Polycomb Group Proteins: Methods and Protocols

ISBN-13: 9781071631423 / Angielski / Twarda / 2023 / 446 str.

Chiara Lanzuolo; Federica Marasca

Opis

This volume explores the latest technologies used in the Polycomb Group of proteins field and helps scientists--working on PcG proteins--investigate all functions of PcG proteins in diverse cellular contexts. The chapters in this book cover topics such the distribution of histone marks by CUT&Tag in Drosophila embryos; Co-IP in mammalian cells; replication timing of gene loci in different cell cycle phases; STORM and electronmicroscopy and relative data analysis; and polycomb mediated epigenetic modification in spheroids. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and comprehensive, Polycomb Group Proteins: Methods and Protocols, Second Edition is a valuable tool for all researchers looking to expand their knowledge of this developing field.

Spis treści

Preface…
Table of Contents…
Contributing Authors…

1. Analyzing the Genome-Wide Distribution of Histone Marks by CUT&Tag in Drosophila Embryos
Fides Zenk, Francesco Cardamone, Dafne Andrea Ibarra Morales, Nazerke Atinbayeva, Yinxiu Zhan, and Nicola Iovino

2. Chromatin Preparation and Chromatin Immuno-Precipitation from Drosophila Heads
Davide Andrenacci and Filippo M. Cernilogar

3. Detecting Cell Compartment Specific PRC2-RNA Interactions via UV-RIP
Francesco Della Valle, Peng Liu, Gabriele Morelli, and Valerio Orlando

4. Chromosome Conformation Capture Followed by Genome-Wide Sequencing (Hi-C) in Drosophila Embryos
Francesco Cardamone, Yinxiu Zhan, Nicola Iovino, and Fides Zenk

5. Physics-Based Polymer Models to Probe Chromosome Structure in Single Molecules
Mattia Conte, Andrea M. Chiariello, Simona Bianco, Andrea Esposito, Alex Abraham, and Mario Nicodemi

6. Co-Immunoprecipitation (Co-IP) in Mammalian Cells
Federica Lo Sardo

7. Site Directed Mutagenesis Protocol to Determine the Role of Amino Acid Residues in Polycomb Group (PcG) Protein Function
Diaa Al-Raawi and Aditi Kanhere

8. Isolation of Chromatin Proteins by Genome Capture
Sergi Aranda and Luciano Di Croce

9. Dynamic Interactome of PRC2-EZH1 Complex Using Tandem-Affinity Purification and Quantitative Mass Spectrometry
Peng Liu, Huoming Zhang, Francesco Della Valle, and Valerio Orlando

10. Replication Timing of Gene Loci in Different Cell Cycle Phases
Irene Cantone

11. Polycomb Bodies Detection in Murine Fibromuscular Stroma from Skin, Skeletal Muscles, and Aortic Tissues
Valentina Rosti, Francesca Gorini, Philina Santarelli, Maria Lucia Sarnicola, Silvia Magnani, and Chiara Lanzuolo

12. Segmentation, 3D Reconstruction, and Analysis of PcG Proteins in Fluorescence Microscopy Images in Different Cell Culture Conditions
Francesco Gregoretti, Federica Lucini, Elisa Cesarini, Gennaro Oliva, Chiara Lanzuolo, and Laura Antonelli

13. STORM Microscopy and Cluster Analysis for PcG Studies
Laura Martin, Álvaro Castells-García, Maria Pia Cosma, and Maria Victoria Neguembor

14. Quantitative Analysis of PcG-Associated Condensates by Stochastic Optical Reconstruction Microscopy (STORM)
Silvia Scalisi, Ali Ahmad, Sarah D’Annunzio, David Rousseau, and Alessio Zippo

15. Immunoelectron Microscopy Methods
Stefano Squarzoni

16. Differentiation of hPSCs to Study PRC2 Role in Cell-Fate Specification and Neurodevelopment
Silvia Brocchetti and Paola Conforti

17. Analysis of Polycomb Epigenetic Marks in HeLa Spheroids
Carlo Di Cristo and Maria Vivo

18. Establishment and Maintenance of Human CRC-Derived Organoids for PcGs Studies
Giulia Della Chiara and Massimiliano Pagani

Subject Index List...