Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI)

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) ...

United Arab Emirates University

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.

Therapeutic potential of Mediator complex subunits in metabolic diseases.

Although the existence of a close relationship between the early maternal developmental environment, fetal size at birth and the risk of developing disease in adulthood has been suggested, most studies, however, employed experimentally induced intrauterine growth restriction as a model to link this with later adult disease. Because embryonic size variation also occurs under normal growth and differentiation, elucidating the molecular mechanisms underlying these changes and their relevance to later adult disease risk becomes important. The birth weight of rat pups vary according to the uterine horn positions. Using birth weight as a marker, we compared two groups of rat pups - lower birth weight (LBW, 5th to 25th percentile) and average birth weight (ABW, 50th to 75th percentile) - using morphological, biochemical and molecular biology, and genetic techniques. Our results show that insulin metabolism, Pi3k/Akt and Pparγ signaling and the genes regulating growth and metabolism are significantly different in these groups. Methylation at the promoter of the  () gene and DNA methyltransferase 1 in LBW pups are both increased. Additionally, the Dnmt1 repressor complex, which includes Hdac1, Rb (Rb1) and E2f1, was also upregulated in LBW pups. We conclude that the Dnmt1 repressor complex, which regulates the restriction point of the cell cycle, retards the rate at which cells traverse the G1 or G0 phase of the cell cycle in LBW pups, thereby slowing down growth. This regulatory mechanism mediated by Dnmt1 might contribute to the production of small-size pups and altered physiology and pathology in adult life.

Identification of early indicators of altered metabolism in normal development using a rodent model system.

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

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.

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

Diabetes mellitus (DM) is a chronic condition characterised by β cell dysfunction and persistent hyperglycaemia. The disorder can be due to the absence of adequate pancreatic insulin production or a weak cellular response to insulin signalling. Among the three types of DM, namely, type 1 DM (T1DM), type 2 DM (T2DM), and gestational DM (GDM); T2DM accounts for almost 90% of diabetes cases worldwide.Epigenetic traits are stably heritable phenotypes that result from certain changes that affect gene function without altering the gene sequence. While epigenetic traits are considered reversible modifications, they can be inherited mitotically and meiotically. In addition, epigenetic traits can randomly arise in response to environmental factors or certain genetic mutations or lesions, such as those affecting the enzymes that catalyse the epigenetic modification. In this review, we focus on the role of DNA methylation, a type of epigenetic modification, in the pathogenesis of T2DM.

The role of DNA methylation in the pathogenesis of type 2 diabetes mellitus.

Embryonic and adult stem cells possess the capability of self-renewal and lineage-specific differentiation. The intricate balance between self-renewal and differentiation is governed by developmental signals and cell-type-specific gene regulatory mechanisms. A perturbed intra/extracellular environment during lineage specification could affect stem cell fate decisions resulting in pathology. Growing evidence demonstrates that metabolic pathways govern epigenetic regulation of gene expression during stem cell fate commitment through the utilization of metabolic intermediates or end products of metabolic pathways as substrates for enzymatic histone/DNA modifications. UDP-GlcNAc is one such metabolite that acts as a substrate for enzymatic mono-glycosylation of various nuclear, cytosolic, and mitochondrial proteins on serine/threonine amino acid residues, a process termed protein O-GlcNAcylation. The levels of GlcNAc inside the cells depend on the nutrient availability, especially glucose. Thus, this metabolic sensor could modulate gene expression through O-GlcNAc modification of histones or other proteins in response to metabolic fluctuations. Herein, we review evidence demonstrating how stem cells couple metabolic inputs to gene regulatory pathways through O-GlcNAc-mediated epigenetic/transcriptional regulatory mechanisms to govern self-renewal and lineage-specific differentiation programs. This review will serve as a primer for researchers seeking to better understand how O-GlcNAc influences stemness and may catalyze the discovery of new stem-cell-based therapeutic approaches.

Stem cell fate determination through protein O-GlcNAcylation.

A recent International Diabetes Federation report suggests that more than 463 million people between 20 and 79 years have diabetes. Of the 20 million women affected by hyperglycemia during pregnancy, 84% have gestational diabetes. In addition, more than 1.1 million children or adolescents are affected by type 1 diabetes. Factors contributing to the increase in diabetes prevalence are complex and include contributions from genetic, environmental, and epigenetic factors. However, molecular regulatory mechanisms influencing the progression of an individual towards increased susceptibility to metabolic diseases such as diabetes are not fully understood. Recent studies suggest that the pathogenesis of diabetes involves epigenetic changes, resulting in a persistently dysregulated metabolic phenotype. This review summarizes the role of epigenetic mechanisms, mainly DNA methylation and histone modifications, in the development of the pancreas, their contribution to the development of diabetes, and the potential employment of epigenetic modulators in diabetes treatment.

Epigenetic modifications in pancreas development, diabetes, and therapeutics.

Although tumourigenesis occurs due to genetic mutations, the role of epigenetic dysregulations in cancer is also well established. Epigenetic dysregulations in cancer may occur as a result of mutations in genes encoding histone/DNA-modifying enzymes and chromatin remodellers or mutations in histone protein itself. It is also true that misregulated gene expression without genetic mutations in these factors could also support tumour initiation and progression. Interestingly, metabolic rewiring has emerged as a hallmark of cancer due to gene mutations in specific metabolic enzymes or dietary/environmental factors. Recent studies report an intricate cross-talk between epigenetic and metabolic reprogramming in cancer. This review discusses the role of epigenetic and metabolic dysregulations and their cross-talk in tumourigenesis with a special focus on gliomagenesis. We also discuss the role of recently developed human embryonic stem cells/induced pluripotent stem cells-derived organoid models of gliomas and how these models are proving instrumental in uncovering human-specific cellular and molecular complexities of gliomagenesis.

The epigenetic-metabolic interplay in gliomagenesis.

Protein -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 -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 -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 -GlcNAc levels also lead to increased Pol II-Ser5 phosphorylation and affect H2BS112-GlcNAc and H2BK120Ub1 on promoters. Using an in vivo rat model of maternal hyperglycemia, we show similarly elevated -GlcNAc levels and epigenetic dysregulations in the developing embryo brains because of hyperglycemia, whereas pharmacological inhibition of -GlcNAc transferase (OGT) restored these molecular changes. Together, our results demonstrate -GlcNAc mediated sensitivity of chromatin to nutrient status, and indicate how metabolic perturbations could affect gene expression during neurodevelopment.

Suraiya Anjum Ansari

Department of Biochemistry and Molecular Biology,
College of Medicine and Health Sciences,
Zayed Center for Health Sciences,
Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences

United Arab Emirates University

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI)

Suraiya Anjum Ansari

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Molecular Biology,College of Medicine and Health Sciences,Zayed Center for Health Sciences,Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences

United Arab Emirates University

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI)

Department of Biochemistry and Molecular Biology,
College of Medicine and Health Sciences,
Zayed Center for Health Sciences,
Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences