Name: correlating gene-specific dna methylation changes with expression and transcriptional activity of astrocytic kcnj10 (kir4.1)
Uploaded: 2015-09-26T15:00:00
Description: Watch this Scientific Journal Video about Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1 at JoVE.com

This work was supported by R01NS075062-01A1. FACS sorting performed at UAB Comprehensive Flow Cytometry Core facility (P30 AR048311, P30 A1027767). Dr. Scott Philips from the UAB Neurobiology Core facility and Dr. Susan Nozell from UAB CDIB assisted with technical aspects of the luciferase assay.

Tutti gli animali sono stati trattati in conformità con le linee guida del National Institutes of Salute. La cura e l&#39;uso Comitato degli animali presso l&#39;Università di Alabama a Birmingham ha approvato l&#39;uso degli animali. 1. Ottenere RNA e DNA da un Arricchita astrocitica Popolazione utilizzando fluorescenti Cellulari Attivato Ordinamento (FACS) di astrociti da tutto il tessuto del cervello <ol><li> Animale sedate con CO 2 per 1 minuto e poi rapidamente decapitarl...

This protocol describes the isolation of an enriched population of astrocytes via FACS as well as a variety of techniques that allow for both correlative and causative studies between DNA methylation and gene expression. These techniques, used in isolation or in combination, are particularly useful for laboratories that work with tissue of high cellular heterogeneity or are interested in the DNA methylation status of a particular gene or gene region versus global DNA methylation changes. One relatively unique challenge i...

Epigenetics is the study of chemical modifications that can affect the transcriptional activity of the genome. Essentially, without a change in the DNA sequence, epigenetic modifications such as DNA methylation, histone acetylation, and histone methylation are sufficient to reversibly alter patterns of gene expression 1. DNA methylation, a potent regulator of gene expression, is the most well characterized epigenetic modification. DNA methylation is the covalent attachment of methyl groups on the C5 position of a cytosine, typically the cytosine of a cytosine-guanine dinucleotide, also known as a CpG site. Areas that contain a high density of CpG sites are known as CpG islands (CGIs). CGIs are frequently associated with transcriptional start sites (TSS) and gene promoters 1-3. Thus, while changes in DNA methylation at CGIs are not always concomitant with changes in cellular expression or function, changes in DNA methylation at CGIs can exert powerful regulation on transcriptional activity 2. 
Historically, DNA methylation was observed to be essential in embryogenesis, imprinting, and development, with little changes in the levels of DNA methylation occurring in post-mitotic cells (with the exception of alterations in cancer-related genes) 4,5. However, the field of neuroepigenetics has highlighted an important non-developmental role for DNA methylation. Specifically, cognitive epigenetics has redefined DNA methylation as a highly plastic mechanism integral in mediating both the transcriptional activation and repression of genes essential for the process of learning and memory 6. Apart from cognitive epigenetics, studies modeling ischemic injury and neuropathic pain characterize DNA methylation as a labile mechanism that responds rapidly to a variety of CNS insults 7-9. In regards to astrocytes, several lines of evidence suggest DNA methylation plays an important role in astrogliogenesis. Fan et al., found that conditional KO of DNMT1 in neural progenitor cells (NPCs) resulted in precocious development of astrocytes concordant with a global state of hypomethylation 10. Additionally, Perisic et al., concluded differential levels of DNA methylation of the GLT-1 promoter mediated differential levels of expression of the glutamate transporter in the cortex and cerebellum, emphasizing a role in DNA methylation in establishing brain-region specific patterns of astrocytic gene expression 11. Overall, numerous studies underscore the dynamic and labile nature of DNA methylation in the CNS as environment, drugs, and injury have all been shown to change DNA methylation and often, gene expression 4,9. Together, these neuroepigenetic studies point to DNA methylation as a feasible therapeutic target with the potential to mitigate a variety of CNS pathologies.
As the field of epigenetics expands its understanding of the role of DNA methylation in neurodevelopment and disease, the challenge of moving DNA methylation towards a therapeutic target is performing not only correlative, but causative studies that define specific gene targets and sites. Additionally, surveying changes in DNA methylation specific to brain region and cell type remains an ongoing and time worthy challenge unique to the field of neuroepigenetics. This protocol utilizes a variety of techniques including fluorescence-activated cell sorting (FACS) of astrocytes, methylation-sensitive high resolution melt analysis (MS-HRM), and a methylation luciferase assay to investigate the DNA methylation status of KCNJ10, a gene that encodes for Kir4.1. Kir4.1 is a glial specific potassium channel that demonstrates both brain region and cell specific patterns of expression in the CNS 12-16. Kir4.1 expression increases moving from rostral to caudal CNS regions, with the highest expression occurring in the spinal cord 15. Although the channel is expressed in ependymal cells, oligodendrocytes and their precursor cells, Kir4.1 is predominantly expressed in astrocytes and thought to be essential for maintaining homeostatic levels of potassium as well as supporting glutamate uptake by setting the astrocytic resting membrane potential at a hyperpolarized -80mV 12,16-19. Importantly, the expression of Kir4.1 is non-static both during development and following multiple forms of CNS injury 20-25. We wished to examine the epigenetic regulation of this channel, specifically in astrocytes during development. The techniques utilized offer gene-specific and targeted CpG site analyses that provide causal evidence for a role of DNA methylation in regulating KCNJ10 gene expression. These techniques can be applied to other genes.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td></td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>80204</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl Primer</td>
			<td>Applied Biosystems</td>
			<td>online</td>
			<td>software to localize CpG Islands</td>
		</tr>
		<tr>
			<td>EZ DNA methylation Kit</td>
			<td>Zymo Research</td>
			<td>D5001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Premixed Calibration Standard</td>
			<td>EpiGenDx</td>
			<td>80-8060R-Premix</td>
			<td></td>
		</tr>
		<tr>
			<td>CpG Methylase (M.Sssl)</td>
			<td>Zymo Research</td>
			<td>E2010</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction&#160;</td>
			<td>QIagen</td>
			<td>28704</td>
			<td>Used for gel extraction and DNA cleanup</td>
		</tr>
		<tr>
			<td>Restriction enzymes</td>
			<td>New England BioLabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NEB cutter</td>
			<td>New England BioLabs</td>
			<td>online</td>
			<td>verify restriction digest sites</td>
		</tr>
		<tr>
			<td>Dual Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>Luc2 vector, pGL4.10</td>
			<td>Promega</td>
			<td>E6651</td>
			<td></td>
		</tr>
		<tr>
			<td>renilla vector, pGL4.74</td>
			<td>Promega</td>
			<td>E2241</td>
			<td></td>
		</tr>
		<tr>
			<td>TD-20/20 Luminometer</td>
			<td>Turner Designs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine LTX and Plus Reagent</td>
			<td>Life Technologies</td>
			<td>A12621</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol, saturated pH 6.6/6.9</td>
			<td>Fisher Scientific</td>
			<td>BP 17501-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000/2000c Spectrophtometer</td>
			<td>ThermoScientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MeltDoctor Master Mix</td>
			<td>Life Technologies</td>
			<td>4415440</td>
			<td></td>
		</tr>
		<tr>
			<td>High Resolution Melt (HRM) Software v2.0</td>
			<td>Life Technologies</td>
			<td>4397808</td>
			<td></td>
		</tr>
		<tr>
			<td>AB SDS software v2.3</td>
			<td>Life Technologies</td>
			<td>online</td>
			<td></td>
		</tr>
		<tr>
			<td>AB High Resolution Melting Getting Started Guide&#160;</td>
			<td>Life Technologies</td>
			<td>online</td>
			<td></td>
		</tr>
		<tr>
			<td>AB 7900HT Fast Real-Time System</td>
			<td>Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

correlating gene-specific dna methylation changes with expression and transcriptional activity of astrocytic kcnj10 (kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Una popolazione arricchita di astrociti è stato acquisito tramite FACS ordinamento di eGFP-S100β animali transgenici 27. A causa della diminuzione della qualità delle cellule e molecole molecolari isolati da animali di età superiore a postnatale giorno 50 (p50), animali di età p0-P40 sono ottimali per tali esperimenti. Tessuto corticale è stato utilizzato per questi esperimenti. Cortecce da due a sei animali sono stati riuniti insieme. FACS è stata eseguita presso lo stabilimento UAB Comprehensive Cito...

Watch this Scientific Journal Video about Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1 at JoVE.com

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

Correlare DNA Modifiche metilazione gene-specifica con espressione e trascrizionale attività di astrociti<em&gt; KCNJ10</em&gt; (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Research

JoVE Journal

Biology

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Ricerca

Biologia

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Determinazione della metilazione del DNA di geni impressa Arabidopsis Dell&#39;endosperma

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

DNA Methylation: Bisulphite Modification and Analysis

Metilazione del DNA: Modifica bisolfito e analisi

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Cella singola Profiling trascrizionale dei cardiomiociti adulti mouse

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Un quantitativo test per studiare proteine: DNA interazioni, Discover trascrizionali regolatori dell&#39;espressione genica, e identificare nuovi agenti anti-tumorali

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

In vitro Methylation Assay to Study Protein Arginine Methylation

In vitro Methylation Assay to Study Protein Arginine Methylation

In vitro metilazione Assay per studiare proteine ​​Arginina Metilazione

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

ENHANCED ridotto Rappresentanza bisolfito sequenziamento per la valutazione della metilazione del DNA in Risoluzione coppia di basi

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

Targeted DNA metilazione Analisi per sequenziamento di prossima generazione

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...