Presents the process of mapping the genetic interactions of protein and DNA via cross-linking.

Contents

• Dissecting Fission Yeast Centromeres via Silencing
  Robin Allshire.
  • Introduction
  • Our Observations
  • Our Current View
  • References
• Localizing Replication Sites and Nuclear Proteins
  Angela Taddei, Danièle Roche, Jean-Baptiste Sibarita, Sébastien Huart, Christèle Maison, Delphine Bailly, and Geneviève Almouzni.
  • Practical Approach
  • Selected References
• Finding Downstream Target DNAs for Chromatin Proteins by X-CHIP in Drosophila
  Nicolas Nègre and Giacomo Cavalli.
  • Section I. General Discussion of the Approach
  • Section II. Detailed Protocol for X-ChIP
  • References
• Emerging Techniques: UV Laser Footprinting and Protein--DNA Crosslinking
  Stefan Dimitrov.
  • Laser Protein--DNA Photofootprinting
  • UV Laser-induced Protein--DNA Crosslinking
  • Conclusions and Perspectives
  • References
• Nuclear Organization and Gene Expression: Visualization of Transcription and Higher Order Chromatin Structure
  Roel van Driel and Pernette J Verschure.
  • Scientific Context
  • Technical Strategies
  • Protocol for Simultaneous Labelling of DNA and Antigens in the Cell Nucleus
  • Literature Cited
• Examining the Distribution of Telomeric and DNA Repair Proteins by ChrIP and Real-Time PCR
  Karine Dubrana.
  • General Principle of Chromatin ImmunoPrecipitation (ChrIP)
  • Important Steps to Control
  • Investing in the Real-Time Technique
  • Appendix: Chromatin-IP (ChrIP) Protocol
  • References
• Nuclear Organisation and Gene Silencing in Lymphocytes
  Amanda Fisher and Matthias Merkenschlager.
• Direct Visualization of Transcription Factor Binding to Regulatory Elements in Living Cells
  Gordon L Hager.
  • Introduction
  • Tagging Nuclear Receptors with Fluorescent Proteins
  • Characterization of the Fusion Chimera
  • Mode of Expression
  • Instrumentation
  • Gene Targets
  • Dynamic Movement of Receptors on Gene Targets
  • Reference List
• Part I. Chromatin Protein Complexes: Dynamic Assemblies in the Nucleus
  Donna Jovin.
  • Nuclear Architecture and Functional Consequences
  • Polycomb-Group Protein Repression Complexes, Cellular Memory
  • The Drosophila X Chromosome and the MSL Dosage Compensation Complex
  • Photophysical Methods to Extract Information about Molecular Proximity, Interactions, and Mobilities
  • References
• Isolation of Homeodomain Engrailed Target Genes by UV Cross-Linking and Chromatin IP
  Florence Maschat.
  • Part 1: General Discussion of the Research and of the Method
  • IMAGE
  • Part 2: A Detailed Method for in Vivo UV Cross-Linking and Chromatin IP
  • References
• Analysis of Chromatin Assembly, Chromatin Domains, and DNA Replication Using Xenopus Systems
  Jean-Marc Lemaitre, Yegor Vassetzky, and Marcel Méchali.
  • Collection of Eggs
  • Low-Speed Egg Extract (LSE)
  • Preparation of High-Speed Extract (HSE)
  • Preparation of Demembranated Sperm Nuclei
  • Assays for DNA Replication in Xenopus Extracts
  • DNA Replication Assay by TCA Precipitation
  • Calculation of the Activity of the Extract
  • Detection of Protein Interaction with Chromatin by Immunolocalisation
  • Detection of Protein Interaction with Chromatin by a Purification Assay
  • Protocol for Single-stranded DNA Replication and Chromatin Assembly
  • Notes
  • Chromatin Assembly
  • Supercoiling Assay (First Method)
  • Supercoiling Assay (Second Method)
  • Micrococcal Nuclease Digestion Assay
  • Notes
  • Studying DNA--Protein Interactions in Vivo Using LM-PCR Coupled with Nuclear Matrix Isolation
  • Protocol for the Nuclear Matrix Isolation
  • Ligation-mediated PCR
  • Appendix: Buffers and Solutions
  • References
• Binding Sights in Chromatin by X-ChIP
  Achim Breiling and Valerio Orlando.
  • Formaldehyde Fixation, Chromatin Purification, and Immunoprecipitation for Drosophila SL2 Tissue Culture Cells (Applicable Also to Mouse and Human Cell Lines)
  • References
• Chromatin Immunoprecipitation in Budding Yeast
  Tomoyuki Tanaka.
  • Experimental Protocol
  • References
• ChIP with Native Chromatin: Advantages and Problems Relative to Methods Using Cross-Linked Material
  Bryan Turner.
  • Part 1. General Discussion
  • References
• Highly Sensitive FISH in Combination with the Visualization of the Mammalian Genome: Architecture
  Daniele Zink.
  • Introduction
  • Results
  • Discussion
  • Materials and Methods
  • References
• Part II. Fly-FISHing: A Protocol to Localize Single Copy Genes inside the Nucleus in Whole Mount Embryos
  Mark J Gemkow.
  • Introduction
  • Protocol and Materials
  • Considerations
  • Acknowledgments
  • References
• Part 2. Detailed Method for Immunoprecipitation of Unfixed Chromatin from Mammalian Cells
  Laura O'Neill and Bryan Turner.
  • A. Chromatin Preparation from Cultured Human Cells
  • B. Analysis of Chromatin
  • C. Immunoprecipitation of Unfixed Chromatin (with Antibodies to Acetylated H4)
  • D. DNA Isolation from Chromatin Fractions
  • E. Protein Isolation from Chromatin Fractions
  • F. Solutions