Enzymes of Epigenetics

 
 
Academic Press
  • 1. Auflage
  • |
  • erschienen am 29. Juni 2016
  • |
  • 528 Seiten
 
E-Book | ePUB mit Adobe DRM | Systemvoraussetzungen
E-Book | PDF mit Adobe DRM | Systemvoraussetzungen
978-0-12-805432-1 (ISBN)
 

Enzymes of Epigenetics, 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 to study epigenetic regulation and includes 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 covering research methods in enzymes of epigenetics
  • Covers topics such 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
  • Saint Louis
  • |
  • USA
Elsevier Science
  • 36,03 MB
978-0-12-805432-1 (9780128054321)
0128054328 (0128054328)
weitere Ausgaben werden ermittelt
  • Front Cover
  • Enzymes of Epigenetics, Part A
  • Copyright
  • Contents
  • Contributors
  • Preface
  • Part I: Chromatin Structure and Histones
  • Chapter One: In Vitro Chromatin Assembly: Strategies and Quality Control
  • 1. Introduction
  • 2. Chromatin Reconstitution Strategies
  • 2.1. Fluorescent Labeling of the Histones and Refolding into Octamer
  • 2.1.1. Equipment
  • 2.1.2. Buffers and Reagents
  • 2.1.3. Procedure
  • 2.1.4. Notes
  • 2.2. End-Labeling of DNA
  • 2.2.1. Equipment
  • 2.2.2. Buffers and Reagents
  • 2.2.3. Procedure
  • 2.2.4. Notes
  • 2.3. Nucleosome Assembly
  • 2.3.1. Procedure
  • 2.3.2. Notes
  • 2.4. Trinucleosome Assembly
  • 2.4.1. Equipment
  • 2.4.2. Buffers and Reagents
  • 2.4.3. Procedure
  • 2.4.4. Notes
  • 2.5. ``Microscale´´ or Step Dilution Method for Nucleosome Reconsitution
  • 3. Quality Control Strategies for In Vitro Reconstituted Chromatin
  • 3.1. Micrococcal Nuclease Digestion
  • 3.1.1. Equipment
  • 3.1.2. Buffers and Reagents
  • 3.1.3. Procedure
  • 3.1.4. Notes
  • 3.2. Analysis of MNase-Digested Products Via Bioanalyzer 2100
  • 3.2.1. Equipment
  • 3.2.2. Buffers and Reagents
  • 3.2.3. Procedure
  • 3.2.4. Notes
  • 3.3. Size-Exclusion Chromatography Coupled with Multiangle Light Scattering
  • 3.3.1. Equipment
  • 3.3.2. Buffer and Reagents
  • 3.3.3. Procedure
  • 3.3.4. Notes
  • 3.4. Analytical Ultracentrifugation
  • 3.4.1. Equipment
  • 3.4.2. Buffers and Reagents
  • 3.4.3. Notes
  • 3.4.4. Procedure
  • 3.5. Atomic Force Microscopy Analysis of Trinucleosomes
  • 3.5.1. Equipment
  • 3.5.2. Buffers and Reagents
  • 3.5.3. Procedure
  • 3.5.4. AFM Data Processing
  • 3.5.5. Notes
  • 3.6. Small-Angle X-ray Scattering
  • 3.6.1. Equipment
  • 3.6.2. Buffers and Reagents
  • 3.6.3. Procedure
  • 3.6.4. Notes
  • 4. Summary and Conclusion
  • References
  • Chapter Two: Preparation, Crystallization, and Structure Determination of Chromatin Enzyme/Nucleosome Complexes
  • 1. Introduction
  • 2. Prepare Nucleosome Core Particles
  • 3. Prepare Chromatin Protein
  • 4. Purify Chromatin Complex
  • 4.1. Analytical Purification
  • 4.2. Preparative Purification
  • 5. Concentrate Chromatin Complex
  • 6. Crystallize Chromatin Complex
  • 6.1. Crystallization
  • 6.2. Fluorescent Labeling of Chromatin Protein for Cocrystallization with the Nucleosome
  • 7. Postcrystallization Soaks to Improve Diffraction
  • References
  • Chapter Three: Preparation of Recombinant Centromeric Nucleosomes and Formation of Complexes with Nonhistone Centromere P ...
  • 1. Introduction
  • 2. Preparation of Histones for CENP-A Nucleosome Assembly
  • 2.1. Expression of His-H2A and H2B Monomers and (CENP-A/H4)2 Heterotetramers
  • 2.1.1. Equipment
  • 2.1.2. Reagents
  • 2.1.3. Buffers
  • 2.1.4. Protocol
  • 2.2. Purification of (CENP-A/H4)2 Heterotetramer
  • 2.2.1. Equipment
  • 2.2.2. Reagents
  • 2.2.3. Buffers
  • 2.2.4. Protocol
  • 2.3. Purification of His-H2A, H2A, and H2B Monomers
  • 2.3.1. Equipment
  • 2.3.2. Reagents
  • 2.3.3. Buffers
  • 2.3.4. Protocol
  • 2.4. Histone H2A/H2B Refolding
  • 2.4.1. Equipment
  • 2.4.2. Buffers
  • 2.4.3. Protocol
  • 3. Preparation of DNA for CENP-A Nucleosome Assembly
  • 3.1. Preparation of DNA by PCR
  • 3.1.1. Equipment
  • 3.1.2. Reagents
  • 3.1.3. Buffers
  • 3.1.4. Protocol
  • 3.2. Preparation of DNA from plasmid excision
  • 4. CENP-A Nucleosome Assembly
  • 4.1. Reconstitution of CENP-A Nucleosomes
  • 4.1.1. Equipment
  • 4.1.2. Buffers
  • 4.1.3. Protocol
  • 4.2. Checking Nucleosome Quality by Native PAGE
  • 4.2.1. Equipment
  • 4.2.2. Reagents
  • 4.2.3. Buffers
  • 4.2.4. Protocol
  • 4.3. Nucleosome Repositioning by Thermal Shifting
  • 4.4. Purification of Nucleosomes by Preparative Native PAGE
  • 4.4.1. Equipment
  • 4.4.2. Buffers
  • 4.4.3. Protocol
  • 5. Complex of CENP-A Nucleosome with CENP-C
  • 5.1. Expression and Purification of CENP-C
  • 5.1.1. Equipment
  • 5.1.2. Reagents
  • 5.1.3. Buffers
  • 5.1.4. Protocol
  • 5.2. Determining Stoichiometry of CENP-A Nucleosome and CENP-C
  • 5.2.1. Reagents
  • 5.2.2. Protocol
  • Acknowledgments
  • References
  • Chapter Four: Functional Characterization of Histone Chaperones Using SNAP-Tag-Based Imaging to Assess De Novo Histone De ...
  • 1. Introduction
  • 2. The SNAP-Tag System to Visualize in vivo New Histone Incorporation into Chromatin: A De Novo Histone Deposition Assay ...
  • 2.1. Establishment of Cell Lines Expressing SNAP-Tagged Histones
  • 2.2. Protocol for the ``Quench-Chase-Pulse´´ Assay
  • 2.2.1. Cell Culture Conditions
  • 2.2.2. Quench
  • 2.2.3. Chase
  • 2.2.4. Pulse
  • 2.2.5. Triton Extraction
  • 2.2.6. Cell Imaging
  • 2.2.7. Controls in the ``Quench-Chase-Pulse´´ Assay
  • 3. De Novo Histone Deposition Assay Coupled with Histone Chaperone Depletion and Labeling of Replicating Cells
  • 4. De Novo Histone Deposition Assay Coupled with Histone Chaperone Depletion in Response to UV-DNA Damage
  • 5. Imaging and Quantifying SNAP Labeling
  • 6. Interpreting the De Novo Histone Deposition Assay Coupled with Histone Chaperone Depletion
  • 7. Conclusion and Perspectives
  • Acknowledgments
  • References
  • Chapter Five: Analysis of Nucleosome Sliding by ATP-Dependent Chromatin Remodeling Enzymes
  • 1. Introduction
  • 2. Reconstituting Nucleosomes for FRET-Based Method
  • 2.1. Histone Labeling
  • 2.2. Visualization of Labeling on an SDS-PAGE Gel (Fig.2)
  • 2.3. DNA Labeling
  • 2.4. Octamer and Nucleosome Assembly
  • 2.5. Purification of Nucleosomes from Free DNA by Glycerol Gradient
  • 2.6. Quantification of Nucleosomes by Gel
  • 3. ATP-Dependent Chromatin Remodeling Assays
  • 3.1. Gel-Based Remodeling Assay
  • 3.2. FRET-Based Remodeling Assay
  • 4. Data Analysis
  • 5. Adaptation to Other Types of Remodeling Reactions
  • Acknowledgments
  • References
  • Part II: Posttranslational Histone Modification Enzymes and Complexes
  • Chapter Six: In Vitro Activity Assays for MYST Histone Acetyltransferases and Adaptation for High-Throughput Inhibitor Sc ...
  • 1. Introduction
  • 2. Preparation of Recombinant MYST Proteins
  • 3. Kinetic Analysis
  • 3.1. Primer on Steady-State Kinetic Analysis
  • 3.2. Specific Kinetic Properties of MYST Acetyltransferases
  • 3.3. Controlling for Chemical Acetylation
  • 3.4. Kinetic Assay Using Radioactive Acetyl-CoA
  • 4. Thermofluor Assay to Investigate Cofactor and Inhibitor Binding
  • 5. Inhibitor Screening
  • 5.1. General Considerations
  • 5.2. Buffer Considerations
  • 5.3. Assays
  • 5.3.1. Radioactivity-Based Assay
  • 5.3.2. ELISA
  • 5.3.3. Other Assays
  • 5.4. Thermofluor Binding Assay
  • 5.5. Eliminating Promiscuous Inhibitors
  • 5.6. Pan-Assay Interference Compounds
  • 5.7. Colloidal Aggregators
  • 6. Prospects and Conclusions
  • Acknowledgments
  • References
  • Chapter Seven: Preparation and Biochemical Analysis of Classical Histone Deacetylases
  • 1. Introduction
  • 2. Generation of HDAC Stable Cell Lines Using Lentivirus
  • 2.1. Initial Titration for Selecting Antibiotic Resistance
  • 2.2. Establishment of Monoclonal Populations
  • 3. Conventional HDAC Activity Assay
  • 3.1. Labeling of H4 Peptide with [3H] for HDAC Assays
  • 3.2. HDAC Assay
  • 4. Assessment of Selective Inhibition of HDACs Using HDAC Knockout Cells
  • 4.1. Quantification of Deacetylase Activity in Multiplate Readers
  • 5. HDAC Chromatin Immunoprecipitation Assay
  • 5.1. Cross-Linking
  • 5.2. Chromatin Preparation
  • 5.3. Sample Sonication
  • 5.4. Shear Sample
  • 5.5. Chromatin Immunoprecipitation
  • 5.6. Sample Wash and Elution
  • 6. Identification of Specific HDAC Substrates
  • Acknowledgments
  • References
  • Chapter Eight: Recombinant Preparation, Biochemical Analysis, and Structure Determination of Sirtuin Family Histone/Prote ...
  • 1. Sirtuins-Evolutionary Conserved Epigenetic Regulators
  • 2. Recombinant Sirtuin Preparation-Constructs and Expression Systems
  • 3. Purification of a Mammalian Sirtuin Recombinantly Expressed in E. coli
  • 3.1. Instruments and Buffers
  • 3.2. Protein Expression
  • 3.3. Preparation of Cleared Lysate
  • 3.4. Affinity Chromatography
  • 3.5. Affinity Tag Removal
  • 3.6. Ion Exchange Chromatography
  • 3.7. Size-Exclusion Chromatography
  • 3.8. Modifications to the Standard Protocol
  • 4. Biochemical Characterizations Using Purified, Recombinant Sirtuins
  • 4.1. Sirtuin Activity
  • 4.2. Sirtuin Binding Studies
  • 4.3. Sirtuin Crystallization
  • 4.4. Further Applications
  • Acknowledgments
  • References
  • Chapter Nine: Preparation, Biochemical Analysis, and Structure Determination of SET Domain Histone Methyltransferases
  • 1. Introduction
  • 2. Experimental Procedures
  • 2.1. Construct Design Strategies
  • 2.2. Protein Sample Preparation
  • 2.3. Preparation of Selenomethionyl-Labeled RcATXR5
  • 2.4. Protein Crystallization
  • 2.5. Data Collection and Structural Determination
  • 2.6. In Vitro MTAs
  • 3. Conclusions
  • Acknowledgments
  • References
  • Chapter Ten: Expression, Purification, and Biochemical Analysis of the LSD1/KDM1A Histone Demethylase
  • 1. Introduction
  • 2. Expression and Purification of LSD1 Proteins
  • 2.1. Expression and Purification of LSD1 in Bacteria
  • 2.2. Expression and Purification of LSD1 from Sf9 Insect Cells
  • 2.3. LSD1 Purification for Structure Determination
  • 2.4. Purification of LSD1 Complex from Mammalian Cells
  • 3. In Vitro Enzymatic Assays
  • 3.1. Enzymatic Assay on Peptides and Histones
  • 3.2. Enzymatic Assay on Nucleosomes
  • 3.3. Enzymatic Assays with LSD1 Complex
  • 4. Analysis of LSD1 Demethylase Activity
  • 4.1. Analysis by Mass Spectrometry
  • 4.2. Analysis by Western Blot
  • 4.3. Additional Methods for Detecting LSD1 Activity
  • 4.4. In vivo Analysis
  • Acknowledgments
  • References
  • Chapter Eleven: LSD1 Histone Demethylase Assays and Inhibition
  • 1. Introduction and Background
  • 2. LSD1 Assays
  • 2.1. Coupled Enzyme Assays
  • 2.2. Radiolabeled Assays
  • 2.3. Antibody-Based Assays
  • 2.4. Mass Spectrometry-Based Assays
  • 3. Inhibitors of LSD1
  • 4. Applications of LSD1 Inhibitors
  • 4.1. Bizine
  • 4.2. NCL1
  • 4.3. GSK2879552
  • 5. Summary and Future Directions
  • Acknowledgments
  • References
  • Chapter Twelve: Purification, Biochemical Analysis, and Structure Determination of JmjC Lysine Demethylases
  • 1. Introduction
  • 2. Purification
  • 2.1. Expression and Purification of Hexahistidine-Tagged JMJD2 KDMs
  • 2.2. Expression and Purification of Strep(II)-Tagged JMJD2 KDMs
  • 2.3. Analysis of the Metal Content of JMJD2 KDMs
  • 3. KDM Assay Methodology
  • 3.1. Design of Histone Peptide Substrates
  • 3.2. Purification of FDH
  • 3.3. FDH-Coupled Fluorescent Assay
  • 4. Structure Determination of JmjC KDMs
  • 4.1. Crystallization of JMJD2 KDMs with Methylated Histone Peptides
  • 4.2. Structure Determination of JMJD2 KDM Peptide Complexes
  • 5. Summary and Future Perspectives
  • Acknowledgment
  • References
  • Chapter Thirteen: Preparation and Analysis of Native Chromatin-Modifying Complexes
  • 1. Introduction
  • 2. Methods
  • 2.1. Endogenous Tagging of EPC1 C-Terminus Using CRISPR/Cas9
  • 2.1.1. Design and Cloning of sgRNA Targeting EPC1
  • 2.1.2. Design and Cloning of Tagging Donor
  • 2.1.3. Gene Targeting in K562 Cells
  • 2.2. Rapid Generation of Isogenic Cell Lines Expressing TAP-Tagged EPC1 from the AAVS1 Safe Harbor Locus
  • 2.3. Large-Scale Expansion of K562 Cells
  • 2.4. Preparation of NEs
  • 2.5. TAP Purification of EPC1
  • 2.5.1. Anti-FLAG M2 Affinity Purification
  • 2.5.2. Strep-Tactin Affinity Purification
  • 2.6. Analysis of EPC1 Complex Subunits by SDS-Polyacrylamide Gel Electrophoresis and Silver Staining
  • 2.7. Sample Preparation for Mass Spectrometry Analysis
  • 2.8. Endogenous Tagging of EZH2 N-Terminus Using TALENs
  • 2.9. Expression of TAP-Tagged EZH2 Variants from the AAVS1 Safe Harbor Locus
  • 2.10. Dox-Inducible Expression of EZH2 from the AAVS1 Safe Harbor Locus
  • 2.11. TAP Purification of EZH2
  • 2.12. HAT/HMT Assays
  • 3. Conclusion
  • Acknowledgments
  • References
  • Part III: Histone Modification Binders (Readers)
  • Chapter Fourteen: Preparation, Biochemical Analysis, and Structure Determination of the Bromodomain, an Acetyl-Lysine Bin ...
  • 1. Introduction
  • 2. General Equipment
  • 3. General Materials
  • 3.1. Bacterial Protein Expression Vector and Host Strain
  • 3.2. Bacteria Growth Medium
  • 3.3. Antibiotics
  • 3.4. Purification Columns and Resins
  • 3.5. Protein Purification Buffers
  • 3.6. Crystallization Reagents and Tools
  • 3.7. Mounting Tools and Shipping Dewar
  • 3.8. NMR Reagents and Tools
  • 3.9. LC/MS Reagents and Tools
  • 4. Preparation of BrDs
  • 4.1. Transformation
  • 4.2. Expression
  • 4.3. Purification
  • 5. Biochemical Analysis of BrDs
  • 5.1. NanoDrop to Determine Protein Concentration and Check Protein Purity
  • 5.2. SDS-PAGE to Check Protein Purity and MW
  • 5.3. LC/MS to Determine Protein MW
  • 5.4. 1H-15N-HSQC to Detect Ligand Binding
  • 5.5. Fluorescence Polarization to Determine Protein-Ligand Binding
  • 5.5.1. Saturation Binding Assay to Determine Binding Affinity, Measured as Dissociation Constant, Kd
  • 5.5.2. Competition Binding Assay to Determine IC50
  • 6. Structural Determination of BrDs
  • 6.1. Structural Determination Using X-Ray Crystallography
  • 6.1.1. Crystallization
  • 6.1.2. Crystal Mounting
  • 6.1.3. Data Collection at Synchrotron
  • 6.1.4. Structure Determination
  • 6.2. Structural Determination Using NMR
  • 6.2.1. Backbone Assignment
  • 6.2.2. Side-Chain Assignment
  • 6.2.3. Restraints Collection
  • 6.2.4. Structure Calculations
  • 7. Conclusions and Perspectives
  • Acknowledgments
  • References
  • Chapter Fifteen: Preparation, Biochemical Analysis, and Structure Determination of Methyllysine Readers
  • 1. Introduction
  • 2. Preparation of Methyllysine Readers
  • 2.1. Construct Design
  • 2.2. Overexpression in E. coli
  • 2.3. Purification
  • 2.3.1. Affinity-Tag Purification (Per Liter of Culture)
  • 2.3.2. FPLC Purification
  • 3. Screening for Methyllysine Recognition
  • 4. Measuring Affinity for Methylated Substrate
  • 5. Analysis of Binding Using NMR Spectroscopy
  • 6. Structure Determination by X-ray Crystallography
  • Acknowledgments
  • References
  • Part IV: DNA Modifications and Nucleic Acid Regulators
  • Chapter Sixteen: Quantification of Oxidized 5-Methylcytosine Bases and TET Enzyme Activity
  • 1. Introduction
  • 2. Analysis of Cytosine Modifications in Cellular DNA
  • 2.1. Preparation of Genomic DNA from TET-Transfected Cells
  • 2.2. Qualitative Analysis by Dot Blotting
  • 2.3. Quantitative Analysis by LC-MS/MS
  • 3. Analysis of Cytosine Modifications In Vitro
  • 3.1. Purification of TET Enzymes from Sf9 Insect Cells
  • 3.2. Synthesis and Isotopic Labeling of TET Substrates
  • 3.3. Chemoenzymatic Activity Assays on Full-Length Oligonucleotides
  • 3.4. Quantitative Activity Assays on Nucleosides
  • Acknowledgments
  • References
  • Chapter Seventeen: Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins
  • 1. Introduction
  • 2. Soluble Expression and Purification of ZnF Proteins
  • 2.1. Expression
  • 2.2. Purification
  • 3. Fluorescence Polarization Assay for Analysis of DNA Binding
  • 4. Crystallization of ZnF Proteins in Complex with DNA
  • Acknowledgments
  • References
  • Chapter Eighteen: Crystallographic Studies of Telomerase
  • 1. Introduction
  • 2. Crystal Structure Determination of the Catalytic Subunit of Triobolium castaneum Telomerase (tcTERT)
  • 2.1. tcTERT Protein Isolation
  • 2.2. tcTERT Crystallization
  • 2.3. tcTERT Structure Determination
  • 3. Crystal Structure Determination of a Partial Telomerase Elongation Complex
  • 3.1. Nucleic Acid Substrate Design
  • 3.2. Activity Assays
  • 3.3. Complex Crystallization
  • 3.4. Structure Determination
  • 4. Conclusions
  • Acknowledgments
  • References
  • Chapter Nineteen: Detection and Analysis of Long Noncoding RNAs
  • 1. Introduction
  • 2. Traditional Methods for lncRNA Detection
  • 3. Genome-Wide Detection of lncRNAs Through Direct RNA Sequencing Approaches
  • 3.1. Total RNA Sequencing
  • 3.2. Chromatin-Associated RNA Sequencing
  • 3.3. Mapping Transcription Start Sites by Direct Cap Sequencing
  • 4. Indirect Sequencing Methods Coupled with Transcription
  • 4.1. Nascent Transcript Global Run-On Sequencing
  • 4.2. Precision Nuclear Run-On and Sequencing Assay (PRO-seq)
  • 5. Protein-Based Approaches for Genomic Characterization of lncRNAs
  • 5.1. RNA Immunoprecipitation and Sequencing (RIP-seq)
  • 5.2. Photoactivatable Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation
  • 5.3. Individual Nucleotide Resolution CLIP
  • 6. Future Directions and Conclusion
  • References
  • Chapter Twenty: Identifying Centromeric RNAs Involved in Histone Dynamics In Vivo
  • 1. Introduction
  • 2. Materials
  • 2.1. Tissue Cell Culture Elements
  • 2.2. RNA Immunoprecipitation Components
  • 2.3. Immunofluorescence/RNA FISH Buffers
  • 3. Methods
  • 3.1. Tissue Cell Culture: Maintenance of HeLa Cells
  • 3.2. RIP-Seq
  • 3.3. Immunofluorescence/RNA FISH
  • 3.4. Computational Analysis
  • 3.4.1. Sequencing Strategy
  • 3.4.2. Read Preprocessing
  • 3.4.3. In Silico rRNA Depletion
  • 3.4.4. Aligning to a Reference Genome
  • 3.4.5. Transcript Abundance Estimation
  • 3.4.6. Assembly of Unaligned Reads
  • Acknowledgments
  • References
  • Author Index
  • Subject Index
  • Color Plate
  • Back Cover

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