases, is required for the activation of REST-responsive genes during neuronal differentiation. We find that INTS11 and INTS9 subunits of Integrator complex interact with BRAT1 as a distinct trimeric complex to activate critical neuronal genes during differentiation. BRAT1 depletion results in persistence of REST residence on critical neuronal genes disrupting the differentiation of NT2 cells into astrocytes and neuronal cells. We identified BRAT1 and INTS11 co-occupying the promoter region of these genes and pinpoint a role for BRAT1 in recruiting INTS11 to their promoters. Disease-causing mutations in BRAT1 diminish its association with INTS11/INTS9, linking the manifestation of disease phenotypes with a defect in transcriptional activation of key neuronal genes by BRAT1/INTS11/INTS9 complex. Finally, loss of Brat1 in mouse embryonic stem cells leads to a defect in neuronal differentiation assay. Importantly, while reconstitution with wild-type BRAT1 restores neuronal differentiation, the addition of a BRAT1 mutant is unable to associate with INTS11/INTS9 and fails to rescue the neuronal phenotype. Taken together, our study highlights the importance of BRAT1 association with INTS11 and INTS9 in the development of the nervous system.

Comprehensive analysis of the REST transcription factor regulatory networks in IDH mutant and IDH wild-type glioma cell lines and tumors

The RE1-silencing transcription factor (REST) acts either as a repressor or activator of transcription depending on the genomic and cellular context. REST is a key player in brain cell differentiation by inducing chromatin modifications, including DNA methylation, in a proximity of its binding sites. Its dysfunction may contribute to oncogenesis. Mutations in IDH1/2 significantly change the epigenome contributing to blockade of cell differentiation and glioma development. We aimed at defining how REST modulates gene activation and repression in the context of the IDH mutation-related phenotype in gliomas. We studied the effects of REST knockdown, genome wide occurrence of REST binding sites, and DNA methylation of REST motifs in IDH wild type and IDH mutant gliomas. We found that REST target genes, REST binding patterns, and TF motif occurrence proximal to REST binding sites differed in IDH wild-type and mutant gliomas. Among differentially expressed REST targets were genes involved in glial cell differentiation and extracellular matrix organization, some of which were differentially methylated at promoters or gene bodies. REST knockdown differently impacted invasion of the parental or IDH1 mutant glioma cells. The canonical REST-repressed gene targets showed significant correlation with the GBM NPC-like cellular state. Interestingly, results of REST or KAISO silencing suggested the interplay between these TFs in regulation of REST-activated and repressed targets. The identified gene regulatory networks and putative REST cooperativity with other TFs, such as KAISO, show distinct REST target regulatory networks in IDH-WT and IDH-MUT gliomas, without concomitant DNA methylation changes. We conclude that REST could be an important therapeutic target in gliomas.

Valproic Acid Treatment after Traumatic Brain Injury in Mice Alleviates Neuronal Death and Inflammation in Association with Increased Plasma Lysophosphatidylcholines.

The histone deacetylase inhibitor (HDACi) valproic acid (VPA) has neuroprotective and anti-inflammatory effects in experimental traumatic brain injury (TBI), which have been partially attributed to the epigenetic disinhibition of the transcription repressor RE1-Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF). Additionally, VPA changes post-traumatic brain injury (TBI) brain metabolism to create a neuroprotective environment. To address the interconnection of neuroprotection, metabolism, inflammation and REST/NRSF after TBI, we subjected C57BL/6N mice to experimental TBI and intraperitoneal VPA administration or vehicle solution at 15 min, 1, 2, and 3 days post-injury (dpi). At 7 dpi, TBI-induced an up-regulation of REST/NRSF gene expression and HDACi function of VPA on histone H3 acetylation were confirmed. Neurological deficits, brain lesion size, blood-brain barrier permeability, or astrogliosis were not affected, and REST/NRSF target genes were only marginally influenced by VPA. However, VPA attenuated structural damage in the hippocampus, microgliosis and expression of the pro-inflammatory marker genes. Analyses of plasma lipidomic and polar metabolomic patterns revealed that VPA treatment increased lysophosphatidylcholines (LPCs), which were inversely associated with interleukin 1 beta (Il1b) and tumor necrosis factor (Tnf) gene expression in the brain. The results show that VPA has mild neuroprotective and anti-inflammatory effects likely originating from favorable systemic metabolic changes resulting in increased plasma LPCs that are known to be actively taken up by the brain and function as carriers for neuroprotective polyunsaturated fatty acids.

Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

BackgroundGlioblastoma (GBM) is an aggressive brain cancer associated with poor prognosis, intrinsic heterogeneity, plasticity, and therapy resistance. In some GBMs, cell proliferation is fueled by a transcriptional regulator, repressor element-1 silencing transcription factor (REST).ResultsUsing CRISPR/Cas9, we identified GBM cell lines dependent on REST activity. We developed new small molecule inhibitory compounds targeting small C-terminal domain phosphatase 1 (SCP1) to reduce REST protein level and transcriptional activity in glioblastoma cells. Top leads of the series like GR-28 exhibit potent cytotoxicity, reduce REST protein level, and suppress its transcriptional activity. Upon the loss of REST protein, GBM cells can potentially compensate by rewiring fatty acid metabolism, enabling continued proliferation. Combining REST inhibition with the blockade of this compensatory adaptation using long-chain acyl-CoA synthetase inhibitor Triacsin C demonstrated substantial synergetic potential without inducing hepatotoxicity.ConclusionsOur results highlight the efficacy and selectivity of targeting REST alone or in combination as a therapeutic strategy to combat high-REST GBM.

The homeostatic effects of the RE-1 silencing transcription factor on cortical networks are altered under ictogenic conditions in the mouse.

The Repressor Element-1 Silencing Transcription Factor (REST) is an epigenetic master regulator playing a crucial role in the nervous system. In early developmental stages, REST downregulation promotes neuronal differentiation and the acquisition of the neuronal phenotype. In addition, postnatal fluctuations in REST expression contribute to shaping neuronal networks and maintaining network homeostasis. Here we investigate the role of the early postnatal deletion of neuronal REST in the assembly and strength of excitatory and inhibitory synaptic connections. We investigated excitatory and inhibitory synaptic transmission by patch-clamp recordings in acute neocortical slices in a conditional knockout mouse model (RestGTi) in which Rest was deleted by delivering PHP.eB adeno-associated viruses encoding CRE recombinase under the control of the human synapsin I promoter in the lateral ventricles of P0-P1 pups. We show that, under physiological conditions, Rest deletion increased the intrinsic excitability of principal cortical neurons in the primary visual cortex and the density and strength of excitatory synaptic connections impinging on them, without affecting inhibitory transmission. Conversely, in the presence of a pathological excitation/inhibition imbalance induced by pentylenetetrazol, Rest deletion prevented the increase in synaptic excitation and decreased seizure severity. The data indicate that REST exerts distinct effects on the excitability of cortical circuits depending on whether it acts under physiological conditions or in the presence of pathologic network hyperexcitability. In the former case, REST preserves a correct excitatory/inhibitory balance in cortical circuits, while in the latter REST loses its homeostatic activity and may become pro-epileptogenic.

Molecular pathology of small cell lung cancer: Overview from studies on neuroendocrine differentiation regulated by ASCL1 and Notch signaling.

Pulmonary neuroendocrine (NE) cells are rare airway epithelial cells. The balance between Achaete-scute complex homolog 1 (ASCL1) and hairy and enhancer of split 1, one of the target molecules of the Notch signaling pathway, is crucial for NE differentiation. Small cell lung cancer (SCLC) is a highly aggressive lung tumor, characterized by rapid cell proliferation, a high metastatic potential, and the acquisition of resistance to treatment. The subtypes of SCLC are defined by the expression status of NE cell-lineage transcription factors, such as ASCL1, which roles are supported by SRY-box 2, insulinoma-associated protein 1, NK2 homeobox 1, and wingless-related integration site signaling. This network reinforces NE differentiation and may induce the characteristic morphology and chemosensitivity of SCLC. Notch signaling mediates cell-fate decisions, resulting in an NE to non-NE fate switch. The suppression of NE differentiation may change the histological type of SCLC to a non-SCLC morphology. In SCLC with NE differentiation, Notch signaling is typically inactive and genetically or epigenetically regulated. However, Notch signaling may be activated after chemotherapy, and, in concert with Yes-associated protein signaling and RE1-silencing transcription factor, suppresses NE differentiation, producing intratumor heterogeneity and chemoresistance. Accumulated information on the molecular mechanisms of SCLC will contribute to further advances in the control of this recalcitrant cancer.

Nuclear KRT19 is a transcriptional corepressor promoting histone deacetylation and liver tumorigenesis.

Epigenetic reprogramming and escape from terminal differentiation are poorly understood enabling characteristics of liver cancer. Keratin 19 (KRT19), classically known to form the intermediate filament cytoskeleton, is a marker of stemness and worse prognosis in liver cancer. This study aimed to address the functional roles of KRT19 in liver tumorigenesis and to elucidate the underlying mechanisms. Using multiplexed genome editing of hepatocytes in vivo, we demonstrated that KRT19 promoted liver tumorigenesis in mice. Cell fractionation revealed a previously unrecognized nuclear fraction of KRT19. Tandem affinity purification identified histone deacetylase 1 and REST corepressor 1, components of the corepressor of RE-1 silencing transcription factor (CoREST) complex as KRT19-interacting proteins. KRT19 knockout markedly enhanced histone acetylation levels. Mechanistically, KRT19 promotes CoREST complex formation by enhancing histone deacetylase 1 and REST corepressor 1 interaction, thus increasing the deacetylase activity. ChIP-seq revealed hepatocyte-specific genes, such as hepatocyte nuclear factor 4 alpha ( HNF4A ), as direct targets of KRT19-CoREST. In addition, we identified forkhead box P4 as a direct activator of aberrant KRT19 expression in liver cancer. Furthermore, treatment of primary liver tumors and patient-derived xenografts in mice suggest that KRT19 expression has the potential to predict response to histone deacetylase 1 inhibitors especially in combination with lenvatinib. Our data show that nuclear KRT19 acts as a transcriptional corepressor through promoting the deacetylase activity of the CoREST complex, resulting in dedifferentiation of liver cancer. These findings reveal a previously unrecognized function of KRT19 in directly shaping the epigenetic landscape in cancer.

Abstract 2864: Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis

Abstract Introduction: Medulloblastoma (MB) is the most common malignant brain tumor in children. It is categorized into four molecular subgroups, each with unique genetic and epigenetic alterations, molecular signatures, and differences in clinical features and treatment responses. Targeted therapy for MB has failed in the clinic, prompting new studies of mechanisms involved in MB tumorigenesis and progression. Experimental Procedure: The transcriptomic information of the MB patients was obtained from the publicly available RNAseq datasets and was analyzed for differential expression and clustering analysis. Patient-derived orthotopic xenografts of medulloblastoma tumors with high- and low-REST expression were histologically and biochemically studied for autophagy markers. Furthermore, the SHH-MB cell lines were studied to get a mechanistic understanding of the REST-driven autophagy in this MB subgroup. Summary: Our data showed that autophagy, a process responsible for cellular recycling, and genes associated with this pathway exhibit distinctive expression patterns in human MB subgroups. Specifically, we demonstrate that autophagy is induced in one of the MB subgroups (Sonic Hedgehog-SHH) in response to elevated expression of the RE1-silencing transcription factor (REST), a transcriptional repressor and a canonical regulator of neuronal differentiation genes. REST is also a driver of SHH-MB metastasis. Pharmacological inhibition of autophagy decreases MB cell growth, suggesting it is a pro-survival pathway in REST-driven SHH MBs. In mechanistic studies, we found REST-dependent autophagy induction to involve upregulation of the hypoxia-inducible factor 1-alpha (HIF1α) due to REST-mediated silencing of the von Hippel-Lindau (VHL) gene. The VHL gene product promotes HIF1α ubiquitination and proteasomal degradation, and its loss in SHH-MBs with elevated REST expression drives HIF1α stabilization and nuclear localization to activate target autophagy-related genes. Conclusion: Our work is the first to link VHL to MB pathology. VHL loss of function in cancers has been mainly attributed to mutational events. Here, we report a novel mechanism of VHL loss, namely its epigenetic silencing by REST. Our findings provide the foundation for future pre-clinical investigations to examine the feasibility of autophagy blockade as a therapeutic strategy for REST-driven SHH MBs. Citation Format: Ashutosh Singh, Donghang Cheng, Jyothishmathi Swaminathan, Yan Zheng, Nancy Gordon, Vidya Gopalakrishnan. Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2864.

Overexpression of REST Represses the Epithelial-Mesenchymal Transition Process and Decreases the Aggressiveness of Prostate Cancer Cells.

The RE-1 silencing transcription factor (REST) is a repressor factor related to neuroendocrine prostate cancer (PCa) (NEPC), a poor prognostic stage mainly associated with castration-resistant PCa (CRPC). NEPC is associated with cell transdifferentiation and the epithelial-mesenchymal transition (EMT) in cells undergoing androgen deprivation therapy (ADT) and enzalutamide (ENZ). The effect of REST overexpression in the 22rv1 cell line (xenograft-derived prostate cancer) on EMT, migration, invasion, and the viability for ENZ was evaluated. EMT genes, Twist and Zeb1, and the androgen receptor (AR) were evaluated through an RT-qPCR and Western blot in nuclear and cytosolic fractions of REST-overexpressing 22rv1 cells (22rv1-REST). The migratory and invasive capacities of 22rv1-REST cells were evaluated via Transwell® assays with and without Matrigel, respectively, and their viability for enzalutamide via MTT assays. The 22rv1-REST cells showed decreased nuclear levels of Twist, Zeb1, and AR, and a decreased migration and invasion and a lower viability for ENZ compared to the control. Results were expressed as the mean + SD of three independent experiments (Mann-Whitney U test, Kruskal-Wallis, Tukey test). REST behaves like a tumor suppressor, decreasing the aggressiveness of 22rv1 cells, probably through the repression of EMT and the neuroendocrine phenotype. Furthermore, REST could represent a response marker to ENZ in PCa patients.

Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex.

REST (Repressor-Element 1 [RE1]-silencing transcription factor) inhibits Na+/Ca2+exchanger-1 (Ncx1) transcription in neurons through the binding of RE1 site on brain promoter (Br) after stroke. We identified a new putative RE1 site in Ncx1 heart promoter (Ht) sequence (Ht-RE1) that participates in neuronal Ncx1 transcription. Because REST recruits DNA-methyltransferase-1 (DNMT1) and MeCP2 (methyl-CpG binding protein 2) on different neuronal genes, we investigated the role of this complex in Ncx1 transcriptional regulation after stroke. Luciferase experiments performed in SH-SY5Y cells demonstrated that Br activity was selectively decreased by REST, whereas Ht activity was reduced by DNMT1, MeCP2, and REST. Notably, site-direct mutagenesis of Ht-RE1 prevented REST-dependent downregulation of Ncx1. Furthermore, in temporoparietal cortex of 8-week-old male wild-type mice (C57BL/6) subjected to transient middle cerebral artery occlusion, DNMT1, MeCP2, and REST binding to Ht promoter was increased, with a consequent DNA promoter hypermethylation. Intracerebroventricular injection of siREST prevented DNMT1/MeCP2 binding to Ht and Ncx1 downregulation, thus causing a reduction in stroke-induced damage. Consistently, in cortical neurons subjected to oxygen and glucose deprivation plus reoxygenation Ncx1 knockdown counteracted neuronal protection induced by the demethylating agent 5-azacytidine. For comparisons between 2 experimental groups, Student's ttest was used, whereas for more than 2 experimental groups, 1-way ANOVA was used, followed by Tukey or Newman Keuls. Statistical significance was set at P<0.05. If the results of this study are confirmed in humans, it could be asserted that DNMT1/MeCP2/REST complex disruption could be a new pharmacological strategy to reduce DNA methylation of Ht in the brain, ameliorating stroke damage.

Astrocyte-induced mGluR1 activates human lung cancer brain metastasis via glutamate-dependent stabilization of EGFR

There are limited methods to stably analyze the interactions between cancer cells and glial cells invitro, which hinders our molecular understanding. Here, we develop a simple and stable culture method of mouse glial cells, termed mixed-glial culture on/in soft substrate (MGS), which serves well as a platform to study cancer-glia interactions. Using this method, we find that human lung cancer cells become overly dependent on metabotropic glutamate receptor 1 (mGluR1) signaling in the brain microenvironment. Mechanistically, interactions with astrocytes induce mGluR1 in cancer cells through the Wnt-5a/prickle planar cell polarity protein 1 (PRICKLE1)/RE1 silencing transcription factor (REST) axis. Induced mGluR1 directly interacts with and stabilizes the epidermal growth factor receptor (EGFR) in a glutamate-dependent manner, and these cells then become responsive to mGluR1 inhibition. Our results highlight increased dependence on mGluR1 signaling as an adaptive strategy and vulnerability of human lung cancer brain metastasis.

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

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

REST-restrained lncRNA EPB41L4A-AS2 Modulates Laryngeal Squamous Cell Carcinoma Development via Regulating miR-1254/HIPK2 Pathway.

LncRNAs have been corroborated to exert crucial effects in malignancies, including laryngeal squamous cell carcinoma (LSCC). Nevertheless, the role and mechanism of EPB41L4A- AS2 in LSCC are inadequately investigated and warrant further exploration. Relevant database was adopted to analyze the relationship between EPB41L4A-AS2 expression level and tumors. The expressions and relationships of EPB41L4A-AS2, RE-1 silencing transcription factor (REST), miR-1254, and homeodomain interacting protein kinase 2 (HIPK2) in LSCC cells were evaluated by qRT-PCR, Pearson's correlation tests, RNA immunoprecipitation, RNA pull-down assay, chromatin immunoprecipitation, database, and dual-luciferase reporter assay. Following the required transfection, the biological behaviors of LSCC cells were examined using cell function experiments. Meanwhile, the levels of Ki-67 and apoptosis-, and epithelial-mesenchymal transition (EMT) pathway-related proteins were quantified with Western blot. Moreover, xenografts in nude mice were constructed, and the tumor volume and weight were measured. Ki-67 positivity was determined by immunohistochemical staining. EPB41L4A-AS2 and HIPK2 were lower-expressed, yet miR-1254 and REST were higher- expressed in LSCC cells. Pearson's correlation assay results exhibited a positive correlation between HIPK2 and EPB41L4A-AS2 and a negative correlation between HIPK2 and miR-1254. Overexpressed EPB41L4A-AS2 diminished the biological behavior, and repressed the levels of Ki-67 and EMT-related markers in LSCC cells whilst enhancing those of apoptosis-related markers. These aforementioned effects were counteracted by miR-1254 mimic. Moreover, EPB41L4A- AS2 overexpression suppressed the growth of tumors and reduced the positive expression of Ki-67 in nude mice. Besides, miR-1254 aggravated the biological behaviors and elevated the levels of Ki-67 and EMT-related proteins in LSCC cells while reducing the levels of apoptosis-related markers via targeting HIPK2. REST-restrained EPB41L4A-AS2 modulates LSCC development via regulating miR-1254/HIPK2 pathway.

REST Is Not Resting: REST/NRSF in Health and Disease.

Chromatin modifications play a crucial role in the regulation of gene expression. The repressor element-1 (RE1) silencing transcription factor (REST), also known as neuron-restrictive silencer factor (NRSF) and X2 box repressor (XBR), was found to regulate gene transcription by binding to chromatin and recruiting chromatin-modifying enzymes. Earlier studies revealed that REST plays an important role in the development and disease of the nervous system, mainly by repressing the transcription of neuron-specific genes. Subsequently, REST was found to be critical in other tissues, such as the heart, pancreas, skin, eye, and vascular. Dysregulation of REST was also found in nervous and non-nervous system cancers. In parallel, multiple strategies to target REST have been developed. In this paper, we provide a comprehensive summary of the research progress made over the past 28 years since the discovery of REST, encompassing both physiological and pathological aspects. These insights into the effects and mechanisms of REST contribute to an in-depth understanding of the transcriptional regulatory mechanisms of genes and their roles in the development and progression of disease, with a view to discovering potential therapeutic targets and intervention strategies for various related diseases.

Abstract P1146: Global Analysis Of Murine Cardiac Gene Program Regulated By Thyroid Hormone And Transcriptional Repressor Rest

Background: During adult heart failure, transcriptional reprogramming of fetal cardiac genes coincides with dysregulated heart functions. However, the underlying mechanisms remain incompletely understood. Thyroid hormone and RE1 silencing transcription factor (REST) are known regulators of cardiac gene reprogramming. Here, we report a global analysis of their co-regulatory functions. Methods: We generated a dual Myh6 and Myh7 mouse reporter line. This line was crossed with a conditional Rest allele to generate myocardial Rest knockout mice. Control and Rest null mice were treated with propylthiouracil (PTU) to inhibit thyroid hormone synthesis. PTU treatment induced heart failure and adult-to-fetal cardiac gene remodeling. Single nuclei multiomic profiling was performed, linking gene expression and gene regulatory network information from each nuclei to elucidate the impact of PTU or REST or both on cardiac gene reprogramming. Samples were collected to validate the newly identified factors involved in reprogramming. Results: Data analysis showed that PTU treatment upregulates transcription factor binding involving circadian gene expression ( Cry2 , Per2 , Per3 ). Downregulated genes are related to voltage-gated channel activity and BMP/TGFB activities involving heart development ( Myh6 , Nrg1 , Bmpr1a ) and SA node action potential ( Scn5a , Hcn2 , Ryr2 ). In contrast, Rest knockout upregulated genes are associated with arrhythmia, i.e. , cAMP catabolism ( Pde4d , Pde7a , Pde7b ), heart conduction ( Cacna1c , Gja1 , Pln ), and ion transport ( Kcnip2 , Pde4d, Esr1 ). Downregulated genes are involved in protein functions involving integrin-mediated signaling ( Itgal , Itga4 , Itga5 ), cytoskeleton organization ( Ctnna2 , Actb , Xirp1 ), and fiber organization ( Tgfb3 , Smad3 , Ptk2b ). Furthermore, a subset of genes was found to be co-regulated by thyroid hormone and REST, including Ccn2 , Myh9 , Nmrk2 , Fgr , Itga4 , Prkcb , Cspg4 which are involved in integrin signaling pathway, immune activation, and angiogenesis. Conclusion: Thyroid hormone and REST have shared function in cardiac gene reprogramming involving cardiac conduction and fibrosis as well as their own newly discovered, unique roles regulating circadian genes and cAMP catabolism, respectively.

A novel GRK3-HDAC2 regulatory pathway is a key direct link between neuroendocrine differentiation and angiogenesis in prostate cancer progression

The mechanisms underlying the progression of prostate cancer (PCa) to neuroendocrine prostate cancer (NEPC), an aggressive PCa variant, are largely unclear. Two prominent NEPC phenotypes are elevated NE marker expression and heightened angiogenesis. Identifying the still elusive direct molecular links connecting angiogenesis and neuroendocrine differentiation (NED) is crucial for our understanding and targeting of NEPC. Here we found that histone deacetylase 2 (HDAC2), whose role in NEPC has not been reported, is one of the most upregulated epigenetic regulators in NEPC. HDAC2 promotes both NED and angiogenesis. G protein-coupled receptor kinase 3 (GRK3), also upregulated in NEPC, is a critical promoter for both phenotypes too. Of note, GRK3 phosphorylates HDAC2 at S394, which enhances HDAC2's epigenetic repression of potent anti-angiogenic factor Thrombospondin 1 (TSP1) and master NE-repressor RE1 Silencing Transcription Factor (REST). Intriguingly, REST suppresses angiogenesis while TSP1 suppresses NE marker expression in PCa cells, indicative of their novel functions and their synergy in cross-repressing the two phenotypes. Furthermore, the GRK3-HDAC2 pathway is activated by androgen deprivation therapy and hypoxia, both known to promote NED and angiogenesis in PCa. These results indicate that NED and angiogenesis converge on GRK3-enhanced HDAC2 suppression of REST and TSP1, which constitutes a key missing link between two prominent phenotypes of NEPC.

Abstract P1015: Functional Investigation Of The Coronary Artery Disease Risk Gene Rest In Atherosclerosis

Background: GWAS discovered two CAD risk variants, rs17087335 and rs72627509, located downstream of REST (RE1-silencing transcription factor). So far, REST has been mainly associated with neurological diseases, while its role in vascular function remains unclear. Here, we aimed to explore the role of REST in atherosclerosis. Methods and Results: Transcriptome data from the GTEx consortium hinted towards an association of reduced REST expression in vascular tissues with the risk alleles of rs17087335 and rs72627509. We performed reporter gene assays in HEK293 cells using constructs that contain the REST promoter upstream and the SNP sequence (200 bp) downstream of the reporter gene. In line with the GTEx data, the luciferase assays showed reduced reporter gene activity with the risk alleles of both variants. We next studied the function of REST in different atherosclerosis-relevant vascular cell types, such as human vascular smooth muscle cells (HVSMC) and human vascular endothelial cells (HVEC). In HVEC, siRNA-mediated REST downregulation experiments revealed no changes in HVEC proliferation or migration. In HVSMC, in contrast, migration was significantly enhanced secondary to REST knockdown. Furthermore, reduced REST led to increased uptake of lipids into HVSMC under inflammatory conditions. To elucidate putative molecular pathways, we performed RNA sequencing in HVSMC secondary to REST downregulation. Surprisingly, we detected downregulation of ATF6 (ActivatingTranscription Factor 6) upon REST knockdown, which was confirmed by qPCR. In silico analysis revealed several REST binding sites at the ATF6 promoter. ATF6 plays a role in endoplasmatic reticulum (ER) stress, which has been reported to contribute to a macrophage-/fibroblast-like lipid-loaden phenotype of SMC. In line with the findings for REST, we could show that decreased ATF6 results in increased lipid uptake by HVSMC. Conclusion and Outlook: We provide first evidence that reduced REST expression may increase the risk of atherosclerosis and CAD via promoting HVSMC migration and a phenotypic switch towards macrophage-/fibroblast-like lipid-loaden SMC. These effects might be mediated via ATF6 . Modulation of REST might be a new strategy for preventing and treating CAD.

Promoter hypermethylation of neural-related genes is compatible with stemness in solid cancers

BackgroundDNA hypermethylation is an epigenetic feature that modulates gene expression, and its deregulation is observed in cancer. Previously, we identified a neural-related DNA hypermethylation fingerprint in colon cancer, where most of the top hypermethylated and downregulated genes have known functions in the nervous system. To evaluate the presence of this signature and its relevance to carcinogenesis in general, we considered 16 solid cancer types available in The Cancer Genome Atlas (TCGA).ResultsAll tested cancers showed significant enrichment for neural-related genes amongst hypermethylated genes. This signature was already present in two premalignant tissue types and could not be explained by potential confounders such as bivalency status or tumor purity. Further characterization of the neural-related DNA hypermethylation signature in colon cancer showed particular enrichment for genes that are overexpressed during neural differentiation. Lastly, an analysis of upstream regulators identified RE1-Silencing Transcription factor (REST) as a potential mediator of this DNA methylation signature.ConclusionOur study confirms the presence of a neural-related DNA hypermethylation fingerprint in various cancers, of genes linked to neural differentiation, and points to REST as a possible regulator of this mechanism. We propose that this fingerprint indicates an involvement of DNA hypermethylation in the preservation of neural stemness in cancer cells.

REST in the Road Map of Brain Development.

Repressor element-1 silencing transcription factor (REST) or also known as neuron-restrictive silencing factor (NRSF), is the key initiator of epigenetic neuronal gene-expression modification. Identification of a massive number of REST-targeted genes in the brain signifies its broad involvement in maintaining the functionality of the nervous system. Additionally, REST plays a crucial role in conferring neuroprotection to the neurons against various stressors or insults during injuries. At the cellular level, nuclear localisation of REST is a key determinant for the functional transcriptional regulation of REST towards its target genes. Emerging studies reveal the implication of REST nuclear mislocalisation or dysregulation in several neurological diseases. The expression of REST varies depending on different types of neurological disorders, which has created challenges in the discovery of REST-targeted interventions. Hence, this review presents a comprehensive summary on the physiological roles of REST throughout brain development and its implications in neurodegenerative and neurodevelopmental disorders, brain tumours and cerebrovascular diseases. This review offers valuable insights to the development of potential therapeutic approaches targeting REST to improve pathologies in the brain. The important roles of REST as a key player in the nervous system development, and its implications in several neurological diseases.

MicroRNA biogenesis machinery activation and lncRNA and REST overexpression as neuroprotective responses to fight inflammation in the hippocampus

Brain Long non-coding RNA (lncRNA) and microRNAs (miRs) play essential roles in the regulation of several important biological processes, including neuronal activity, cognitive processes, neurogenesis, angiogenesis, and neuroinflammation. In this context, the transcriptional repressor, RE1 silencing transcription factor (Rest), acts regulating the expression of neuronal genes as well as of lncRNAs and multiple miRNAs in the central nervous system. Nevertheless, its role in neuroinflammation was less explored. Here, we demonstrate, using an in vivo model of neuroinflammation induced by i.p. injection of LPS (0.33 mg/kg), that neuroinflammation increases gene expression of pro-inflammatory cytokines concomitant with the native and truncated forms of Rest and of non-coding RNAs. Additionally, the increased expression of enzymes Drosha ribonuclease III) (Drosha), Exportin 5 (Xpo5) and Endoribonuclease dicer (Dicer), associated with high expression of neuroprotective miRs 22 and 132 are indicative that the activation of biogenesis of miRs in the hippocampal region is a Central Nervous System (CNS) protective mechanism for the deleterious effects of neuroinflammation. Our results indicate that positive regulation of Rest gene expression in the hippocampal region by neuroinflammation correlates directly with the expression of miRs 22 and 132 and inversely with miR 335. In parallel, the confirmation of the possible alignment between the lncRNAs with miR 335 by bioinformatics corroborates with the sponge effect of Hottip and Hotair hybridizing and inhibiting the pro-inflammatory action of miR 335. This suggests the existence of a possible correlation between the activation of miR biogenesis machinery with increased expression of the transcription factor Rest, contributing to neuroprotection.