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time-ChIP: A Method to Determine Long-Term Locus-Specific Nucleosome Inheritance

  • Wojciech Siwek
  • Mariluz Gómez-Rodríguez
  • Daniel Sobral
  • Ivan R. CorrêaJr
  • Lars E. T. JansenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1832)

Abstract

Understanding chromatin dynamics is essential to define the contribution of chromatin to heritable gene silencing and the long-term maintenance of gene expression. Here we present a detailed protocol for time-ChIP, a novel method to measure histone turnover at high resolution across long timescales. This method is based on the SNAP-tag, a self-labeling enzyme that can be pulse labeled with small molecules in cells. Upon pulse biotinylation of a cohort of SNAP-tagged histones we can determine their abundance and fate across a chase period using a biotin-specific chromatin pulldown followed by DNA sequencing or quantitative PCR. This method is unique in its ability to trace the long-term fate of a chromatin bound histone pool, genome wide. In addition to a step by step protocol, we outline advantages and limitations of the method in relation to other existing techniques. time-ChIP can define regions of high and low histone turnover and identify the location of pools of long lived histones.

Key words

Epigenetic memory Histone variants Chromatin dynamics SNAP-tag time-ChIP Turnover 

Notes

Acknowledgments

We thank João Mata (IGC) for technical support, Brenda Baker (New England Biolabs) for assistance with synthesis of CP-Biotin. Bianka Baying and Vladimir Benes (EMBL genecore) for NGS support. We are grateful to Alekos Athanasiadis (IGC) and EpiLab members for helpful comments on the manuscript.

This work is supported by Fundação para a Ciência e a Tecnologia (FCT) postdoctoral fellowship SFRH/BPD/117179/2016 (to WS) and doctoral fellowship SFRH/BD/33567/2008 (to MRG). Salary support to LETJ is provided by an “Investigador FCT” grant. Further support was provided by FCT grants BIA-BCM/100557/2008 and BIA-PRO/100537/2008, European Commission FP7 Program, Marie Curie Reintegration grant, an EMBO installation grant and an ERC consolidator grant ERC-2013-CoG-615638 to LETJ.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Wojciech Siwek
    • 1
  • Mariluz Gómez-Rodríguez
    • 1
    • 2
  • Daniel Sobral
    • 1
  • Ivan R. CorrêaJr
    • 3
  • Lars E. T. Jansen
    • 1
    Email author
  1. 1.Instituto Gulbenkian de CiênciaOeirasPortugal
  2. 2.Departamento de Ciencias Naturales and MatemáticasPontificia Universidad JaverianaCaliColombia
  3. 3.New England BiolabsIpswichUSA

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