Detection and quantitation of acetylated histones on replicating DNA using in situ proximity ligation assay and click-it chemistry

Pavlo Lazarchuk, Sunetra Roy, Katharina Schlacher, Julia Sidorova

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

Abstract

Histone acetylation plays important roles in the regulation of DNA transcription, repair, and replication. Here we detail a method for quantitative detection of specific histone modifications in the nascent chromatin at or behind replication forks in vivo in cultured cells. The method involves labeling DNA with EdU, using Click chemistry to biotinylate EdU moieties in DNA, and then using in situ proximity ligation assay (PLA) to selectively visualize co-localization of EdU with a modified histone of choice recognized by a modification-specific antibody. We focus on detection of acetylated histones H3 and H4 in the nascent chromatin of cultured human cells as a specific example of the method's application. Notably, the method is fully applicable to studies of histones or nonhistone proteins expected to be present on nascent DNA or at replication forks, and has been successfully used in model organisms and human tissue culture.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages29-45
Number of pages17
DOIs
StatePublished - 2019

Publication series

NameMethods in Molecular Biology
Volume1983
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Click-It
  • EdU
  • HDAC inhibitor
  • Histone acetylation
  • Human cells
  • In situ
  • PLA
  • Replication fork
  • SIRF
  • p53

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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