Nucleosomes, Histones and Chromatin Part A

Nucleosomes, Histones and Chromatin Part A

1st Edition - August 31, 2012

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  • Editors: Carl Wu, C. Allis
  • Hardcover ISBN: 9780123919403
  • eBook ISBN: 9780123919410

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Description

This new volume of Methods in Enzymology continues the legacy of this premier serial by containing quality chapters authored by leaders in the field. The first of 2 volumes covering nucleosomes, histones and chromatin, it has chapters on methods applied to the study of protein arginine methylation, high-resolution identification of intra- and interchromosomal DNA interactions by 4C technology, and peptide arrays to interrogate the binding specificity of chromatin-binding proteins.

Key Features

  • Continues the legacy of this premier serial by containing quality chapters authored by leaders in the field
  • The first of 2 volumes covering nucleosomes, histones and chromatin
  • Chapters on methods applied to the study of protein arginine methylation, high-resolution identification of intra- and interchromosomal DNA interactions by 4C technology, and peptide arrays to interrogate the binding specificity of chromatin-binding proteins

Readership

Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists

Table of Contents

  • 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

Product details

  • No. of pages: 390
  • Language: English
  • Copyright: © Academic Press 2012
  • Published: August 31, 2012
  • Imprint: Academic Press
  • Hardcover ISBN: 9780123919403
  • eBook ISBN: 9780123919410

About the Serial Volume Editors

Carl Wu

Affiliations and Expertise

National Cancer Institute, Bethesda, MD

C. Allis

Affiliations and Expertise

The Rockefeller University, New York, NY

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