Main description:

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.

Contents:

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-FormylcytosineMichael J. Booth and Shankar Balasubramanian
2) Aba-Seq: High-Resolution Enzymatic Mapping of Genomic 5-HydroxymethylcytosineZhiyi 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 LevelsNelly N. Olova
5) Avidin-Biotin ELISA-Based Detection of 5hmCNelly N. Olova
6) Quantification of DNA Methylation and Its Oxidized Derivatives using LC-MSFranziska 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 MicroarraysRochelle L. Tiedemann, Hope E. Eden, Zhijun Huang, Keith D. Robertson, and Scott B. Rothbart
8) Bioinformatic Estimation of DNA Methylation and Hydroxymethylation ProportionsSamara Flamini Kiihl
9) TAB-seq and ACE-seq Data Processing for Genome-Wide DNA Hydroxymethylation ProfilingKsenia Skvortsova and Ozren Bogdanovic
Part III Precise Epigenome Manipulation using TET Enzymes
10) Genomic Targeting of TET Activity for Targeted Demethylation using CRISPR/Cas9Trung Viet Nguyen and Ryan Lister
11) High-Fidelity CRISPR/Cas9-Based Gene-Specific HydroxymethylationXingbo Xu and Elisabeth M. Zeisberg
Part IV TET and DNA Hydroxymethylation Biochemistry
12) Identifying Protein-(hydroxy)methylated DNA Interactions using Quantitative Interaction ProteomicsVelin Marita Sequeira and Michiel Vermeulen
13) Purification of TET ProteinsZhijun Huang, Jiyoung Yu, Jennifer Johnson, Seung-Gi Jin, and Gerd P. Pfeifer
14) Uncovering Sequence Specific Transcription Factors Interacting with TET2Tian V. Tian and Jose Luis Sardina
15) ChIP-Sequencing of TET ProteinsKasper 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...