Epigenetics and Gene Expression in Cancer, Inflammatory and Immune Diseases (Methods in Pharmacology and Toxicology)

This practical collection examines methodologies originating from the benefits of genome-wide approaches to studying epigenetics, which has opened the emerging field of epigenomics. Focusing on the areas of cancer, inflammatory and autoimmune disorders, chapters discuss three main components of the epigenome and their role in the regulation of gene expression and present a detailed method section specific to studying each component, including data analyses, troubleshooting, and feasibility in different experimental settings. The main topics are high-throughput and targeted methods for DNA methylation analysis, nucleosome position mapping, studying epigenetic effects of gut microbiota, optical imaging for detection of epigenetic aberrations in living cells, methods for microRNA, and histone code profiling. Written for the __Methods in Pharmacology and Toxicology__ series, the book includes the kind of detail and implementation advice to encourage success in the lab. Authoritative and easily applicable, Epigenetics and Gene Expression in Cancer, Inflammatory and Immune Diseases aims to provide pharmacologists, molecular biologists, bioinformaticians, and toxicologists with a vital background on epigenetics and state-of-the-art techniques in epigenomics. Preface 5 Contents 8 Contributors 9 Chapter 1: High-Throughput Techniques for DNA Methylation Profiling 11 1 Introduction 11 2 Methylated DNA Immunoprecipitation (MeDIP) 13 2.1 MeDIP-ChIP 15 2.2 MeDIP-Seq 16 3 Reduced Representation Bisulfite Sequencing 16 4 Infinium HumanMethylation450 BeadChip Array 18 4.1 Technical Requirement 18 4.2 Illumina 450 K Array Overview 19 4.3 Illumina 450 K Data Analysis 20 5 Comparison of Methods 21 6 Conclusion 22 References 23 Chapter 2: Reduced Representation Bisulfite Sequencing (RRBS) and Cell Sorting Prior to DNA Methylation Analysis in Psychiatric Disorders 26 1 Introduction 26 2 Epigenetic Mechanisms in Psychiatric Disorders 27 3 Reduced Representation Bisulfite Sequencing (RRBS) 28 4 Comparison of RRBS and Other Methods for Methylome Analysis 28 5 Application of RRBS 29 6 RRBS: Laboratory Procedures 30 6.1 Restriction Digest 30 6.1.1 Summary 30 6.1.2 Sample Protocol 31 6.2 End Repair and A-tailing 31 6.2.1 Summary 31 6.2.2 Sample Protocol 32 6.3 Adapter Ligation and Bisulfite Conversion 32 6.3.1 Summary 32 6.3.2 Sample Protocol 32 6.4 Size Selection 32 6.4.1 Summary 32 6.4.2 Sample Protocol 32 6.5 Library Preparation Using PCR 33 6.5.1 Summary 33 6.5.2 Sample Protocol 33 7 RRBS: Sequencing and Analysis 34 8 Limitations of RRBS 34 9 Cell Type Selection, Heterogeneity, and Its Effect on Epigenomic Data 35 10 FACS: Laboratory Procedures 36 10.1 Antibody Selection 36 10.1.1 Summary 36 10.2 Staining Cells for FACS 37 10.2.1 Summary 37 10.2.2 Sample Protocol 37 10.3 Cell Sorting 37 10.3.1 Summary 37 11 Computational Alternative to Cell Sorting 38 12 Conclusion 38 References 39 Chapter 3: Targeted DNA Methylation Analysis Methods 42 1 Introduction 42 2 DNA Methylation Analysis by Pyrosequencing 43 2.1 Pyrosequencing Overview 43 2.2 Genomic DNA Preparation and DNA Concentration Measurement 44 2.3 DNA Bisulfite Treatment 45 2.4 Quantifying Bisulfite-­Treated DNA 46 2.5 Primer and Assay Design 47 2.6 PCR of Bisulfite-­Treated DNA and Pyrosequencing 48 2.7 Quantitative Methylated DNA Immunoprecipitation (qMeDIP) 50 2.8 Primer Design 52 2.9 Analysis 52 2.10 High Resolution Melting for Data Validation of Genome-­Wide DNA Methylation Studies 52 2.11 Protocol 53 2.12 Primer Design 54 2.13 Input and Setup 55 2.14 Data Analysis 56 2.15 Conclusion for MS-HRM 57 References 58 Chapter 4: Analyzing Targeted Nucleosome Position and Occupancy in Cancer, Obesity, and Diabetes 60 1 Introduction 60 1.1 Role of Epigenetics and Chromatic Structure in Cancer, Obesity, and Diabetes 60 2 Materials 62 2.1 Equipment 62 2.2 General Materials for All Applications 62 2.3 Materials 63 2.3.1 Mononucleosomal DNA Isolation and Purification 63 2.3.2 Scanning qPCR 63 2.4 Buffers 63 2.4.1 Sucrose Buffer (100 mL). 63 2.4.2 Digestion Buffer (50 mL) 63 2.4.3 Lysis Buffer (50 mL) 63 2.4.4 0.2 M EDTA (1 mL) 63 3 Methods 64 3.1 Sucrose Buffer Preparation 64 3.2 Digestion Buffer Preparation 64 3.3 Lysis Buffer Preparation 64 3.4 Mono­nucleosomal DNA Isolation and Purification 65 3.4.1 Frozen Tissue Sample Preparation 65 3.4.2 Nuclear and Chromatin Isolation 65 3.4.3 Mononucleosomal DNA Fragmentation and Purification 67 3.5 Scanning qPCR 68 4 Notes 68 References 70 Chapter 5: Synthesis and Application of Cell-Permeable Metabolites for Modulating Chromatin Modifications Regulated by α-Ketoglutarate-Dependent Enzymes 72 1 Introduction 72 2 Materials 75 2.1 Synthesis of Octyl-R-2-­hydroxyglutarate or Octyl-L-2-­ hydroxyglutarate 75 2.2 Cell Culture and Treatment with Octyl-R-2-­hydroxyglutarate or Octyl-L-2-­hydroxyglutarate 76 2.3 Mammalian Whole Cell Extracts 77 2.4 Protein Blotting 77 3 Methods 78 3.1 Synthesis of Octyl-R-2-­hydroxyglutarate 78 3.1.1 Synthesis of (R)-2-amino-5-(benzyloxy)-5-­oxopentanoic Acid 78 3.1.2 Synthesis of 5-benzyl 1-octyl (R)-2- 79 3.1.3 Synthesis of (R)-4-hydroxy-­5-(octyloxy)-5-­oxopentanoic Acid 80 3.2 Application of Octyl-R-2-­hydroxyglutarate or Octyl-L-2-­hydroxyglutarate to Inhibit Histone Demethylation in Mammalian Cells 81 3.3 Protein Extraction from Mammalian Cells 82 3.4 Monitoring Histone Methylation by Immunoblotting 82 3.4.1 Electrophoresis and Protein Transfer 82 3.4.2 Immunoblotting 83 3.4.3 Stripping Membranes 84 4 Notes 85 References 86 Chapter 6: High-Throughput Screening of Small Molecule Transcriptional Regulators in Embryonic Stem Cells Using qRT-PCR 89 1 Small Molecule-Mediated Regulation of Transcription 89 2 Designing the qRT-PCR Assay 91 2.1 Selection of a Reporter Gene 91 2.2 Validation of the Reporter Gene 91 3 Optimizing the Cells-to-CTTM qRT-PCR Assay 91 3.1 Determine Timeline 92 3.2 Confirming Robust Readout Using HTS System 92 3.3 Selection of the Housekeeping Gene and Multiplexing 92 3.4 Primer Efficiency 92 3.5 Determine Assay Range 94 3.6 Taqman Probe Concentration 94 3.7 Measuring the Robustness of the Assay 94 3.8 Pilot Screen 95 4 Cell Culture 95 4.1 Making Feeder Free ES cell lines 96 4.2 Plating Feeder Free ES Cells for HTS Efforts 97 5 Compound Library and Screening 98 5.1 Data Analysis 100 5.2 High-Throughput qRT-PCR Screen 100 6 Confirmatory and Secondary Screens 100 6.1 Confirmation of Hits Using the Primary qRT-PCR Assay 100 6.2 Secondary Screen Using SYBR qRT-PCR 101 6.3 Validation of Additional Transcriptional Targets 101 7 Conclusions 102 8 Resources 102 References 103 Chapter 7: Methods for MicroRNA Profiling in Cancer 104 1 Introduction 104 2 Materials for In Situ Hybridization 109 3 Method for In Situ Hybridization 112 3.1 Deparaffinization and Rehydration of Tissue 112 3.2 Proteinase K Digestion, Fixation, Acetylation, and Permeabilization of Tissues 113 3.3 Hybridization 113 3.4 TSA Green Reaction 114 3.5 TSA Red Reaction 114 3.6 TSA Blue Reaction 115 3.7 Nuclear Marker Detection and Mounting of Slides 115 4 Notes 116 References 118 Chapter 8: Microbiota and Epigenetic Regulation of Inflammatory Mediators 121 1 Analysis of Microbiota and Microbial Epigenetic Active Products 122 1.1 Sample Preparation: DNA and RNA Extraction 123 1.2 Purity Control of DNA and RNA 125 1.3 Real-Time Polymerase Chain Reaction (qPCR) 125 1.4 Denaturing Gradient Gel Electrophoresis (DGGE) 126 1.4.1 Amplification 127 1.4.2 Ethanol Precipitation and Resuspension 127 1.4.3 DGGE Gel Preparation 127 1.4.4 DGGE 128 2 Analysis of Gene Expression 128 2.1 Sample Processing: RNA 129 3 Analysis of Epigenetic Modifications 130 3.1 Sample Processing: DNA 131 3.2 Detection of Promoter-­Specific Methylation Status: Pyrosequencing 132 3.3 High-Throughput Analysis of Epigenetic Modifications 135 3.3.1 HIFI Technology Shows Several Assets 136 4 Whole Genome Approaches 136 4.1 Combination of Methylation and Histone Analyses 137 4.2 Pathway Analysis and Statistical Modelling 138 References 138 Chapter 9: Optical Microscopy and Spectroscopy for Epigenetic Modifications in Single Living Cells 141 1 Complexity of Probing Epigenetic Modifications in Single Living Cells 141 2 Instrumentation of Modern Optical Microscopy 142 3 General Rules in Live-Cell Imaging 143 3.1 Phototoxicity 143 3.2 Environmental Factors 145 4 Epigenetic Information in Single Living Cells Enabled by Optical Microscopy 146 4.1 Localization and Interaction 147 4.1.1 Super-Resolution Microscopy 147 4.1.2 Förster Resonance Energy Transfer (FRET) 148 4.1.3 Gene-Specific Epigenetic Profiling and 3D Imaging 148 4.2 Quantity and Stoichiometry 149 4.2.1 Fluorescence Fluctuation Spectroscopy 149 4.3 Dynamics and Kinetics 151 5 Representative Applications in Probing Real-Time Epigenetics 151 5.1 DNA Methylation 152 5.1.1 Practical Advice for Live-Cell FCS Experiments 154 5.2 Histone Modification 155 5.2.1 Practical Advice for FRET Analysis 155 5.3 Chromatin Dynamics 157 6 Future Prospects 158 References 159 Chapter 10: MicroRNAs in Therapy and Toxicity 161 1 Introduction 161 1.1 Role of miRNAs 161 1.2 miRNAs in Human Disease 162 1.3 miRNAs in Models of Drug-Induced Toxicity 165 2 Materials 168 3 Methods 168 3.1 Identifying miRNA Expression 168 3.2 miRNA Regulation 169 3.3 Manipulating miRNA Expression 170 4 Notes 171 References 171 Chapter 11: Genetics and Epigenetics of Multiple Sclerosis 174 1 Introduction 174 2 Genome-Wide Genetic Surveys in Multiple Sclerosis 175 3 Whole-Exome Sequencing in Familiar Multiple Sclerosis 178 4 Transcriptome Studies in Multiple Sclerosis 179 5 Epigenetics in Multiple Sclerosis 184 6 Integrative View of Results from Omic Studies in Multiple Sclerosis 188 7 Conclusion 190 References 191 Index 198 This practical collection examines methodologies originating from the benefits of genome-wide approaches to studying epigenetics, which has opened the emerging field of epigenomics. Focusing on the areas of cancer, inflammatory and autoimmune disorders, chapters discuss three main components of the epigenome and their role in the regulation of gene expression and present a detailed method section specific to studying each component, including data analyses, troubleshooting, and feasibility in different experimental settings. The main topics are high-throughput and targeted methods for DNA methylation analysis, nucleosome position-mapping, studying epigenetic effects of gut microbiota, optical imaging for detection of epigenetic aberrations in living cells, methods for microRNA, and histone code-profiling. Written for the Methods in Pharmacology and Toxicology series, the book includes the kind of detail and implementation advice to encourage success in the lab. Authoritative and easily applicable, Epigenetics and Gene Expression in Cancer, Inflammatory and Immune Diseases aims to provide pharmacologists, molecular biologists, bioinformaticians, and toxicologists with a vital background on epigenetics and state-of-the-art techniques in epigenomics.-- Source other than Library of Congress "This practical collection examines methodologies originating from the benefits of genome-wide approaches to studying epigenetics, which has opened the emerging field of epigenomics. Focusing on the areas of cancer, inflammatory and autoimmune disorders, chapters discuss three main components of the epigenome and their role in the regulation of gene expression and present a detailed method section specific to studying each component, including data analyses, troubleshooting, and feasibility in different experimental settings. The main topics are high-throughput and targeted methods for DNA methylation analysis, nucleosome position mapping, studying epigenetic effects of gut microbiota, optical imaging for detection of epigenetic aberrations in living cells, methods for microRNA, and histone code profiling. Written for the Methods in Pharmacology and Toxicology series, the book includes the kind of detail and implementation advice to encourage success in the lab. Authoritative and easily applicable, Epigenetics and Gene Expression in Cancer, Inflammatory and Immune Diseases aims to provide pharmacologists, molecular biologists, bioinformaticians, and toxicologists with a vital background on epigenetics and state-of-the-art techniques in epigenomics." Springer