Enzymes of Epigenetics Part B

 
 
Academic Press
  • 1. Auflage
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  • erschienen am 14. Juli 2016
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  • 440 Seiten
 
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978-0-12-811902-0 (ISBN)
 

Enzymes of Epigenetics: Part B, one of two new volumes in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field.

This volume covers research methods that are employed in the study of epigenetic regulation, including structural, biochemical, molecular, biological, cellular, computational, and systems approaches.

Topics include chromatin structure and histones, posttranslational histone modification enzymes and complexes, histone modification binders, DNA modifications and nucleic acid regulators, epigenetic technologies, and small molecule epigenetic regulators and biological connections.

  • Continues the legacy of this premier serial with quality chapters authored by leaders in the field
  • Contains two new volumes that cover research methods in enzymes of epigenetics
  • Covers such topics as chromatin structure and histones, posttranslational histone modification enzymes and complexes, histone modification binders, DNA modifications and nucleic acid regulators, epigenetic technologies and small molecule epigenetic regulators, and biological connections
0076-6879
  • Englisch
  • San Diego
  • |
  • USA
Elsevier Science
  • 23,45 MB
978-0-12-811902-0 (9780128119020)
0128119020 (0128119020)
weitere Ausgaben werden ermittelt
  • Front Cover
  • Enzymes of Epigenetics, Part B
  • Copyright
  • Contents
  • Contributors
  • Preface
  • Part I: Epigenetic Technologies
  • Chapter One: Identification and Quantification of Histone PTMs Using High-Resolution Mass Spectrometry
  • 1. Introduction
  • 2. Histone Extraction from Cells
  • 2.1. Materials and Buffer Recipes
  • 2.2. Cell Harvest
  • 2.2.1. Cell Harvest: Tissue Samples
  • 2.2.2. Cell Harvest: Cell Cultures
  • 2.3. Nuclei Isolation
  • 2.4. Acid Extraction
  • 3. Bottom-Up Mass Spectrometry
  • 3.1. Materials and Buffer Recipes
  • 3.2. Derivatization and Digestion
  • 3.3. Desalting
  • 3.4. Online RP-HPLC and MS Acquisition
  • 3.5. Data Analysis
  • 3.5.1. Software-Based Peak Area Extraction and Abundance Calculation
  • 4. Offline Fractionation of Histone Species
  • 4.1. Materials and Buffer Recipes
  • 4.2. Histone Variant Purification
  • 5. Middle-Down Mass Spectrometry
  • 5.1. Materials and Buffer Recipes
  • 5.2. Digestion
  • 5.2.1. GluC
  • 5.2.2. AspN (Alternative to GluC)
  • 5.3. WCX-HILIC and MS
  • 5.4. Data Analysis
  • 6. Top-Down Mass Spectrometry
  • 6.1. Materials and Buffer Recipes
  • 6.2. Top-Down MS Using Direct Infusion
  • 6.3. Data Analysis
  • References
  • Chapter Two: Substrate Specificity Profiling of Histone-Modifying Enzymes by Peptide Microarray
  • 1. Introduction
  • 2. Assay Optimization
  • 2.1. Design of Custom Print Formats
  • 2.2. Detection Reagent Considerations
  • 2.2.1. Radioisotopes
  • 2.2.2. Antibodies
  • 3. Assay Methodology
  • 3.1. Microarray-Based Lysine Methyltransferase (KMT) Assays with G9a
  • 3.2. Solution-Based KMT Assays with G9a
  • 3.3. Microarray-Based Lysine Demethylase (KDM) Assays with JMJD2A
  • 4. Enzyme Specificity Profiling by Microarray
  • 4.1. High-Throughput Profiling of G9a KMT Activity
  • 4.2. High-Throughput Profiling of JMJD2A KDM Activity
  • 5. Summary and Perspectives
  • Acknowledgments
  • References
  • Chapter Three: ArrayNinja: An Open Source Platform for Unified Planning and Analysis of Microarray Experiments
  • 1. Introduction
  • 2. ArrayNinja
  • 2.1. Obtaining and Running ArrayNinja
  • 2.2. Overview of the ArrayNinja Database
  • 3. Planning Custom Microarrays with ArrayNinja
  • 4. Data Analysis Features of ArrayNinja
  • 4.1. Microarray Image Preparation
  • 4.2. Quantification of Microarray Data
  • 5. Benchmarking ArrayNinja Against ImageQuant TL
  • 5.1. Homogeneous Noise
  • 5.2. Inhomogeneous Noise
  • 5.3. Implicit Assumption of Local Noise Correction
  • 6. Methodological Details
  • 6.1. Default (Whole Spot) Quantification
  • 6.2. Nonlocal Noise Thresholding
  • 6.3. Local Noise Thresholding
  • 6.4. Variegated Spot Morphology
  • 7. Limitations, Assumptions, Other Features, and Future Development
  • 7.1. Limitations and Assumptions
  • 7.2. Other Features
  • 7.3. Future Development
  • 8. Summary
  • Acknowledgments
  • References
  • Chapter Four: Chemical Biology Approaches for Characterization of Epigenetic Regulators
  • 1. Introduction
  • 1.1. Chemical Probes and Their Use in Biology
  • 1.2. Epigenetics and Protein Methyltransferases
  • 2. Validation of Chemical Probes for Use in Cell-Based Experiments
  • 2.1. Requirements for Chemical Probes
  • 2.2. Biomarker Assays: How to Ensure Your Chemical Probe Is Active in Cells
  • 2.3. Assay Readout Choice
  • 2.4. Biomarker Assays for PMTs
  • 2.4.1. Protocol 1-In-Cell Western Assay for Histone/Protein Mark Detection
  • 2.4.2. Protocol 2-Exogenous PMT Cell-Based Biomarker Assay
  • 2.4.3. Example1: Enzyme Overexpression Assay
  • 2.4.4. Example2: Enzyme/Substrate Co-Overexpression Assay
  • 2.5. Timing and Other Considerations for Cell-Based Overexpression Assays
  • 3. Inhibitor Enabled Discovery
  • 3.1. Inhibitor Handling and Inhibitor Libraries
  • 3.2. Phenotypic Assays Using Chemical Probes
  • 3.3. Demonstration of On-Target Phenotypic Effects
  • 3.3.1. Protocol 3: Measuring Early Toxicity/Apoptosis Response to Chemical Probes Using Incucyte
  • 3.3.2. Example 3: Target Validation with Multiple Probes Per Target
  • 4. Conclusions
  • Acknowledgments
  • References
  • Chapter Five: Mapping Lysine Acetyltransferase-Ligand Interactions by Activity-Based Capture
  • 1. Introduction
  • 2. Technical Aspects
  • 2.1. Synthesis of KAT Capture Probes
  • 2.1.1. Materials
  • 2.1.2. Synthesis of Alkyne-Modified KAT Bisubstrate Inhibitors
  • 2.1.3. Synthesis of Biotinylated KAT Bisubstrate Inhibitors
  • 2.2. Preparation of Proteomes
  • 2.2.1. Materials
  • 2.2.2. Cell Lysate Preparation
  • 2.3. Activity-Based Capture of KATs and Competitive Profiling of KAT-Ligand Interactions
  • 2.3.1. Materials
  • 2.3.2. Enrichment of KATs in the Presence or Absence of Active Site Competitive Ligands
  • 2.4. Analysis of KAT-Ligand Interactions by Immunoblot
  • 2.4.1. Materials
  • 2.4.2. Elution and Western Blot Sample Preparation
  • 2.5. Analysis of KAT Capture by LC-MS/MS
  • 2.5.1. Materials
  • 2.5.2. On-Bead Trypsin Digest and LC-MS/MS Sample Preparation
  • 3. Discussion
  • 3.1. Critical Parameters and Troubleshooting
  • 3.2. Future Applications and Directions
  • References
  • Chapter Six: Investigating Histone Acetylation Stoichiometry and Turnover Rate
  • 1. Introduction
  • 2. Labeling and Methods for Sample Preparation
  • 2.1. General Experimental Design
  • 2.2. Incubating Cells with Isotopically Labeled Precursors
  • 2.3. Extracting Metabolites
  • 2.4. Extracting Histones
  • 3. Sample Analysis
  • 3.1. Analyzing Metabolite Labeling
  • 3.2. Analyzing Overall Histone Acetylation
  • 3.2.1. Digesting Histones into Single Amino Acids
  • 3.2.2. Analyzing Histone Digest by HPLC-MS
  • 3.3. Analyzing Site-Specific Acetylation Stoichiometry
  • 3.3.1. Acetylation Stoichiometry Sample Preparation for LC-MS/MS
  • 3.3.2. Peptide Cleanup Prior to MS Analysis
  • 3.3.2.1. Generating Desalting Tips
  • 3.3.2.2. Desalting Procedure
  • 3.3.3. LC-MS/MS Data Analysis
  • 3.4. Analyzing Site-Specific Acetylation-Labeling Kinetics
  • 3.4.1. Sample Preparation for LC-MS/MS
  • 3.4.2. LC-MS/MS Analysis for Histone Acetylation-Labeling Kinetics
  • 4. Data Analysis and Kinetic Modeling
  • 4.1. Analyzing Small-Molecule Data
  • 4.2. Analyzing Site-Specific Histone Acetylation
  • 4.3. Quantifying Histone Acetylation Turnover Rate
  • 5. Discussion and Perspective
  • Acknowledgments
  • References
  • Chapter Seven: Rapid Semisynthesis of Acetylated and Sumoylated Histone Analogs
  • 1. Introduction
  • 2. Materials and Methods
  • 2.1. General Materials and Methods
  • 3. Semisynthesis of Sumoylated Histone H4
  • 3.1. Overall Design of the Semisynthesis
  • 3.2. Preparation of Recombinant Histone H4 K12C
  • 3.3. Preparation of Recombinant SUMO-3-Aminoethanethiol
  • 3.4. Generation of Sumoylated Histone H4
  • 4. Preparation of Acetylated Histone H3 Analogs
  • 4.1. Overall Design of the Semisynthesis
  • 4.2. Preparation of Recombinant Histone H3
  • 4.3. Generation of Thialysine Analogs of Acetylated Histone H3
  • 5. Generation of Designer MNs
  • 5.1. Octamer Formation Using Modified Histones
  • 5.2. Generation of 147bp 601 DNA
  • 5.3. Reconstitution of MNs
  • 5.4. Preparation of 177bp Repeat 601 DNA
  • 5.5. Generation of 12-mer Nucleosome Arrays
  • 6. Summary and Conclusions
  • Acknowledgments
  • References
  • Chapter Eight: An IF-FISH Approach for Covisualization of Gene Loci and Nuclear Architecture in Fission Yeast
  • 1. Introduction
  • 2. Case Studies in the Application of the IF-FISH Approach
  • 2.1. Scoring Associations Between Two Gene Loci
  • 2.2. Visualizing Clusters of Centromeres and Telomeres
  • 2.3. Coordinating Gene Positioning to the Nuclear Architecture
  • 3. Supplies
  • 3.1. Equipment
  • 3.2. Materials
  • 3.3. Buffers
  • 4. Protocol
  • 4.1. Preparation of the FISH Probe Templates
  • 4.2. Fluorescent Labeling of the Templates
  • 4.3. Fixation of the Fission Yeast Cells
  • 4.4. Permeabilization of the Cells
  • 4.5. Antigen-Antibody Reactions (IF) and Fixation
  • 4.6. Hybridization (FISH)
  • 4.7. Preparation of the Cells for Microscopy
  • 5. Notes
  • Acknowledgments
  • References
  • Part II: Small Molecule Epigenetic Regulators
  • Chapter Nine: Biology, Chemistry, and Pharmacology of Sirtuins
  • 1. Introduction
  • 2. Sirtuins and Metabolism
  • 3. Sirtuins and Regulation of Cellular NAD+ Levels
  • 4. Sirtuin Functions
  • 5. Sirtuins and Metabolic Disorders
  • 6. Sirtuins and Cancer
  • 7. Sirtuin Activity Assays
  • 8. Identification of First-Generation Sirtuin Inhibitors
  • 9. Second-Generation Splitomicin Inhibitors
  • 10. Other Sirtuin Inhibitors
  • 11. Concluding Remarks
  • References
  • Chapter Ten: Synthesis and Assay of SIRT1-Activating Compounds
  • 1. Introduction
  • 2. Materials
  • 2.1. Synthesis of SIRT1-Activating Compounds
  • 2.2. Expression and Purification of Recombinant His-Tagged SIRT1
  • 2.3. Assay of SIRT1 Activators Using the PNC1-OPT Assay
  • 2.4. Assay of SIRT1 Activators Using the RapidFire Mass Spectrometry Assay
  • 3. Methods
  • 3.1. Synthesis of SIRT1-Activating Compounds
  • 3.1.1. Preparation of a Imidazo[1,2-b]thiazole STAC (Synthesis Adapted from Milne et al., 2007)
  • 3.1.2. Preparation of a Thiazolopyridine STAC (Synthesis Adapted from Dai et al., 2010)
  • 3.1.3. Preparation of an Imidazo[4,5-c]pyridine STAC (Synthesis Adapted from Hubbard, Gomes, et al., 2013)
  • 3.1.4. Preparation of a Bridged-Urea STAC (Synthesis Adapted from Dai et al., 2015)
  • 3.2. Expression and Purification of Recombinant His-Tagged SIRT1
  • 3.3. Assay of SIRT1 Activators Using the PNC1-OPT Assay
  • 3.4. Assay of SIRT1 Activators Using the RapidFire O-Ac-ADPR Detection Assay
  • 4. Notes
  • Acknowledgments
  • References
  • Chapter Eleven: Synthesis and Assays of Inhibitors of Methyltransferases
  • 1. Introduction to Methyltransferases
  • 2. Designing and Synthesizing Inhibitors of Methyltransferases
  • 2.1. Overview of Methyltransferase Inhibitors
  • 2.2. Pan-Inhibitors
  • 2.3. Target-Selective Inhibitors of Methyltransferases
  • 2.3.1. 5'-Alkylthio SAM Analogs as Protein and DNA Methyltransferase Inhibitors
  • 2.3.2. 5'-Alkoxy SAM Analogs as COMT Inhibitors
  • 2.3.3. 5'-Alkyl/Alkenyl SAM Analogs as Methyltransferase Inhibitors
  • 2.3.4. 5'-Aminoalkyl SAM Analogs (Sinefungin Analogs) as PKMT Inhibitors
  • 2.3.5. 5'-Amino SAM Analogs as PMT Inhibitors
  • 3. Evaluating Methyltransferase Inhibitors
  • 3.1. Assay Formats
  • 3.2. Radiometric Assays
  • 3.2.1. Filter Paper-Based Scintillation Assay
  • 3.2.2. Scintillation Proximity Assay
  • 3.3. Fluorescence-Based Detection of Methyltransferase Activity
  • 3.4. Directly Monitoring SAH Formation
  • 3.5. Mass Spectrometry-Based Detection of Methylated Product
  • 3.6. Detailed Protocol for Radiometric Filter Paper Assay
  • 3.6.1. Materials
  • 3.6.2. Preliminary Test for Methyltransferase Activity
  • 3.6.3. Measurement of Steady-State Kinetic Parameters
  • 3.6.4. Evaluating Potency of Inhibitors In Vitro
  • 4. Conclusion
  • References
  • Part III: Epigenetics and Biological Connections
  • Chapter Twelve: Exploring the Dynamic Relationship Between Cellular Metabolism and Chromatin Structure Using SILAC-Mass S ...
  • 1. Introduction
  • 2. Analyzing the Turnover Dynamics of Histone Modifications
  • 2.1. SILAC-Mass Spec Methodology
  • 2.2. Methyl-SILAC
  • 2.3. Analysis of Histone Methylation Dynamics Using Methyl-SILAC
  • 2.3.1. Heavy-Labeled Medium Preparation
  • 2.3.2. Mammalian Cell Culture and Methyl-SILAC Labeling
  • 2.3.3. Methyl-SILAC Peptide Quantification and Kinetics Modeling
  • 2.4. Acetyl-SILAC
  • 2.5. Limitations of SILAC-Mass Spec
  • 3. Genome-Wide Mapping of Histone Modifications
  • 3.1. Charting the Epigenetic Landscape by ChIP-seq
  • 3.2. ChIP-Materials and Buffer Recipes
  • 3.3. ChIP-Cell Harvest and Cross-linking
  • 3.4. ChIP Day 1 | Bead Preparation, Cell Lysis, and Sonication, Setting Up the IP
  • 3.4.1. Bead Preparation
  • 3.4.2. Cell Lysis
  • 3.4.3. Setting Up the IP
  • 3.5. ChIP Day 2 | Washes, Elution, and Reverse Cross-Linking
  • 3.5.1. Washes
  • 3.5.2. Elution
  • 3.5.3. Reverse Cross-linking
  • 3.6. ChIP Day 3 | DNA Purification
  • 3.7. ChIP-seq Library Preparation
  • 3.8. Computational Pipeline and Considerations in ChIP-seq
  • 4. Summary
  • References
  • Chapter Thirteen: Current Proteomic Methods to Investigate the Dynamics of Histone Turnover in the Central Nervous System
  • 1. Introduction
  • 2. Early Methods to Study Histone Turnover in Brain
  • 2.1. The Introduction of Radioactive Tracers
  • 2.2. Limitations of Radioactive Labeling
  • 3. Current Proteomic Methods to Study Histone Turnover in Brain
  • 3.1. Mass Spectrometry: A Brief Introduction
  • 3.1.1. Bottom Up vs Top Down
  • 3.2. Sample Preparation Considerations
  • 3.3. Label Free vs Stable Isotope Incorporation
  • 3.4. Acquisition Types
  • 3.5. Mass Spectrometry-Based Methods to Study Histone Turnover in Brain
  • 4. Retrospective Birth Dating of Histones in Human Postmortem Brain
  • 4.1. ``Bomb Pulse Labeling´´ Coupled to Accelerator Mass Spectrometry
  • 4.2. Bomb Pulse Labeling: Analytical Considerations
  • 5. Conclusion
  • 6. Methodology: Preparing Chromatin from Neurons for Mass Spectrometry Analysis of Histone Variants and Turnover
  • Acknowledgments
  • References
  • Chapter Fourteen: ChIP-Sequencing to Map the Epigenome of Senescent Cells Using Benzonase Endonuclease
  • 1. Introduction
  • 2. Buffer Compositions
  • 3. Protocol
  • 3.1. Antibody Bead Preparation
  • 3.2. Cell Culture, Lysis, and ChIP
  • 4. Conclusion
  • References
  • Chapter Fifteen: Exploiting Chromatin Biology to Understand Immunology
  • 1. Introduction: Design Principle of Immune Responses
  • 2. Epigenome: Our Software
  • 3. Mapping DNA Methylation
  • 4. Mapping Histone Modifications by Chromatin Immunoprecipitation and Sequencing
  • 5. Limitations of ChIP-seq
  • 6. MNase-seq for Nucleosome Positioning
  • 7. DNase-seq and ATAC-seq for DNA Accessibility
  • 8. Analysis of NGS Data
  • 9. Painting an Enhancer Landscape
  • 10. Chromatin Biology to Understand Gene Regulation in Immune Cells
  • 11. Role of Intrinsic and Extrinsic Signals on Enhancer Formation
  • 12. Conclusions
  • Acknowledgments
  • References
  • Author Index
  • Subject Index
  • Color Plate
  • Back Cover

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