TET Proteins and DNA Demethylation: Methods and Protocols

ISBN-13: 9781071612965 / Angielski / Miękka / 2021

Bogdanovic, Ozren

Opis

This volume explores the latest methods used to study various aspects of TET proteins and their biology. Chapters in this book are divided into five parts. Part One describes technologies aimed at detecting and quantifying DNA methylation turnover using massively parallel sequencing, ELISA, and mass spectrometry approaches. Part Two looks at data analyses protocols for distinguishing acting versus passive DNA demethylation and estimation of 5mC and 5hmC levels. Part Three deals with a new topic that takes advantage of modified CRISPR/Cas9 genome editing systems to target DNA demethylation activity to genomic loci of interest. Part Four discusses protocols that detail how to purify TET proteins and unravel their protein interactions, and Part Five looks at the assessment of TET protein function and activity in vivo and in vitro. 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 thorough, TET Proteins and DNA Demethylation: Methods and Protocols is a valuable resource that aims to help research scientists at all levels working in the fields of DNA demethylation dynamics.

Chapters 3, 7 and 17 are available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

Spis treści

Preface…

Table of Contents…

Contributing Authors…

Part I Detection and Quantification of DNA Methylation Turnover

1) Reduced Bisulfite-Sequencing: Quantitative Base-Resolution Sequencing of 5-Formylcytosine

Michael J. Booth and Shankar Balasubramanian

2) Aba-Seq: High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine

Zhiyi Sun, Jolyon Terragni, Zhenyu Zhu, Yu Zheng, and Sriharsa Pradhan

3) Estimating Global Methylation and Erasure Using Low-Coverage Whole Genome Bisulfite Sequencing (WGBS)

Oscar Ortega-Recalde, Julian R. Peat, Donna M. Bond, and Timothy A. Hore

4) ELISA-Based Quantitation of Global 5hmC Levels

Nelly N. Olova

5) Avidin-Biotin ELISA-Based Detection of 5hmC

Nelly N. Olova

6) Quantification of DNA Methylation and Its Oxidized Derivatives using LC-MS

Franziska R. Traube, Sarah Schiffers, and Thomas Carell

Part II Bioinformatic Analysis of DNA Methylation and Hydroxymethylation

7) Distinguishing Active Versus Passive DNA Demethylation using Illumina MethylationEPIC BeadChip Microarrays

Rochelle L. Tiedemann, Hope E. Eden, Zhijun Huang, Keith D. Robertson, and Scott B. Rothbart

8) Bioinformatic Estimation of DNA Methylation and Hydroxymethylation Proportions

Samara Flamini Kiihl

9) TAB-seq and ACE-seq Data Processing for Genome-Wide DNA Hydroxymethylation Profiling

Ksenia Skvortsova and Ozren Bogdanovic

Part III Precise Epigenome Manipulation using TET Enzymes 

10) Genomic Targeting of TET Activity for Targeted Demethylation using CRISPR/Cas9

Trung Viet Nguyen and Ryan Lister

11) High-Fidelity CRISPR/Cas9-Based Gene-Specific Hydroxymethylation

Xingbo Xu and Elisabeth M. Zeisberg

Part IV TET and DNA Hydroxymethylation Biochemistry

12) Identifying Protein–(hydroxy)methylated DNA Interactions using Quantitative Interaction Proteomics

Velin Marita Sequeira and Michiel Vermeulen

13) Purification of TET Proteins

Zhijun Huang, Jiyoung Yu, Jennifer Johnson, Seung-Gi Jin, and Gerd P. Pfeifer

14) Uncovering Sequence Specific Transcription Factors Interacting with TET2

Tian V. Tian and José Luis Sardina

15) ChIP-Sequencing of TET Proteins

Kasper D. Rasmussen and Kristian Helin

Part V Assessing TET Protein Activity and Function 

16) Harnessing Alternative Substrates to Probe TET Family Enzymes 

Uday Ghanty, Juan C. Serrano, and Rahul M. Kohli

17) Generation and Molecular Characterization of Transient tet1/2/3 Zebrafish Knockouts 

Samuel E. Ross and Ozren Bogdanovic

Subject Index List…