Histone Methyltransferases: Methods and Protocols (Methods in Molecular Biology, 2529)

✍ Scribed by Raphaël Margueron (editor), Daniel Holoch (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 490
• Edition: 1st ed. 2022
• Category: Library

✦ Synopsis

This volume provides methods used to investigate histone methyltransferase function. Chapters guide readers through a comprehensive set of approaches that detail phylogenetic diversity, histone demethylase activities in vitro, generating chromatin substrates, auto-methylation, quantification of metabolites, protein purification, crystallization, X-ray structure, cryogenic electron microscopy, assessing genome-wide patterns, CUT&Tag in mouse embryonic tissues, chemical biology approaches, peptide SPOT arrays, nascent chromatin capture, ectopic protein tethering, computational models, and development of methyltransferase inhibitors. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

Authoritative and cutting-edge, Histone Methyltransferases: Methods and Protocols aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.

✦ Table of Contents

Preface
References
Contents
Contributors
Part I: Introduction and Biological Context
Chapter 1: Not all Is SET for Methylation: Evolution of Eukaryotic Protein Methyltransferases
1 Introduction
2 Meet the Organisms: Deep Phylogenetic Sequencing Makes New Models
3 Classes of Histone Methyltransferases
3.1 Protein Arginine Methyltransferases (PRMTs)
3.1.1 Distribution of PRMTs in the Eukaryotes
3.1.2 Histone Methylation Catalyzed by PRMTs
3.2 Non-SET Domain Histone Lysine Methyltransferases (KMTs)
3.3 SET Domain KMTs
3.3.1 Distribution of SET Domain Proteins in Eukaryotes
3.3.2 Function of Selected SET Domain-Containing Protein Complexes
3.3.3 The Relationship Between H3K36 and H3K27 Methylation
3.3.4 A Single Enzyme that Methylates both H3K9 and H3K27
4 Summary
References
Part II: In Vitro Enzymatic Assays
Chapter 2: Detection and Quantification of Histone Methyltransferase Activity In Vitro
1 Introduction
2 Materials
2.1 HMT Reaction
2.2 Radiometric Detection of HMT Activity Based on Incorporation of Tritiated Methyl Groups
2.3 Antibody-Based Detection of HMT Activity
2.4 Detection of HMT Activity Via SAH Quantification
3 Methods
3.1 HMT Reaction
3.2 Radiometric Detection of HMT Activity Based on Incorporation of Tritiated Methyl Groups
3.3 Detection of Methylated Peptides or Histones Using Antibodies
3.4 Detection of HMT Activity Via SAH Quantification
4 Notes
References
Chapter 3: In Vitro Histone Demethylase Assays
1 Introduction
2 Materials
2.1 Enzyme Purification
2.1.1 Purification of Recombinant GST Fusion Proteins from E. coli
2.1.2 Purification of Recombinant Enzymes from Yeast
2.1.3 Recombinant Enzymes and Complexes Purification from Sf9 Cells and Mammalian Cells
2.2 Substrate Preparation
2.3 Demethylation Assays
3 Methods
3.1 Enzyme Purification
3.1.1 Purification of Recombinant GST Fusion Proteins from E. coli
3.1.2 Purification of Recombinant Enzymes from Yeast
3.1.3 Purification of Recombinant Enzymes and Complexes from Sf9 Cells
3.1.4 Purification of Histone Demethylases and Enzyme Complexes from Mammalian Cells
3.2 Substrate Preparation
3.2.1 Peptide Substrates
3.2.2 Prepare Recombinant Octamer and Nucleosome
3.2.3 Methylation of Histone Octamers by In Vitro HMT Reactions
3.2.4 Histones with Methyl-Lysine Analog
3.3 Demethylation Assays
3.3.1 Peptide Mass Spectrometry Demethylase Assay
3.3.2 FDH (Formaldehyde Dehydrogenase) Assay
3.3.3 Demethylation with Radioactivity Assay
3.3.4 Demethylation with Western Blots
4 Notes
References
Part III: Exploring the Regulation of Histone Methyltransferase Enzymatic Activity
Chapter 4: Preparation and Characterization of Chromatin Templates for Histone Methylation Assays
1 Introduction
2 Materials
2.1 Expression and Purification of Recombinant Histones
2.2 Purification of Different DNA Templates
2.3 Reconstitutions and Purifications of Histone Octamers
2.4 Nucleosome and Chromatin Fiber Assembly
2.5 Characterizing the Chromatin Structure Using Electron Microscopy (EM)
2.6 Analyzing Chromatin Structure Using Sedimentation Velocity of Analytical Ultracentrifugation (AUC)
2.7 HMT Assay
3 Methods
3.1 Expression and Purification of Recombinant Histones
3.1.1 For the Preparation of Canonical Histones and H2A/H3 Variants
3.1.2 For the Preparation of H1e, the Expression Condition Is the Same as Canonical Histones (Subheading 3.1.1, steps 1-5): Th...
3.2 Purification of Different DNA Templates
3.2.1 12x 177 bp 601 DNA
3.2.2 4x 177 bp 601 DNA for Sequential Ligation
3.2.3 177 bp DNA with 601 Sequence
3.3 Reconstitution and Purification of Histone Octamers
3.4 Nucleosome and Chromatin Fiber Assembly
3.5 Characterizing the Chromatin Structure Using Electron Microscopy (EM)
3.5.1 Metal Shadowing with Tungsten to Analyze the Mono-Nucleosomes and Nucleosome Array
3.5.2 The Negative Staining Method Is Applied to Analyze the Chromatin Fibers with Histone H1e Incorporation
3.5.3 The Samples Are Evaluated Under a FEI Tecnai G2 Spirit 120 kV Transmission Electron Microscope (Fig. 7)
3.6 Analyzing Chromatin Structure Using Sedimentation Velocity of Analytical Ultracentrifugation (AUC)
3.7 HMT Assay
4 Notes
References
Chapter 5: Techniques to Study Automethylation of Histone Methyltransferases and its Functional Impact
1 Introduction
2 Materials
2.1 Histone Methyltransferase Assay
2.2 SDS-PAGE Gel Running, Western Blot, and Autoradiography
2.3 Immunoprecipitation
2.4 Antibody Generation and Testing
3 Methods
3.1 Identification of Methylation Substrate Using Methyltransferase Assay
3.2 Identification of Automethylation Site
3.2.1 In Vitro Methylation and Mass Spectrometry
3.2.2 In Vivo Methylation and Mass Spectrometry
3.3 Generation and Confirmation of Automethylation Antibody
3.4 Investigate the Function of Automethylation
3.4.1 Compare Automethylated Recombinant Methyltransferase Versus Recombinant Methyltransferase (Fig. 3a)
3.4.2 Wild-Type Versus Automethylation-Deficient Methyltransferase (Fig. 3b)
4 Notes
References
Chapter 6: Profiling the Regulation of Histone Methylation and Demethylation by Metabolites and Metals
1 Introduction
2 Materials
2.1 Metabolomics
2.2 Quantitative Proteomics
2.3 Inductively Coupled Mass Spectrometry
2.4 Nano SIMS Imaging
3 Methods
3.1 Metabolomics
3.2 Quantitative Proteomics
3.3 Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
3.4 NanoSIMS
4 Notes
References
Part IV: Structural Investigation of Histone Methyltransferases
Chapter 7: Determination of Histone Methyltransferase Structure by Crystallography
1 Introduction
2 Materials
2.1 Molecular Biology and Protein Purification
2.2 Protein Crystallography
2.3 Data Collection and Structure Determination
3 Methods
3.1 Construct Design
3.2 Protein Production
3.3 Screening Crystallization Conditions to Obtain Well-Diffracting Crystals
3.4 Data Collection and Structure Solution
4 Notes
References
Chapter 8: Determination of Histone Methyltransferase Structures in Complex with the Nucleosome by Cryogenic Electron Microsco...
1 Introduction
2 Materials
2.1 Reconstitution
2.2 Crosslinking
2.3 Grid Freezing
2.4 Grid Screening and Data Collection
2.5 Data Analysis and Model Building
3 Methods
3.1 Reconstitute, Purify, and Concentrate Methyltransferase-Nucleosome Complex
3.2 Crosslink Methyltransferase Complex
3.3 Prepare Cryo-EM Grids
3.4 Grid Screening and Data Collection
3.5 Data Analysis
3.6 Model Building and Validation
4 Notes
References
Part V: Analysis of Histone Methylation in Cells and Genomes
Chapter 9: Development and Validation of Antibodies Targeting Site-Specific Histone Methylation
1 Introduction
2 Materials
2.1 Antibody Preparation and Preliminary Specificity Tests
2.2 Affinity Purification
2.2.1 Peptide Coupling to Beads
2.2.2 Affinity Purification Schemes
2.2.3 Validation of Antibody Specificity
Native Versus Recombinant Histone Analysis by Protein Immunoblot
Protein Immunoblot and Peptide Competition
2.3 Verification of the General Antibody Specificity and Activity In Vitro and In Vivo
2.3.1 HMT Assays Using Nuclear Extracts and Recombinant K-Mutant Histones as Substrates
2.3.2 Expression of Tagged K/R-Mutant Histones in Mammalian Cells
2.3.3 Native Antigen Recognition by Immunofluorescence
2.3.4 Native Antigen Recognition by Nucleosome Immunoprecipitation (IP)
3 Methods
3.1 Peptide Design, Antibody Preparation, and Preliminary Specificity Tests
3.1.1 Peptide Design and Aqueous Solubilization
3.1.2 Peptide Conjugation to Carrier Protein and Rabbit Immunization
3.1.3 Bleed Handling and Initial Serum Characterization in Peptide Immuno-Dot Blot
3.2 Affinity Purification
3.2.1 Coupling of Peptides to Beads
3.2.2 Affinity Purification Schemes and Preliminary Assessment of Purified Antibody Specificity
3.2.3 Validation of Antibody Specificity
Native Versus Recombinant Histone Analysis by Protein Immunoblot
Protein Immunoblot and Peptide Competition
3.3 Verification of the General Antibody Specificity and Activity In Vitro and In Vivo
3.3.1 HMT Assays Using Nuclear Extracts and Recombinant K-Mutant Histones as Substrates
3.3.2 Expression of Tagged K/R-Mutant Histones in Mammalian Cells to Assess Antibody Specificity
3.3.3 Native Antigen Recognition by Immunofluorescence
3.3.4 Native Antigen Recognition by Nucleosome Immunoprecipitation
3.4 Concluding Remarks
4 Notes
References
Chapter 10: Genetic, Genomic, and Imaging Approaches to Dissect the Role of Polycomb Group Epigenetic Regulators in Mice
1 Introduction
2 Materials
2.1 Generation of PcG Mutant Mice
2.2 Derivation of Embryonic Stem Cells from Blastocysts
2.3 Epigenomic Profiling of Mouse Embryonic Tissues by Using Quantitative CUT&Tag
2.3.1 Cell Dissociation and Cell Freezing
2.3.2 Preparation of pAG-Tn5 or Tn5 Adapter Complex
2.3.3 Spike-in CUT&Tag
2.3.4 Spike-in Control Genomic DNA Library Preparation
2.3.5 Library Amplification for Spike-in CUT&Tag and Spike-in Control Genomic DNA
2.4 Immunofluorescence and Fluorescence In Situ Hybridization (Immuno-FISH)
3 Methods
3.1 Generation of PcG Mutant Mice
3.1.1 How to Search for Mutant Mice from the Resource
3.1.2 Breeding Condition for Generation of Conditional Knockout Embryos/Mice
3.1.3 Tamoxifen Treatment for Obtaining Mutant Embryos
3.2 Derivation of Embryonic Stem Cells from Blastocysts
3.2.1 Mating
3.2.2 Collecting Blastocysts from the Uterus
3.2.3 Removal of the Zona Pellucida
3.2.4 Culture of Blastocysts
3.2.5 Passaging of Blastocyst Outgrowth
3.2.6 Genotyping for Identification of Male ES Cells
3.3 Epigenomic Profiling of Mouse Embryonic Tissues by Using Quantitative CUT&Tag
3.3.1 Cell Dissociation from Mouse Embryonic Tissues and Cell Freezing
3.3.2 Harvest 293T Cells and Cryopreservation
3.3.3 Prepare pAG-Tn5 or Tn5 Adapter Complex
3.3.4 Prepare Precoated 96-Well Plate and Concanavalin A-Coated Magnetic Beads
3.3.5 Defrost Cells Derived from Embryonic Tissues and 293T Cells
3.3.6 Light Fixation
3.3.7 Bind Cells to Concanavalin A-Coated Magnetic Beads
3.3.8 Bind Primary Antibody
3.3.9 Bind Secondary Antibody
3.3.10 Bind the Protein AG-Tn5 Adapter Complex
3.3.11 Tagmentation and De-Crosslinking
3.3.12 DNA Extraction
3.3.13 Library DNA Amplification
3.3.14 Library DNA Quantification for Sequencing
3.3.15 Genomic DNA Extraction for Spike-in Control Genomic DNA Library Preparation
3.3.16 Tagmentation
3.3.17 Spike-in Control Genomic DNA Library Amplification
3.4 Immunofluorescence and Fluorescence In Situ Hybridization (Immuno-FISH)
3.4.1 Probe Design
3.4.2 Probe Preparation
3.4.3 Section Preparation
3.4.4 Section Treatment for Antigen Retrieval and DNA Hybridization
3.4.5 Immunohistochemistry
3.4.6 Hybridization
4 Notes
References
Chapter 11: Profiling Histone Methylation in Low Numbers of Cells
1 Introduction
2 Materials
2.1 Sample Isolation and Storage
2.2 Chromatin Preparation and Antibody-Bead Complex Preparation
2.3 Chromatin Immunoprecipitation
2.4 DNA Purification
2.5 Library Construction
2.6 Library Pooling and Size Selection
3 Methods
3.1 Sample Isolation and Storage (Select Appropriate Section for Tissue)
3.1.1 FACS-Sorted Cells
3.1.2 Oocytes and Early Embryos
3.1.3 Dissected Tissue or Postimplantation Embryo (<5000 Cell-Equivalents)
3.2 Chromatin Preparation
3.2.1 Pooling of Samples Stored in Multiple Tubes
3.2.2 Preparation of Antibody-Bead Complexes
3.2.3 Micrococcal Nuclease (MNAse) Digestion
3.3 Chromatin Immunoprecipitation
3.3.1 Immunoprecipitation
3.3.2 Washes and Elution
3.4 DNA Purification and Elution
3.5 Low Input DNA Library Construction
3.5.1 End Prep and Adaptor Ligation
3.5.2 DNA Purification and Size Enrichment
3.5.3 PCR Amplification of Low-Input Libraries
3.5.4 Quantitation and Quality Control of Constructed Libraries
3.6 Pooling and Size Selection of Multiplexed Low-Input Libraries for Sequencing
4 Notes
References
Chapter 12: Automated CUT & RUN Using the KingFisher Duo Prime
1 Introduction
2 Materials
2.1 Cells
2.2 Buffers
2.3 Reagents
2.4 Antibodies
2.5 Equipment and Supplies
3 Methods
3.1 Setting Up the Automated KingFisher Protocol
3.2 Cell Preparation
3.3 Concanavalin A Bead Preparation
3.4 Cell Binding to Concanavalin A Beads and Antibody Binding
3.5 pA-MNase Binding
3.6 MNase Digestion
3.7 Digestion Stop and Chromatin Recovery
3.8 DNA Extraction and Downstream Analysis
4 Notes
References
Chapter 13: Bioinformatics Methods for ChIP-seq Histone Analysis
1 Experimental Design
2 Pre-analysis Steps
2.1 Quality Controls on Raw Data and Adapter Trimming
2.2 Alignment to a Reference Genome
2.3 Duplicate Removal
2.4 ChIP-seq Quality Metrics
2.4.1 Cumulative Enrichment
2.4.2 Enrichment Around Transcriptionally Active Regions
2.4.3 ENCODE Guidelines
3 Data Visualization
4 The Value of Spike-in Data
5 Enrichment Calling
5.1 The Concept of Peak Calling
5.2 Peak Quality Controls
5.3 Functional Annotation of Peaks
6 Differential Analysis
6.1 Working on a Common Set of Prespecified Regions
6.2 Working on Genomic Windows
7 Analysis of Cut&Run and Cut&Tag Data
8 Conclusion
References
Part VI: Discovery of Histone Methyltransferase Substrates and Methylated Histone Interactors
14: Characterization of SET-Domain Histone Lysine Methyltransferase Substrates Using a Cofactor S-Adenosyl-l-Methionine Surrog...
Abbreviations
1 Introduction
2 Materials
3 Methods
3.1 Design and Characterization of the HKMT Mutants
3.2 Evaluation of the Processing of SAM Analogs by the Wild-Type or Engineered HKMT
3.3 Enzymatic Functionalization with SAM Analog in the Presence of Cell Extracts
3.4 Click Chemistry Reaction
3.5 On-Bead Protein Enrichment
3.6 On-Bead Protein Digestion and Mass Spectrometry
3.7 Data Processing and Label-Free Quantification
3.8 Proteomic Data Analysis
4 Notes
References
Chapter 15: Specificity Analysis of Protein Methyltransferases and Discovery of Novel Substrates Using SPOT Peptide Arrays
1 Introduction
2 Materials
2.1 Synthesis of Peptide SPOT Arrays
2.2 Peptide Array Methylation
2.3 Data Analysis
3 Methods
3.1 Design of the Substrate Sequence Specificity Array
3.2 Preparation of the MultiPep RS Spotter
3.3 Spotting of the First Amino Acid
3.4 Blocking of the Free Amino Acids
3.5 Peptide Chain Elongation
3.6 Terminating the Peptide Synthesis and Quality Control
3.7 Side Chain Deprotection
3.8 Peptide Array Methylation
3.9 Analysis of the Substrate Specificity Array
3.10 Search for Novel Histone Methyltransferase Substrates
4 Notes
References
Chapter 16: Identifying Specific Protein Interactors of Nucleosomes Carrying Methylated Histones Using Quantitative Mass Spect...
1 Introduction
2 Materials
2.1 Expression of Core Histones
2.2 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.3 Coomassie Stain
2.4 Histone Inclusion Body Preparation
2.5 Purification of Histones
2.6 Native Chemical Ligation of Modified Histones
2.7 Histone Octamer Refolding
2.8 Nucleosome Assembly
2.9 Nuclear Extract Preparation
2.10 Nucleosome Affinity Purification
2.11 Proteomic Analysis of Nucleosome Pull-Down Samples
3 Methods
3.1 Expression and Purification of Recombinant Human Core Histones
3.1.1 Expression of Histones in Bacteria
3.1.2 Histone Inclusion Body Preparation
3.1.3 Purification of Histones
3.1.4 Alternative Protocol for Capturing Histone H2A on Cation-Exchange Beads
3.1.5 Alternative Protocol for Purifying Histone H4
3.2 Generation of Post-translationally Modified Histones by Native Chemical Ligation
3.2.1 Expression and Purification of N-Terminally Truncated Histones
3.2.2 Native Chemical Ligation and Purification of Modified Histones
3.3 Refolding of Histone Octamers
3.4 Assembly of Biotinylated Dinucleosomes
3.4.1 Preparation of Biotinylated 601 Dinucleosome DNA
3.4.2 Preparation of MMTV-A Competitor DNA
3.4.3 Reconstitution of Dinucleosomes for Affinity Purifications
3.4.4 Quality Control of Nucleosomal Assembly Reactions by Native PAGE
3.5 Preparation of HeLa S3 Nuclear Extracts
3.6 Nucleosome Affinity Purification
3.6.1 Immobilization of Nucleosomes on Streptavidin-Coated Beads
3.6.2 Nucleosome Affinity Purification from Nuclear Extract
3.7 Proteomic Analysis of Nucleosome Pull-Down Samples
3.7.1 Sample Preparation for Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Analysis
3.7.2 Mass Spectrometric Measurements
3.7.3 Processing and Analysis of Mass Spectrometry Data
4 Notes
References
Part VII: Inheritance of Histone Methylation Patterns
Chapter 17: Investigating Mitotic Inheritance of Histone Posttranslational Modifications by Triple pSILAC Coupled to Nascent C...
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 18: Investigating Mitotic Inheritance of Histone Modifications Using Tethering Strategies
1 Introduction
2 Materials
2.1 General Reagents and Equipment
2.2 Plasmid Cloning and Transformation
2.3 Silencing Assays
2.4 Quantitative RT-PCR
2.5 Chromatin Immunoprecipitation
3 Methods
3.1 Construction of a TetR-Clr4 Expression Plasmid
3.2 Construction of 10XtetO-Reporter Gene Plasmid
3.3 Strain Construction
3.4 Methods for Tracking Reporter Gene Silencing
3.4.1 Colorimetric Output (ade6+ Reporter Silencing)
3.4.2 GFP-Based Maintenance Assay
3.5 Quantitative RT-PCR Analysis to Measure RNA Levels
3.6 Chromatin Immunoprecipitation Analysis to Measure Protein Occupancy
3.7 Expanding the Scope of the Ectopic Tethering System
3.7.1 Methods for a Dual Chromatin-Modifying Enzyme Tethering Approach
4 Notes
References
Chapter 19: Investigating Histone Modification Dynamics by Mechanistic Computational Modeling
1 Introduction
Box 1 Modelling to Investigate Cold-Induced H3K27me3 Nucleation at FLC: Angel et al. [6]
Box 2 Modelling to Investigate Timing of Heterochromatin Formation in Yeast MatingType Region: Obersriebnig et al. [3]
2 Materials
2.1 Building Blocks of the Model
2.1.1 Histone Modifications
2.1.2 Read-Write Enzymes
2.1.3 Transcription
2.1.4 Replication
2.1.5 Noise
2.2 Spatiotemporal Context
2.2.1 Spatial Extent and Boundaries
2.2.2 Timescales and Kinetics
2.3 Choice of Stochastic Histone Level Model
2.4 Model Structure
2.4.1 Transcriptional Feedback Model
2.4.2 M-U-A Type Model
3 Methods
3.1 Programming the Simulation Algorithm
3.1.1 Transcriptional Feedback Type Model Using a Gillespie Exact Stochastic Simulation Algorithm
3.1.2 Transcriptional Feedback Type Model Using a Monte-Carlo Approach
3.1.3 M-U-A Type Model Using a Monte-Carlo Approach
3.2 Initial Conditions and Simulation Output
3.3 Model Analysis: Time Averaging, Quantifying Bistability, First Passage Times
3.3.1 Time Averaging
3.3.2 Quantifying Bistability
3.3.3 Computing First Passage Times
3.4 Parameterizing the Model
4 Notes
References
Part VIII: Finding Inhibitors of Histone Methyltransferases
Chapter 20: Screening for Small-Molecule Inhibitors of Histone Methyltransferases
1 Introduction
2 Materials
2.1 EZH2 Biochemical Assay
2.2 TSA Assay
2.3 H3K27me3 Cell Assay
2.4 Equipment
3 Methods
3.1 Identification of EZH2 SMIs Using Biochemical Assays and Compound High Throughput Screening
3.2 Thermal Shift Analysis
3.3 H3K27me3 Cell Assay
3.3.1 Cell Assay, Day 0: Plating and Compound Treatment
3.3.2 Cell Assay, Day 3: AlphaLISA H3K27me3 Readout
3.3.3 Cell Assay, Day 3: Cell Viability
4 Notes
References
Index