Detection and Quantitation of Acetylated Histones on Replicating DNA Using In Situ Proximity Ligation Assay and Click-It Chemistry
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.
KeywordsHistone 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.
- 6.Nagarajan P, Ge Z, Sirbu B, Doughty C, Agudelo Garcia PA, Schlederer M, Annunziato AT, Cortez D, Kenner L, Parthun MR (2013) Histone acetyl transferase 1 is essential for mammalian development, genome stability, and the processing of newly synthesized histones H3 and H4. PLoS Genet 9:e1003518CrossRefGoogle Scholar
- 13.Alabert C, Bukowski-Wills J-C, Lee S-B, Kustatscher G, Nakamura K, de Lima Alves F, Menard P, Mejlvang J, Rappsilber J, Groth A (2014) Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components. Nat Cell Biol 16:281–293CrossRefGoogle Scholar
- 17.Sirbu BM, McDonald WH, Dungrawala H, Badu-Nkansah A, Kavanaugh GM, Chen Y, Tabb DL, Cortez D (2013) Identification of proteins at active, stalled, and collapsed replication forks using isolation of proteins on nascent DNA (iPOND) coupled with mass spectrometry. J Biol Chem 288:31458–31467CrossRefGoogle Scholar
- 21.Petruk S, Cai J, Sussman R, Sun G, Kovermann SK, Mariani SA, Calabretta B, McMahon SB, Brock HW, Iacovitti L et al (2017) Delayed accumulation of H3K27me3 on nascent DNA is essential for recruitment of transcription factors at early stages of stem cell differentiation. Mol Cell 66:247–257.e245CrossRefGoogle Scholar