O-GlcNAc transferase promotes glioblastoma by modulating genes responsible for cell survival, invasion, and inflammation.

Metabolic reprogramming has emerged as one of the key hallmarks of cancer cells. Various metabolic pathways are dysregulated in cancers, including the hexosamine biosynthesis pathway. Protein O-GlcNAcylation is catalyzed by the enzyme O-GlcNAc transferase (OGT), an effector of hexosamine biosynthesis pathway that is found to be upregulated in most cancers. Posttranslational O-GlcNAcylation of various signaling and transcriptional regulators could promote cancer cell maintenance and progression by regulating gene expression, as gene-specific transcription factors and chromatin regulators are among the most highly O-GlcNAcylated proteins. Here, we investigated the role of OGT in glioblastoma. We demonstrate that OGT knockdown and chemical inhibition led to reduced glioblastoma cell proliferation and downregulation of many genes known to play key roles in glioblastoma cell proliferation, migration, and invasion. We show that genes downregulated due to OGT reduction are also known to be transcriptionally regulated by transcriptional initiation/elongation cofactor BRD4. We found BRD4 to be O-GlcNAcylated in glioblastoma cells; however, OGT knockdown/inhibition neither changed its expression nor its chromatin association on promoters. Intriguingly, we observed OGT knockdown led to reduced Pol II-Ser2P chromatin association on target genes without affecting other transcription initiation/elongation factors. Finally, we found that chemical inhibition of BRD4 potentiated the effects of OGT inhibition in reducing glioblastoma cell proliferation, invasion, and migration. We propose BRD4 and OGT act independently in the transcriptional regulation of a common set of genes and that combined inhibition of OGT and BRD4 could be utilized therapeutically for more efficient glioblastoma cell targeting than targeting of either protein alone.

STAU2 binds a complex RNA cargo that changes temporally with production of diverse intermediate progenitor cells during mouse corticogenesis.

STAU2 is a double-stranded RNA-binding protein enriched in the nervous system. During asymmetric divisions in the developing mouse cortex, STAU2 preferentially distributes into the intermediate progenitor cell (IPC), delivering RNA molecules that can impact IPC behavior. Corticogenesis occurs on a precise time schedule, raising the hypothesis that the cargo STAU2 delivers into IPCs changes over time. To test this, we combine RNA-immunoprecipitation with sequencing (RIP-seq) over four stages of mouse cortical development, generating a comprehensive cargo profile for STAU2. A subset of the cargo was 'stable', present at all stages, and involved in chromosome organization, macromolecule localization, translation and DNA repair. Another subset was 'dynamic', changing with cortical stage, and involved in neurogenesis, cell projection organization, neurite outgrowth, and included cortical layer markers. Notably, the dynamic STAU2 cargo included determinants of IPC versus neuronal fates and genes contributing to abnormal corticogenesis. Knockdown of one STAU2 target, Taf13, previously linked to microcephaly and impaired myelination, reduced oligodendrogenesis in vitro. We conclude that STAU2 contributes to the timing of corticogenesis by binding and delivering complex and temporally regulated RNA cargo into IPCs.

The RSC complex localizes to coding sequences to regulate Pol II and histone occupancy.

ATP-dependent chromatin remodelers regulate chromatin structure during multiple stages of transcription. We report that RSC, an essential chromatin remodeler, is recruited to the open reading frames (ORFs) of actively transcribed genes genome wide, suggesting a role for RSC in regulating transcription elongation. Consistent with such a role, Pol II occupancy in the ORFs of weakly transcribed genes is drastically reduced upon depletion of the RSC catalytic subunit Sth1. RSC inactivation also reduced histone H3 occupancy across transcribed regions. Remarkably, the strongest effects on Pol II and H3 occupancy were confined to the genes displaying the greatest RSC ORF enrichment. Additionally, RSC recruitment to the ORF requires the activities of the SAGA and NuA4 HAT complexes and is aided by the activities of the Pol II CTD Ser2 kinases Bur1 and Ctk1. Overall, our findings strongly implicate ORF-associated RSC in governing Pol II function and in maintaining chromatin structure over transcribed regions.

The metabolic sensor PASK is a histone 3 kinase that also regulates H3K4 methylation by associating with H3K4 MLL2 methyltransferase complex.

The metabolic sensor Per-Arnt-Sim (Pas) domain-containing serine/threonine kinase (PASK) is expressed predominantly in the cytoplasm of different cell types, although a small percentage is also expressed in the nucleus. Herein, we show that the nuclear PASK associates with the mammalian H3K4 MLL2 methyltransferase complex and enhances H3K4 di- and tri-methylation. We also show that PASK is a histone kinase that phosphorylates H3 at T3, T6, S10 and T11. Taken together, these results suggest that PASK regulates two different H3 tail modifications involving H3K4 methylation and H3 phosphorylation. Using muscle satellite cell differentiation and functional analysis after loss or gain of Pask expression using the CRISPR/Cas9 system, we provide evidence that some of the regulatory functions of PASK during development and differentiation may occur through the regulation of these histone modifications.

Neuroprotective Effects of Thymol, a Dietary Monoterpene Against Dopaminergic Neurodegeneration in Rotenone-Induced Rat Model of Parkinson's Disease.

Parkinson's disease (PD), a multifactorial movement disorder that involves progressive degeneration of the nigrostriatal system affecting the movement ability of the patient. Oxidative stress and neuroinflammation both are shown to be involved in the etiopathogenesis of PD. The aim of this study was to evaluate the therapeutic potential of thymol, a dietary monoterpene phenol in rotenone (ROT)-induced neurodegeneration in rats that precisely mimics PD in humans. Male Wistar rats were injected ROT at a dose of 2.5 mg/kg body weight for 4 weeks, to induce PD. Thymol was co-administered for 4 weeks at a dose of 50 mg/kg body weight, 30 min prior to ROT injection. The markers of dopaminergic neurodegeneration, oxidative stress and inflammation were estimated using biochemical assays, enzyme-linked immunosorbent assay, western blotting and immunocytochemistry. ROT challenge increased the oxidative stress markers, inflammatory enzymes and cytokines as well as caused significant damage to nigrostriatal dopaminergic system of the brain. Thymol treatment in ROT challenged rats appears to significantly attenuate dopaminergic neuronal loss, oxidative stress and inflammation. The present study showed protective effects of thymol in ROT-induced neurotoxicity and neurodegeneration mediated by preservation of endogenous antioxidant defense networks and attenuation of inflammatory mediators including cytokines and enzymes.

Attomole quantification and global profile of RNA modifications: Epitranscriptome of human neural stem cells.

Exploration of the epitranscriptome requires the development of highly sensitive and accurate technologies in order to elucidate the contributions of the more than 100 RNA modifications to cell processes. A highly sensitive and accurate ultra-high performance liquid chromatography-tandem mass spectrometry method was developed to simultaneously detect and quantify 28 modified and four major nucleosides in less than 20 min. Absolute concentrations were calculated using extinction coefficients of each of the RNA modifications studied. A comprehensive RNA modifications database of UV profiles and extinction coefficient is reported within a 2.3-5.2 % relative standard deviation. Excellent linearity was observed 0.99227-0.99999 and limit of detection values ranged from 63.75 attomoles to 1.21 femtomoles. The analytical performance was evaluated by analyzing RNA modifications from 100 ng of RNA from human pluripotent stem cell-derived neural cells. Modifications were detected at concentrations four orders of magnitude lower than the corresponding parental nucleosides, and as low as 23.01 femtograms, 64.09 attomoles. Direct and global quantitative analysis of RNA modifications are among the advantages of this new approach.

Distinct role of Mediator tail module in regulation of SAGA-dependent, TATA-containing genes in yeast.

The evolutionarily conserved Mediator complex is required for transcription of nearly all RNA Pol II-dependent promoters, with the tail module serving to recruit Mediator to active promoters in current models. However, transcriptional dependence on tail module subunits varies in a gene-specific manner, and the generality of the tail module requirement for transcriptional activation has not been explored. Here, we show that tail module subunits function redundantly to recruit Mediator to promoters in yeast, and transcriptome analysis shows stronger effects on genome-wide expression in a double-tail subunit deletion mutant than in single-subunit deletion mutants. Unexpectedly, TATA-containing and SAGA-dependent genes were much more affected by impairment of tail module function than were TFIID-dependent genes. Consistent with this finding, Mediator and preinitiation complex association with SAGA-dependent promoters is substantially reduced in gal11/med15Δ med3Δ yeast, whereas association of TBP, Pol II, and other Mediator modules with TFIID-dependent genes is largely independent of the tail module. Thus, we have identified a connection between the Mediator tail module and the division of promoter dependence between TFIID and SAGA.

Mediator, TATA-binding protein, and RNA polymerase II contribute to low histone occupancy at active gene promoters in yeast.

Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction.

Mechanisms of Mediator complex action in transcriptional activation.

Mediator is a large multisubunit complex that plays a central role in the regulation of RNA Pol II transcribed genes. Conserved in overall structure and function among eukaryotes, Mediator comprises 25-30 protein subunits that reside in four distinct modules, termed head, middle, tail, and CDK8/kinase. Different subunits of Mediator contact other transcriptional regulators including activators, co-activators, general transcription factors, subunits of RNA Pol II, and specifically modified histones, leading to the regulated expression of target genes. This review is focused on the interactions of specific Mediator subunits with diverse transcription regulators and how those interactions contribute to Mediator function in transcriptional activation.

Extensive role of the general regulatory factors, Abf1 and Rap1, in determining genome-wide chromatin structure in budding yeast.

The packaging of eukaryotic DNA into chromatin has profound consequences for gene regulation, as well as for other DNA transactions such as recombination, replication and repair. Understanding how this packaging is determined is consequently a pressing problem in molecular genetics. DNA sequence, chromatin remodelers and transcription factors affect chromatin structure, but the scope of these influences on genome-wide nucleosome occupancy patterns remains uncertain. Here, we use high resolution tiling arrays to examine the contributions of two general regulatory factors, Abf1 and Rap1, to nucleosome occupancy in Saccharomyces cerevisiae. These factors have each been shown to bind to a few hundred promoters, but we find here that thousands of loci show localized regions of altered nucleosome occupancy within 1 h of loss of Abf1 or Rap1 binding, and that altered chromatin structure can occur via binding sites having a wide range of affinities. These results indicate that DNA-binding transcription factors affect chromatin structure, and probably dynamics, throughout the genome to a much greater extent than previously appreciated.

Mediator complex association with constitutively transcribed genes in yeast.

Mediator is a large, multisubunit complex that is essential for transcription of mRNA by RNA Pol II in eukaryotes and is believed to bridge transcriptional activators and the general transcription machinery. However, several recent studies suggest that the requirement for Mediator during transcriptional activation is not universal, but rather activator dependent, and may be indirect for some genes. Here we have investigated Mediator association with several constitutively transcribed genes in yeast by comparing a yeast strain that harbors a temperature-sensitive mutation in an essential Mediator subunit, Srb4, with its wild-type (WT) counterpart. We find modest association of Mediator with constitutively active genes and show that this association is strongly decreased in srb4 ts yeast, whereas association with a nontranscribed region or repressed gene promoters is lower and unaffected in the mutant yeast. The tail module of Mediator remains associated with ribosomal protein (RP) gene promoters in srb4 ts yeast, while subunits from the head and middle modules are lost. Tail module association at Rap1-dependent gene promoters is lost in rap1 ts yeast, indicating that Rap1 is required for Mediator recruitment at these gene promoters and that its recruitment occurs via the tail module. Pol II association is also rapidly and severely affected in srb4 ts yeast, indicating that Mediator is directly required for pol II association at constitutively transcribed genes. Our results are consistent with Mediator functioning as a general transcription factor in yeast.

Nutrient sensitive protein O-GlcNAcylation modulates the transcriptome through epigenetic mechanisms during embryonic neurogenesis.

Protein O-GlcNAcylation is a dynamic, nutrient-sensitive mono-glycosylation deposited on numerous nucleo-cytoplasmic and mitochondrial proteins, including transcription factors, epigenetic regulators, and histones. However, the role of protein O-GlcNAcylation on epigenome regulation in response to nutrient perturbations during development is not well understood. Herein we recapitulated early human embryonic neurogenesis in cell culture and found that pharmacological up-regulation of O-GlcNAc levels during human embryonic stem cells' neuronal differentiation leads to up-regulation of key neurogenic transcription factor genes. This transcriptional de-repression is associated with reduced H3K27me3 and increased H3K4me3 levels on the promoters of these genes, perturbing promoter bivalency possibly through increased EZH2-Thr311 phosphorylation. Elevated O-GlcNAc levels also lead to increased Pol II-Ser5 phosphorylation and affect H2BS112O-GlcNAc and H2BK120Ub1 on promoters. Using an in vivo rat model of maternal hyperglycemia, we show similarly elevated O-GlcNAc levels and epigenetic dysregulations in the developing embryo brains because of hyperglycemia, whereas pharmacological inhibition of O-GlcNAc transferase (OGT) restored these molecular changes. Together, our results demonstrate O-GlcNAc mediated sensitivity of chromatin to nutrient status, and indicate how metabolic perturbations could affect gene expression during neurodevelopment.

Developmental modeling of hepatogenesis using obese iPSCs-hepatocyte differentiation uncovers pathological features.

Obesity is a multigene disorder. However, in addition to genetic factors, environmental determinants also participate in developing obesity and related pathologies. Thus, obesity could be best described as a combination of genetic and environmental perturbations often having its origin during the early developmental period. Environmental factors such as energy-dense food and sedentary lifestyle are known to be associated with obesogenicity. However, the combinatorial effects of gene-environment interactions are not well understood. Understanding the role of multiple genetic variations leading to subtle gene expression changes is not practically possible in monogenic or high-fat-fed animal models of obesity. In contrast, human induced pluripotent stem cells (hiPSCs) from individuals with familial obesity or an obesogenic genotype could serve as a good model system. Herein, we have used hiPSCs generated from normal and genetically obese subjects and differentiated them into hepatocytes in cell culture. We show that hepatocytes from obese iPSCs store more lipids and show increased cell death than normal iPSCs. Whole transcriptome analyses in both normal and obese iPSCs treated with palmitate compared to control revealed LXR-RXR and hepatic fibrosis pathways were enriched among other pathways in obese iPSCs compared to normal iPSCs. Among other genes, increased CD36 and CAV1 expression and decreased expression of CES1 in obese iPSCs could have been responsible for excess lipid accumulation, resulting in differential expression of genes associated with hepatic fibrosis, a key feature of non-alcoholic fatty liver disease (NAFLD). Our results demonstrate that iPSCs derived from genetically obese subjects could serve as an excellent model to understand the effects of this multigene disorder on organ development and may uncover pathologies of NAFLD, which is highly associated with obesity.

A T9G mutation in the prototype TATA-box TCACTATATATAG determines nucleosome formation and synergy with upstream activator sequences in plant promoters.

We had earlier reported that mutations to G and C at the seventh and eighth positions in the prototype TATA-box TCACTATATATAG inhibited light-dependent activation of transcription from the promoter. In this study, we characterized mutations at the ninth position of the prototype TATA-box. Substitution of T at the ninth position with G or C enhanced transcription from the promoter in transgenic tobacco (Nicotiana tabacum) plants. The effect of T9G/C mutations was not light dependent, although the 9G/C TATA-box showed synergy with the light-responsive element (lre). However, the 9G/C mutants in the presence of lre failed to respond to phytochromes, sugar, and calcium signaling, in contrast to the prototype TATA-box with lre. The 9G/C mutation shifted the point of initiation of transcription, and transcription activation was dependent upon the type of activating element present upstream. The synergy in activation was noticed with lre and legumin activators but not with rbcS, Pcec, and PR-1a activators. The 9G mutation resulted in a micrococcal nuclease-sensitive region over the TATA-box, suggesting a nucleosome-free region, in contrast to the prototype promoter, which had a distinct nucleosome on the TATA-box. Thus, the transcriptional augmentation with mutation at the ninth position might be because of the loss of a repressive nucleosomal structure on the TATA-box. In agreement with our findings, the promoters containing TATAGATA as identified by genome-wide analysis of Arabidopsis (Arabidopsis thaliana) are not tightly repressed.

The Role of Insulin Resistance and Protein O-GlcNAcylation in Neurodegeneration.

Metabolic syndrome including obesity and type 2 diabetes is increasing at an alarming rate worldwide. Similarly, there has been an increase in the cases of neurodegenerative diseases such as Alzheimer's disease (AD) possibility due to increase in elderly population in the past few decades. Both, metabolic diseases and AD have one common feature that is insulin resistance. Recent studies suggest a link between the regulatory functions of insulin in the brain and AD. Hypoglycemia, a characteristic feature of AD may be a result of impaired insulin signaling in the affected regions of the brain. O-GlcNAcylation is a post-translational protein modification, the levels of which are dependent on the availability of glucose inside the cells. Hyperphosphorylation of Tau is a major molecular feature, which leads to its aggregation and neurotoxicity in AD. In addition, impaired processing of Amyloid precursor protein (APP) leading to toxic amyloid ß (Aß) aggregation is also implicated in the pathogenesis of AD. Both APP and Tau are also found to be O-GlcNAcylated. Reduced O-GlcNAcylation of APP and Tau due to hypoglycemia is found to be associated with their pathological features in AD brain. Recent studies have also identified perturbed O-GlcNAcylation/phosphorylation of several other proteins important for normal neuronal function, which may be contributing to the neuropathological development in AD. Herein, we discuss about the uptake and distribution of insulin inside the brain, brain insulin signaling and insulin resistance as well as its relation to neurodegenerative diseases with a special focus on protein O-GlcNAcylation and its potential role in the treatment of AD.

Fine Tuning of Hepatocyte Differentiation from Human Embryonic Stem Cells: Growth Factor vs. Small Molecule-Based Approaches.

Human embryonic stem cells (hESCs) are being utilized in diverse areas of studies such as development and disease modeling, cell replacement therapy, or drug toxicity testing because of their potential to be differentiated into any cell type in the body. The directed differentiation of hESCs into hepatocytes could provide an invaluable source of liver cells for various liver-based applications. Therefore, several protocols have been established in the past for hESC-hepatocyte differentiation based on the knowledge of signaling pathways and growth factors involved in different stages of embryonic hepatogenesis. Although successful derivation of hepatocytes has been achieved through these protocols, the efficiency is not always ideal. Herein, we have tested several combinations of published protocols, for example, growth factor vs. small molecule and different time durations of treatment for definitive endoderm (DE) induction and further hepatocyte differentiation to develop an efficient DE induction and hepatocyte differentiation in a highly reproducible manner based on the stage-specific marker expression and functional analysis.

Therapeutic potential of Mediator complex subunits in metabolic diseases.

The multisubunit Mediator is an evolutionary conserved transcriptional coregulatory complex in eukaryotes. It is needed for the transcriptional regulation of gene expression in general as well as in a gene specific manner. Mediator complex subunits interact with different transcription factors as well as components of RNA Pol II transcription initiation complex and in doing so act as a bridge between gene specific transcription factors and general Pol II transcription machinery. Specific interaction of various Mediator subunits with nuclear receptors (NRs) and other transcription factors involved in metabolism has been reported in different studies. Evidences indicate that ligand-activated NRs recruit Mediator complex for RNA Pol II-dependent gene transcription. These NRs have been explored as therapeutic targets in different metabolic diseases; however, they show side-effects as targets due to their overlapping involvement in different signaling pathways. Here we discuss the interaction of various Mediator subunits with transcription factors involved in metabolism and whether specific interaction of these transcription factors with Mediator subunits could be potentially utilized as therapeutic strategy in a variety of metabolic diseases.

Saturated fatty acid alters embryonic cortical neurogenesis through modulation of gene expression in neural stem cells.

A perturbed maternal metabolic environment such as chronically elevated circulating free fatty acids have been shown to affect stem cell fate during embryonic neurogenesis. However, molecular mechanisms behind this are not well defined, especially in human. Here in using directed differentiation of human embryonic stem cells (hESCs) into cortical neurons as model, we show that chronically elevated saturated fatty acid (palmitate) results in decreased proliferation of neural stem cells and increased differentiation into neurons. This phenotype could be due to palmitate mediated increased expression of key genes needed for neuronal differentiation such as EOMES, TBR1, NEUROD1 and RELN and reduced expression of SREBP regulated lipogenic genes at early stages of cortical differentiation. Furthermore, palmitate treatment increased histone acetylation globally and at select gene promoters among affected genes. We also found differential expression of several lncRNAs associated with cellular stress and metabolic diseases in the presence of palmitate including BDNF-AS suggesting the contribution of additional epigenetic regulatory mechanisms. Together, our results show that saturated fatty acid affects developmental neurogenesis through modulation of gene expression and through epigenetic regulatory mechanisms.

Saturated fatty acid regulated lncRNA dataset during in vitro human embryonic neurogenesis.

Human embryonic stem cells (hESCs) were used as a model of embryonic neurogenesis to identify the effect of excess fat uptake on neurodevelopment (Ardah et al., 2018). Herein, by directed differentiation of hESCs into neurons using established protocols, this data was generated for expression profiles of select lncRNAs during in vitro embryonic neurogenesis and their differential expression due to excess fat (palmitate) uptake. The undifferentiated hESCs were treated with 250 µM palmitate after identifying it as the highest concentration which is non-toxic to these cells. The palmitate treated hESCs were differentiated towards neurons keeping the levels of palmitate consistent throughout the differentiation process and fat uptake was confirmed by Oil Red O staining. The expression analysis of lncRNAs was performed by RT-qPCR on vehicle control and palmitate treated cells from 4 stages of differentiation, D0 (undifferentiated hESCs), D12 (neural stem cells), D44 (neural progenitors) and D70 (neurons) using lncRNAs array plates from Arraystar Inc. which contains 372 functionally identified lncRNAs found to be associated with lipid metabolism and other pathways (Cat# AS-NR-004).