Histone Deacetylases: Methods and Protocols (Methods in Molecular Biology, 1436)

Preface
Contents
Contributors
Part I: Class I, II, and, IV Histone Deacetylases
	Chapter 1: A Sensitive and Flexible Assay for Determining Histone Deacetylase 1 (HDAC1) Activity
		1 Introduction
		2 Materials
			2.1 Expression of Flag-HDAC1 in Mammalian Cells
			2.2 Cell Extract Preparation
			2.3 Immunoblotting and Immunoaffinity Purification
			2.4 Deacetylation Assay
		3 Methods
			3.1 Expression of Flag-tagged HDAC1 in 293T Cells
			3.2 Preparation of Cell Extract for Immunoblotting and Immunoaffinity Purification
			3.3 Detect the Ectopic Expression of Flag-HDAC1 by Immunoblotting
			3.4 Immunoaffinity Purification of Flag-HDAC1
			3.5 Deacetylation Assay Using Purified Flag-HDAC1
				3.5.1 Deacetylation Assay Using Core Histone as Substrate and Immunoblotting as Detection Method
				3.5.2 Colorimetric Deacetylation Assay
		4 Notes
		References
	Chapter 2: Detection of Sumo Modification of Endogenous Histone Deacetylase 2 (HDAC2) in Mammalian Cells
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Transfection
			2.3 Cell Lysis
			2.4 Immunoblotting
		3 Methods
			3.1 Transfection of Human Cancer Cell Lines HeLa, MCF7, and U2OS (See Note 4)
			3.2 Cell Lysis
			3.3 Immunoblotting
		4 Notes
		References
	Chapter 3: Analysis of Epigenetic Regulation of Hypoxia-Induced Epithelial–Mesenchymal Transition in Cancer Cells by Quantitative Chromatin Immunoprecipitation of Histone Deacetylase 3 (HDAC3)
		1 Introduction
		2 Materials
			2.1 Chromatin Immunoprecipitation Components
			2.2 DNA Extraction
		3 Methods
			3.1 Chromatin Immunoprecipitation Protocol
			3.2 qPCR Protocol
		4 Notes
		References
	Chapter 4: Molecular and Functional Characterization of Histone Deacetylase 4 (HDAC4)
		1 Introduction
		2 Materials
			2.1 Cell Lines and Animals
			2.2 Reagents and Materials
			2.3 Solutions and Buffers
			2.4 Equipment
		3 Methods
			3.1 Development of Antibodies Specific to Regular and Phosphorylated HDAC4
				3.1.1 Rabbit Immunization for Antiserum Production
				3.1.2 Antibody Affinity Purification on CNBr-Activated Sepharose
				3.1.3 Antibody Affinity Purification on Sulfhydryl Coupling Resin
			3.2 HDAC4 Shuttling Between the Cytoplasm and Nucleus
				3.2.1 Analysis of Subcellular Localization of HDAC4 by Fractionation
				3.2.2 Analysis of Subcellular Localization of HDAC4 by Nuclear Export Inhibition
			3.3 Interaction of 14-3-3 Proteins with HDAC4
				3.3.1 Co-immuno-­precipitation of HDAC4 with 14-3-3
				3.3.2 Mapping 14-3-3-Binding Sites on HDAC4
			3.4 Determination of HDAC4 Deacetylase Activity
				3.4.1 Preparation of [3H] Acetyl-Histones
				3.4.2 Determination of Deacetylase Activity of HDAC4
			3.5 Transcriptional Repression by HDAC4
		4 Notes
		References
	Chapter 5: Approaches for Studying the Subcellular Localization, Interactions, and Regulation of Histone Deacetylase 5 (HDAC5)
		1 Introduction
		2 Materials and Equipment
			2.1 Studying Endogenous and Tagged HDAC5 in Cell Culture
				2.1.1 Generating Cell Lines Expressing GFP-­Tagged HDAC5
				2.1.2 Cell Culture for Studies of Endogenous or Tagged HDAC5
			2.2 Assessing Subcellular Localization of HDAC5
				2.2.1 Fixed Cell Imaging
				2.2.2 Live Cell Imaging
			2.3 Defining HDAC5 Protein Interactions
				2.3.1 Harvesting Cells
				2.3.2 Cryogenic Cell Lysis
				2.3.3 Conjugation of Magnetic Beads to Antibodies
				2.3.4 Immunoaffinity Purifications of Endogenous or Tagged HDAC5
				2.3.5 In-Gel Digestion of Isolated Protein Complexes
				2.3.6 Peptide Extraction and Sample Desalting (StageTips)
			2.4 Investigating Regulation of HDAC5 by Posttranslational Modification
				2.4.1 Identification of Posttranslational Modifications
				2.4.2 Characterizing PTM Functions
				2.4.3 Structure Predictions
			2.5 Cell Cycle Dependent Functions of HDAC5
				2.5.1 G1/S, G2/M, and Cytokinesis Arrests
				2.5.2 Flow Cytometry
				2.5.3 Kinase Inhibition Studies
					Small-Molecule Kinase Inhibitors
					siRNA-Mediated Knockdown of Kinases
			2.6 HDAC5 Enzymatic Activity
		3 Methods
			3.1 Studying Endogenous and Tagged HDAC5 in Cell Culture
				3.1.1 HDAC5 Cell Line Generation
					Generation of Stable HDAC5-Flag-EGFP Cell Lines via Retroviral Transduction
				3.1.2 Cell Culture
			3.2 Assessing the Subcellular Localization of HDAC5
				3.2.1 Preparing Cells for Visualization of Fixed Cells
				3.2.2 Preparing Cells for Live Cell Imaging by Immunofluorescence Microscopy
					Immunofluorescence Microscopy by Live Cell Imaging
			3.3 Defining HDAC5 Protein Interactions
				3.3.1 Harvesting and Freezing Cells
				3.3.2 Cryogenic Cell Lysis
				3.3.3 Conjugation of Magnetic Beads
				3.3.4 Immunoaffinity Purification of HDAC5 With Interacting Partners
				3.3.5 In-gel Digestion of HDAC5 Co-isolated Proteins (See Note 10)
				3.3.6 Peptide Extraction and Sample Desalting (StageTips)
			3.4 Investigating Regulation of HDAC5 by Posttranslational Modification
				3.4.1 Identification of Posttranslational Modifications (PTMs)
				3.4.2 Characterizing PTM Functions
					Amino Acid Substitutions
				3.4.3 Motif Searching
				3.4.4 Structure Predictions
			3.5 Cell Cycle-­Dependent Functions of HDAC5
				3.5.1 Arresting Cells in G1/S, G2/M, and Cytokinesis
				3.5.2 Analysis of Cell Cycle Stage by Flow Cytometry
				3.5.3 Kinase Inhibition Studies
				3.5.4 Small-Molecule Kinase Inhibitors
				3.5.5 siRNA-Mediated Knockdown of Kinases
			3.6 Enzymatic Activity of HDAC5
				3.6.1 Fluorometric Deacetylase Assay
		4 Notes
		References
	Chapter 6: Analysis of Expression and Functions of Histone Deacetylase 6 (HDAC6)
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Cell Lysis
			2.3 Immuno-precipitation
			2.4 Immunoblotting
			2.5 Primary and Secondary Antibodies for Immuno-precipitation and Immunoblot
		3 Methods
			3.1 Cell Lysis
			3.2 Immuno-precipitation
			3.3 Immunoblotting
		4 Notes
		References
	Chapter 7: Analysis of Histone Deacetylase 7 (HDAC7) Alternative Splicing and Its Role in Embryonic Stem Cell Differentiation Toward Smooth Muscle Lineage
		1 Introduction
		2 Materials
			2.1 Cell Culture and ES Cell Differentiation
			2.2 PCR and Western Blot
			2.3 Plasmid Construction
			2.4 Transient Transfection and Nucleofection
		3 Method
			3.1 Detection and Verification of HDAC7 Alternative Splicing
				3.1.1 ES Cell Differentiation
				3.1.2 RNA Extraction
				3.1.3 Reverse Transcriptase-­Polymerase Chain
				3.1.4 Expression Plasmid Construction
					HDAC7 Expression Plasmid Construction
					Adenoviral Vector Construction
				3.1.5 Transient Transfection and Adenoviral Infection
					Transient Transfection
					Adenoviral Infection
				3.1.6 Western Blot Analysis
			3.2 To Analyze the Potential Role of HDAC7 Splicing in SMC Differentiation
				3.2.1 Report Gene Plasmid Construction
				3.2.2 Transient Transfection
				3.2.3 Luciferase Reporter Assay
			3.3 To Explore the Underlying Mechanisms
				3.3.1 Histone Deacetylase Activity Assay
				3.3.2 Indirect Immunofluorescence Staining
				3.3.3 Cellular Fraction
				3.3.4 Co-immuno-precipitation
		References
	Chapter 8: Large-Scale Overproduction and Purification of Recombinant Histone Deacetylase 8 (HDAC8) from the Human-Pathogenic Flatworm Schistosoma mansoni
		1 Introduction
		2 Materials
			2.1 Cell Transformation
			2.2 Cell Cultures
			2.3 Purification Steps
		3 Methods
			3.1 Cell Transformation
			3.2 Large-Scale Cultures
			3.3 Purification Procedure
		4 Notes
		References
	Chapter 9: Visualization of HDAC9 Spatiotemporal Subcellular Localization in Primary Neuron Cultures
		1 Introduction
		2 Materials
			2.1 PCR
			2.2 EGFP Fusion Protein Construction
			2.3 Cortical Neuron Dissociation Culture
			2.4 Transfection
			2.5 Pharmacological Treatment
			2.6 Imaging Systems
		3 Methods
			3.1 Hdac9 cDNA Cloning
			3.2 Primary Cortical Neuron Cultures
			3.3 Transfection
			3.4 Live Cell Imaging
			3.5 Pharmacological Treatment
		4 Notes
		References
	Chapter 10: Expression and Function of Histone Deacetylase 10 (HDAC10) in B Cell Malignancies
		1 Introduction
		2 Materials
			2.1 Animal Models
			2.2 General Reagents and Buffers
			2.3 Cell Culture
			2.4 General Equipment
		3 Methods
			3.1 Cell Cycle Analysis
			3.2 High-
			3.3 Overexpression of HDAC10 by Recombinant Adenovirus
				3.3.1 Testing and Large Scale Preparation of Adenovirus
				3.3.2 Gradient Assembling
				3.3.3 Cell Transduction
			3.4 Western Blot Protocol: Expression of Recombinant HDAC10 Protein
		4 Notes
		References
	Chapter 11: Functional Analysis of Histone Deacetylase 11 (HDAC11)
		1 Introduction
		2 Materials
			2.1 Animal Models
			2.2 General Reagents and Buffers
			2.3 Cell Culture
			2.4 Equipment
		3 Methods
			3.1 PCR Genotyping of HDAC11 Knockout (HDAC11KO) Tg-HDAC11 eGFP Reporter (Tg-HDAC11-­eGFP) Mice
				3.1.1 Mouse Tail Genomic DNA Preparation
				3.1.2 PCR
			3.2 Phenotyping of Tg-HDAC11-­eGFP Reporter Mice
				3.2.1 Peripheral Blood Preparation
			3.3 Multiparameter Flow Cytometric Analysis to Dynamically Visualize the HDAC11 Expression Profile in Myelopoiesis
				3.3.1 Extraction of Bone Marrow Cells
				3.3.2 Flow Cytometry (Staining and Analysis)
			3.4 Quantitative Real-Time PCR of HDAC11KO Mouse Model
				3.4.1 Total mRNA Isolation and cDNA Synthesis
				3.4.2 Quantitative Real-Time PCR
			3.5 Chromatin Immunoprecipitation (ChIP) to Determine the Regulatory Role of HDAC11
				3.5.1 Cells Cross-Linking
				3.5.2 Chromatin Preparation
				3.5.3 Chromatin Immunoprecipitation
		4 Notes
		References
Part II: Class III Deacetylases
	Chapter 12: Sirtuin1 (SIRT1) in the Acetylation of Downstream Target Proteins
		1 Introduction
		2 Materials
			2.1 Chemicals, Reagents, and Equipment Used for Immunoprecipi
			2.2 Chemicals, Reagents, and Equipment Used for Western Blotting
				2.2.1 SDS Polyacrylamide Gel Electrophoresis
				2.2.2 Protein Transfer: Western Blot
				2.2.3 Anti-Acetyl-
			2.3 Chemicals, Reagents, and Equipment Used for Site-­Directed Mutagenesis
		3 Methods
			3.1 Immunopreci
				3.1.1 Lysate Preparation (See Notes 1–3)
				3.1.2 Pre-clearing (See Note 4)
				3.1.3 Antibody–Antigen incubation
				3.1.4 Immunopreci
			3.2 Western Blotting
				3.2.1 SDS-
				3.2.2 Protein Transfer: Western Blot
				3.2.3 Anti-Acetyl-
			3.3 Site-Directed Mutagenesis
				3.3.1 Mutant Strand Synthesis
				3.3.2 DpnI Digestion of the Amplification Products
				3.3.3 Transformation of XL10-Gold Ultracompetent Cells (See Note 8)
		4 Notes
		References
	Chapter 13: Protocols for Cloning, Expression, and Functional Analysis of Sirtuin2 (SIRT2)
		1 Introduction
		2 Materials
			2.1 Cloning of the Sirt2 Gene
			2.2 Sirt2 Expression in E. coli
			2.3 Sirt2 Overexpression and Knockdown in Mammalian Cells
		3 Methods
			3.1 Cloning of the Sirt2 Gene
			3.2 Sirt2 Expression in E. coli
			3.3 Sirt2 Overexpression and Knockdown in Mammalian Cells
		4 Notes
		References
	Chapter 14: Cloning and Characterization of Sirtuin3 (SIRT3)
		1 Introduction
		2 Materials
			2.1 Chemicals and Reagents for Gene Cloning
			2.2 Media and Buffers for Mammalian Cell Culture and Subcellular Fractionation
			2.3 Media, Buffers, and Reagents for Affinity Purification
		3 Methods
			3.1 Cloning of Human SIRT3
			3.2 Expression and Purification of Recombinant SIRT3
			3.3 Pull-Down Purification of SIRT3-­Interacting Proteins
			3.4 Overexpression and Co-immuno­precipitation in Mammalian Cells
		4 Notes
		References
	Chapter 15: Identification of Sirtuin4 (SIRT4) Protein Interactions: Uncovering Candidate Acyl-Modified Mitochondrial Substrates and Enzymatic Regulators
		1 Introduction
		2 Materials
			2.1 Generation of Fibroblasts Stably Expressing SIRT4-EGFP
			2.2 Conjugation of Anti-GFP Antibody to Magnetic Beads
			2.3 Isolation of Mitochondria from Cultured Human Cells
			2.4 Characterization of SIRT4 Interacting Proteins by Immunoaffinity Isolation
			2.5 SDS-PAGE and In-Gel Digestion of SIRT4 Interacting Proteins
			2.6 Peptide Desalting Using StageTips
			2.7 Nanoliquid Chromatography Tandem Mass Spectrometry Analysis
			2.8 Mass Spectrometry Data Analysis and Interpretation
		3 Methods
			3.1 Generation of Fibroblasts Stably Expressing SIRT4-EGFP
				3.1.1 Day 1
				3.1.2 Day 2
				3.1.3 Day 3
				3.1.4 Day 4
				3.1.5 Day 5
				3.1.6 Day 6
				3.1.7 Day 7
			3.2 Conjugation of Antibodies to Magnetic Bead for SIRT4 Immunoaffinity Purifications
			3.3 Isolation of Mitochondria from Cultured Cells
			3.4 Immunoaffinity Isolation of SIRT4 Interacting Proteins
			3.5 SDS-PAGE and In-Gel Digestion of SIRT4 Interacting Proteins
				3.5.1 Day 1
				3.5.2 Day 2
				3.5.3 Day 3
			3.6 Peptide Desalting Using StageTips
			3.7 Nanoliquid Chromatography-Tandem Mass Spectrometry Analysis
			3.8 Data Analysis and Interpretation
				3.8.1 Peptide Identification and Protein Assignment
				3.8.2 SAINT Interaction Specificity Analysis Using Label-free Spectral Counting
			3.9 Conclusion
		4 Notes
		References
	Chapter 16: Generation and Purification of Catalytically Active Recombinant Sirtuin5 (SIRT5) Protein
		1 Introduction
		2 Materials
			2.1 Insertion of H158Y Mutation into SIRT5 CodingSequence and Preparation of SIRT5 and SIRT5-H158Y Bacterial Expression Plasmids with N-Terminal His-Tag
			2.2 Bacterial Expression, Solubility Determination, and Purification of His Tagged SIRT5 and SIRT5-­H158Y Proteins
			2.3 In Vitro Desuccinylation Assay
		3 Methods
			3.1 Engineering H158Y Mutation into the SIRT5 Coding Sequence
			3.2 Preparation of SIRT5 and SIRT5-H158Y Bacterial Expression Plasmids with N-Terminal His-Tag
			3.3 Bacterial Expression of His Tagged SIRT5 and SIRT5-H158Y Proteins
			3.4 Assessment of Solubility of Tagged SIRT5 and SIRT5-H158Y Proteins
			3.5 Purification of His Tagged SIRT5 and SIRT5-­H158Y Proteins
			3.6 In Vitro Desuccinylation Assay to Assess the Activity of Purified SIRT5 and SIRT5-H158Y Proteins
		4 Notes
		References
	Chapter 17: Sirtuin 6 (SIRT6) Activity Assays
		1 Introduction
		2 Materials
			2.1 SIRT6 Deacetylation Activation Assay [13]
			2.2 SIRT6 Deacetylation Fluorogenic Assay [9]
			2.3 SIRT6-Magnetic Beads Deacetylation Assay [11]
			2.4 SIRT6 Frontal Chromatographic Analysis [15]
			2.5 SIRT6 In Silico-­Screen [7]
		3 Methods
			3.1 SIRT6 Deacetylation Assay [13]
				3.1.1 Acitvation Assay
				3.1.2 Inhibition Assay [13]
			3.2 SIRT6 Deacetylation Fluorogenic Assay [9]
			3.3 SIRT6-Magnetic Beads Deacetylation Assay [11]
			3.4 SIRT6 Frontal Chromatographic Analysis [15]
			3.5 SIRT6 In Silico-­Screen [7]
		4 Notes
		References
	Chapter 18: Molecular, Cellular, and Physiological Characterization of Sirtuin 7 (SIRT7)
		1 Introduction
		2 Materials
			2.1 Molecular and Cellular Characterization of SIRT7
				2.1.1 Endogenous Co-immunoprecipitation
			2.2 Physiological Characterization of SIRT7
				2.2.1 Physiological Function of SIRT7 in the Liver
					Triglyceride (TG) Quantification in the Livers of Wild Type (WT) and SIRT7−/− Mice
					Histological Analyses of the Livers of WT and SIRT7−/− Mice
				2.2.2 Functional Assessment of SIRT7 in Hematopoietic Stem Cell (HSC) Aging
					Infection of HSCs with Control or SIRT7 Overexpression Lentivirus
		3 Methods
			3.1 Molecular and Cellular Characterization of SIRT7
				3.1.1 Endogenous Co-immunoprecipitation
				3.1.2 Overexpression of ΔOTC in SIRT7 Knockdown Cells
			3.2 Physiological Characterization of SIRT7
				3.2.1 Physiological Function of SIRT7 in Liver
					TG Quantification in the Livers of WT and SIRT7−/− Mice
					Histological Analyses of the Livers of WT and SIRT7−/− Mice
						Oil Red O Staining
						Immunohistochemistry for F4/80
				3.2.2 Functional Assessment of SIRT7 in Hematopoietic Stem Cell Aging
					Infection of Enriched HSCs with Control and SIRT7 Overexpression Lentivirus
		4 Notes
		References
Part III: Histone Deacetylase Inhibitors
	Chapter 19: HDAC Inhibitors
		1 Introduction
		2 Materials
			2.1 HDAC Activity Assay
			2.2 Analysis of Acetylated Marker Proteins Upon HDAC Inhibition in Human Cells
				2.2.1 Cell Culture
				2.2.2 Drug Treatment
				2.2.3 Cell Lysis
				2.2.4 Protein Concentration Determination by Bradford Assay
				2.2.5 Immunopre
				2.2.6 SDS-PAGE and Western Blot
			2.3 IHC to Determine Protein Levels in Individual Cells Upon HDI Treatment
			2.4 shRNA Screen for the Identification of HDI Sensitivity Determinants
				2.4.1 Cell Culture
				2.4.2 Drug Treatment
				2.4.3 shRNA (in pRetroSuper) Library and PCR of Genomic DNA
		3 Methods
			3.1 HDAC Activity Assay
				3.1.1 Determination of the Linear Range of HDAC Activity
				3.1.2 Determination of HDAC Inhibitor Potency
			3.2 Analysis of Acetylated Marker Proteins Upon HDAC Inhibition in Human Cells
				3.2.1 Cell Culture
				3.2.2 Drug Treatments
				3.2.3 Cell Lysis
				3.2.4 Protein Concentration Determination by Bradford Assay
				3.2.5 Immunopre
				3.2.6 SDS-PAGE and Western Blot
			3.3 IHC to Determine Protein Levels in Individual Cells Upon HDI Treatment
			3.4 shRNA Screen for the Identification of HDI Sensitivity Determinants
		4 Notes
				1.	Store HDAC assay components and substrates at −20 °C. Store the cell extracts and nuclear extracts at −70 °C. For best performance, the assay buffer, substrate and developer should be used on the day prepared. If the reagents cannot be use
				8.	Calcium and magnesium ions can interfere with the EDTA in trypsin–EDTA solution.
				25.	Heating the water bath can take up to an hour. While this reaches preset temperature, prepare slides.
				31.	Viral pools are produced by transfection of Phoenix packaging cells with the pRetroSuper NKI shRNA library using calcium phosphate precipitation.
		References
	Chapter 20: Assessment of the Antiproliferative Activity of a BET Bromodomain Inhibitor as Single Agent and in Combination in Non-Hodgkin Lymphoma Cell Lines
		1 Introduction
		2 Materials and Methods
			2.1 Instruments
			2.2 Material
		3 Proliferation Assay for a Single Agent
			3.1 Protocol
				3.1.1 Day 1
				3.1.2 Day 2: Experimental Plate
				3.1.3 Day 2: Time Zero (t0) Plate
				3.1.4 Day 2: Dilution Plate
				3.1.5 Day 4
				3.1.6 Day 5
				3.1.7 Day 7
		4 Proliferation Assay for the Combination of Two Compounds
			4.1 Protocol
				4.1.1 Day 1
				4.1.2 Day 2: Experimental Plate
				4.1.3 Day 2: Dilution Plate
				4.1.4 Day 5
				4.1.5 Day 7
		5 Data Analysis
			5.1 Single Agent Proliferation Assay
			5.2 Combination of Two Compounds Proliferation Assay
		References
	Chapter 21: Screening the Impact of Sirtuin Inhibitors on Inflammatory and Innate Immune Responses of Macrophages and in a Mouse Model of Endotoxic Shock
		1 Introduction
		2 Materials
			2.1 Stock Solutions of Stimuli and Sirtuin Inhibitors
			2.2 Production and Culture of Bone-Marrow Derived Macrophages (BMDM)
			2.3 Culture of RAW 264.7 Mouse Macrophages
			2.4 Testing Inhibitors on Cultures of BMDM and RAW 264.7 Macrophages
			2.5 RNA Purification and cDNA Synthesis
			2.6 Cytokine Measurement by RT-PCR
			2.7 Cytokine Measurement by ELISA
			2.8 Cell Viability Assay Using MTT
			2.9 Mouse Model of Endotoxemia
		3 Methods
			3.1 Preparation of BMDMs
			3.2 Culture of RAW 264.7 Macrophages
			3.3 Testing Inhibitors on BMDM and RAW 264.7 Macrophages
			3.4 RNA Extraction
			3.5 RNA Precipitation and cDNA Synthesis
			3.6 Real-Time PCR (RT-PCR)
			3.7 ELISA
			3.8 Cell Viability Assay Using MTT
			3.9 Mouse Model of Endotoxemia
		4 Notes
		References
ERRATUM TO
Index