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Isolation of Proteins on Nascent Chromatin and Characterization by Quantitative Mass Spectrometry

  • Paula A. Agudelo Garcia
  • Miranda Gardner
  • Michael A. Freitas
  • Mark R. ParthunEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1983)

Abstract

Replication-coupled chromatin assembly is a very dynamic process that involves not only the replication fork machinery but also chromatin-related factors such as histones, histone chaperones, histone-modifying enzymes, and chromatin remodelers which ensure not only that the genetic information is properly replicated but also that the epigenetic code is reestablished in the daughter cell. Of the histone modifications associated with chromatin assembly, acetylation is the most abundant. Determining how newly synthesized histones get acetylated and what factors affect this modification is vital to understanding how cells manage to properly duplicate the epigenome. Here we describe a combination of the iPOND, quantitative mass spectrometry, and SILAC methodologies to study the protein composition of newly assembled chromatin and the modification state of the associated histones.

Keywords

Nascent chromatin Acetylation SILAC Mass spectrometry Histone 

Notes

Acknowledgment

This work was supported by a grant from the National Institutes of Health (GM062970) to M.R.P.

References

  1. 1.
    Serra-Cardona A, Zhang Z (2018) Replication-coupled nucleosome assembly in the passage of epigenetic information and cell identity. Trends Biochem Sci 43(2):136–148CrossRefGoogle Scholar
  2. 2.
    Audia JE, Campbell RM (2016) Histone modifications and cancer. Cold Spring Harb Perspect Biol 8(4):a019521CrossRefGoogle Scholar
  3. 3.
    Alabert C, Groth A (2012) Chromatin replication and epigenome maintenance. Nat Rev Mol Cell Biol 13(3):153–167CrossRefGoogle Scholar
  4. 4.
    Almouzni G, Cedar H (2016) Maintenance of epigenetic information. Cold Spring Harb Perspect Biol 8(5):a019372CrossRefGoogle Scholar
  5. 5.
    Sirbu BM et al (2011) Analysis of protein dynamics at active, stalled, and collapsed replication forks. Genes Dev 25(12):1320–1327CrossRefGoogle Scholar
  6. 6.
    Sirbu BM, Couch FB, Cortez D (2012) Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA. Nat Protoc 7(3):594–605CrossRefGoogle Scholar
  7. 7.
    Agudelo Garcia PA et al (2017) Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly. Nucleic Acids Res 45(16):9319–9335CrossRefGoogle Scholar
  8. 8.
    Keshavan P, Schwemberger SJ, Smith DL, Babcock GF, Zucker SD (2004) Unconjugated bilirubin induces apoptosis in colon cancer cells by triggering mitochondrial depolarization. Int J Cancer 112(3):433–445CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Paula A. Agudelo Garcia
    • 1
  • Miranda Gardner
    • 2
  • Michael A. Freitas
    • 2
  • Mark R. Parthun
    • 1
    Email author
  1. 1.Department of Biological Chemistry and PharmacologyThe Ohio State UniversityColumbusUSA
  2. 2.Department of Cancer Biology and GeneticsThe Ohio State UniversityColumbusUSA

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