Epigenetic Methodologies for the Study of Celiac Disease.
Epigenetic regulation of gene expression is an important event for normal cellular homeostasis. Gene expression may be "switched" on or "turned" off via epigenetic means through adjustments in the architecture of DNA. These structural alterations result from histone posttranslation modifications such as acetylation and methylation on key arginine and lysine residues, or by alterations to DNA methylation. Other known epigenetic mechanisms invoke histone variant exchange or utilize noncoding RNAs (lncRNA/miRNA). Drugs which can target the epigenetic regulatory machinery are currently undergoing clinical trials in a wide variety of autoimmune diseases and cancer. Here we describe RNA isolation and the subsequent Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) methods, post-epigenetic drug treatment, to identify genes, which may be responsive to such epigenetic targeting agents. In addition, we depict a chromatin immunoprecipitation (ChIP) assay to determine the association between chromatin transcription markers and DNA following pretreatment of cell cultures with a histone deacetylase inhibitor (HDi). This assay allows us to determine whether treatment with an HDi dynamically remodels the promoter region of genes, as judged by the differences in the PCR product between our treated and untreated samples. Finally we describe two commonly used methodologies for analyzing DNA methylation. The first, methylation-sensitive high resolution melt analysis (MS-HRM) is used for methylation screening of regions of interest, to identify potential epigenetic "hotspots." The second, quantitative methylation specific PCR (qMSP) is best applied when these hotspots are known, and offers a high-throughput, highly sensitive means of quantifying methylation at specific CpG dinucleotides.
Prostate Molecular Oncology, Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland.
Antoinette S
AS
Perry
Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland.|Thoracic Oncology Research Group, Institute of Molecular Medicine, Trinity Centre for Health Science, St. James's Hospital, Dublin 8, Ireland.|Cancer & Ageing Research Program, Queensland University of Technology, Brisbane, QLD, Australia.
Anne-Marie
AM
Baird
Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland. sgray@stjames.ie.|Thoracic Oncology Research Group, Institute of Molecular Medicine, Trinity Centre for Health Science, St. James's Hospital, Dublin 8, Ireland. sgray@stjames.ie.|HOPE Directorate, St. James's Hospital, Dublin, Ireland. sgray@stjames.ie.
Steven G
SG
Gray
D002446:Celiac Disease / Q000235:genetics*; D018899:CpG Islands; D044127:Epigenesis, Genetic; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction
Chromatin immunoprecipitation; DNA methylation; Histone deacetylase; RNA; RT-PCR