Nucleosomes, Histones and Chromatin Part A

Series Page

Contributors

Preface

Volume in series

Chapter One Examining Histone Posttranslational Modification Patterns by High-Resolution Mass Spectrometry

1 Introduction

2 Materials

3 Nuclei Isolation

4 Acid Extraction

5 Offline Reverse-Phase High-Performance Liquid Chromatography for Histone Variant Purification (Optional)

6 Chemical Derivatization of Histones

7 Stage-Tip Cleanup of Peptide Samples Prior to MS Analysis

8 Enrichment for Phosphorylated Peptides Using Titanium Dioxide (TiO2) Beads (Optional)

9 Online RP-HPLC and MS

Chapter Two Investigating the Cell Cycle-Associated Dynamics of Histone Modifications Using Quantitative Mass Spectrometry

1 Introduction

2 Experimental Design

3 Cell Culture

4 Extraction and Separation of Histones

5 Preparation of Propionic Anhydride-Derivatized Tryptic Histone Peptides

6 Mass Spectrometric Analysis and Quantification of Histone PTMs

Chapter Three A Method to Site-Specifically Incorporate Methyl-Lysine Analogues into Recombinant Proteins

1 Introduction

2 Protocol to Install MLA into Recombinant Proteins

Chapter Four Methods Applied to the Study of Protein Arginine Methylation

1 Introduction

2 In Vitro Arginine-Methylation Assays

3 Enzyme Sources for In Vitro Methylation Reactions

4 Substrate Sources for In Vitro Methylation Assays

5 Cell-Based Methylation Assays

6 Methyl-Specific Antibodies

7 Using Global-Methylation Inhibitors

8 Using Cell-Based Reporter Systems to Screen for PRMT Inhibitors

9 Summary

Chapter Five Enzymatic Analysis of Tet Proteins: Key Enzymes in the Metabolism of DNA Methylation

1 Introduction

2 Expression and Purification of Tet Proteins

3 Tet Activity Assays

4 Analysis of the Cytosine Derivatives by 2D-TLC

5 Analysis of the Cytosine Derivatives by Mass Spectrometry

6 Analysis of the Endogenous Level of Cytosine Derivatives by Mass Spectrometry

Chapter Six Peptide Microarrays to Interrogate the “Histone Code”

1 Introduction

2 Histone Peptide Library

3 Microarray Printing

4 Analysis of Antibodies, Effector Proteins, and Enzymes

5 Data Analysis

6 Summary and Perspectives

Chapter Seven Identifying Chromatin Readers Using a SILAC-Based Histone Peptide Pull-Down Approach

1 Introduction

2 Histone Peptide Synthesis

3 SILAC Labeling

4 Incorporation Check

5 Preparation of Nuclear Extract

6 Histone Peptide Pull-Down

7 In-gel Digestion

8 Mass Spectrometry

9 Data Analysis

10 Outlook

Chapter Eight High-Throughput Strategy to Identify Inhibitors of Histone-Binding Domains

1 Introduction

2 AlphaScreen Assay Principles

3 Materials and Instrumentation

4 Design and Preparation of Histone Peptides and Histidine Tag Fusion Proteins

5 Initial Optimization of an AlphaScreen-Based Histone-Binding Assay

6 Primary Screening

7 Hit Selection and Secondary Screening

8 Concentration-Dependence Validation Studies

9 Assay-Specific Compound Interference

10 Additional Assays for Validation

11 Summary

Chapter Nine Validation of Histone-Binding Partners by Peptide Pull-Downs and Isothermal Titration Calorimetry

1 Introduction

2 Initial Screening: Discovering Putative Interactions Between Histone Peptides and Potential Binding Partners

3 Peptide Pull-downs: Establishing Candidates for Quantitative Assessment

4 Isothermal Titration Calorimetry (ITC): Defining Histone PTM Binding Affinities

5 Reagent Considerations

6 Summary: Peptide Pull-Downs and ITC in the Chromatin Field

Chapter Ten Analysis of Histone Chaperone Antisilencing Function 1 Interactions

1 Introduction

2 Preparation of Asf1

3 Preparation of Histone Components

4 Fluorescence Spectroscopy Analysis of Asf1 Binding Interactions with Histones

5 Electrophoretic Mobility Shift Analysis of Asf1 Binding Interactions with Histones

6 Summary and Implications

Chapter Eleven Quantifying Chromatin-Associated Interactions

1 Introduction to Chromatin and the HI-FI System

2 Fluorescent Labeling and Preparation of Nucleosome Components

3 Imaging Requirements and Reaction Conditions

4 HI-FI Binding Affinity Assay Setup and Execution

5 HI-FI Stoichiometric Assays Setup and Execution

6 HI-FI Competition Assays with FRET

7 Summary

Chapter Twelve Biochemical Assay for Histone H2A.Z Replacement by the Yeast SWR1 Chromatin Remodeling Complex

1 Introduction

2 Preparation of Reagents

3 In Vitro Htz1 Replacement Assay by Chromatin Remodeling Complex SWR1

4 Special Notes

5 Concluding Remarks

Chapter Thirteen Experimental Analysis of the Mechanism of Chromatin Remodeling by RNA Polymerase II

1 Introduction

2 Materials and Methods

3 DNASE I Footprinting of ECs Formed by E. coli RNAP During Transcription Through a Nucleosome

4 Mapping of ECs Formed by Pol II During Transcription Through a Nucleosome Using Restriction Endonucleases

Author Index

Subject Index