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 SidorovaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1983)


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.


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



This work was supported by NIH grants GM115482 and CA215647 to J.S. and Cancer Prevention Research Institution of Texas grant R1312 to K.S. K.S. was also supported by fellowships from Rita Allen Foundation and Andrew Sabin Family Foundation, and Cancer Prevention Research Institution of Texas Scholarship in Cancer Biology.


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Copyright information

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

Authors and Affiliations

  • Pavlo Lazarchuk
    • 1
  • Sunetra Roy
    • 2
  • Katharina Schlacher
    • 2
  • Julia Sidorova
    • 1
    Email author
  1. 1.Department of PathologyUniversity of WashingtonSeattleUSA
  2. 2.Department of Cancer BiologyUniversity of Texas MD Anderson Cancer CenterHoustonUSA

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