Correleren Gen-specifieke DNA-methylatie Wijzigingen met Expression en transcriptionele activiteit van de astrocyten KCNJ10 (Kir4.1)

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

Alle dieren werden behandeld volgens de National Institutes of Health richtlijnen. De Animal Care en gebruik Comite aan de Universiteit van Alabama in Birmingham goedgekeurde gebruik van dieren.

1. Het verkrijgen van RNA en DNA van een verrijkte astrocyten Bevolking met behulp van TL Activated Cell Sorting (FACS) van Astrocyten van Whole Brain Tissue

1. Bezadigd dier met CO ₂ gedurende 1 minuut en vervolgens snel onthoofden. Ontleden cortex zoals beschreven in Albuquerque et al, ^26.; Het is niet noodzakelijk hersenvliezen verwijderd.
   Opmerking: Transgene ratten uitdrukken eGFP onder de S100β (....

Results

Een verrijkte populatie van astrocyten werd verworven via FACS sortering van eGFP-S100β transgene dieren ^27. Door afnemende kwaliteit van cellen en moleculair molecules die uit dieren ouder dan postnatale dag 50 (p50), dieren p0-p40 ouder zijn optimaal voor dergelijke experimenten. Corticaal weefsel werd gebruikt voor deze experimenten. Cortex vanaf 2 om 6 dieren werden samengevoegd. FACS werd uitgevoerd bij UAB Comprehensive flowcytometrie Core faciliteit. Sorteren werd uitgevoerd op Becton Dickinson FACSAri.......

Discussion

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.......

Materials

List of materials used in this article

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