Advertisement

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

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.

Keywords

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

Notes

Acknowledgments

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.

References

  1. 1.
    Alabert C, Groth A (2012) Chromatin replication and epigenome maintenance. Nat Rev Mol Cell Biol 13:153–167CrossRefGoogle Scholar
  2. 2.
    Gong F, Miller KM (2013) Mammalian DNA repair: HATs and HDACs make their mark through histone acetylation. Mutat Res 750:23–30CrossRefGoogle Scholar
  3. 3.
    Galvani A, Thiriet C (2015) Nucleosome dancing at the tempo of histone tail acetylation. Genes (Basel) 6:607–621CrossRefGoogle Scholar
  4. 4.
    Gong F, Chiu LY, Miller KM (2016) Acetylation reader proteins: linking acetylation signaling to genome maintenance and cancer. PLoS Genet 12:e1006272CrossRefGoogle Scholar
  5. 5.
    Alabert C, Jasencakova Z, Groth A (2017) Chromatin replication and histone dynamics. In: Masai H, Foiani M (eds) DNA replication: from old principles to new discoveries. Springer Singapore, Singapore, pp 311–333CrossRefGoogle Scholar
  6. 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
  7. 7.
    Ge Z, Nair D, Guan X, Rastogi N, Freitas MA, Parthun MR (2013) Sites of acetylation on newly synthesized histone H4 are required for chromatin assembly and DNA damage response signaling. Mol Cell Biol 33:3286–3298CrossRefGoogle Scholar
  8. 8.
    Bhaskara S, Jacques V, Rusche JR, Olson EN, Cairns BR, Chandrasekharan MB (2013) Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression. Epigenetics Chromatin 6:27CrossRefGoogle Scholar
  9. 9.
    Kehrli K, Phelps M, Lazarchuk P, Chen E, Monnat R Jr, Sidorova JM (2016) Class I histone deacetylase HDAC1 and WRN RECQ helicase contribute additively to protect replication forks upon hydroxyurea-induced arrest. J Biol Chem 291:24487–24503CrossRefGoogle Scholar
  10. 10.
    Annunziato AT, Seale RL (1983) Histone deacetylation is required for the maturation of newly replicated chromatin. J Biol Chem 258:12675–12684PubMedGoogle Scholar
  11. 11.
    Benson LJ, Gu Y, Yakovleva T, Tong K, Barrows C, Strack CL, Cook RG, Mizzen CA, Annunziato AT (2006) Modifications of H3 and H4 during chromatin replication, nucleosome assembly, and histone exchange. J Biol Chem 281:9287–9296CrossRefGoogle Scholar
  12. 12.
    Sirbu BM, Couch FB, Feigerle JT, Bhaskara S, Hiebert SW, Cortez D (2011) Analysis of protein dynamics at active, stalled, and collapsed replication forks. Genes Dev 25:1320–1327CrossRefGoogle Scholar
  13. 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
  14. 14.
    Filippakopoulos P, Knapp S (2014) Targeting bromodomains: epigenetic readers of lysine acetylation. Nat Rev Drug Discov 13:337–356CrossRefGoogle Scholar
  15. 15.
    Chiu LY, Gong F, Miller KM (2017) Bromodomain proteins: repairing DNA damage within chromatin. Philos Trans R Soc Lond Ser B Biol Sci 372:20160286CrossRefGoogle Scholar
  16. 16.
    Fujisawa T, Filippakopoulos P (2017) Functions of bromodomain-containing proteins and their roles in homeostasis and cancer. Nat Rev Mol Cell Biol 18:246–262CrossRefGoogle Scholar
  17. 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
  18. 18.
    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 Protocols 7:594–605CrossRefGoogle Scholar
  19. 19.
    Roy S, Luzwick JW, Schlacher K (2018) SIRF: quantitative in situ analysis of protein interactions at DNA replication forks. J Cell Biol 217:1521–1536CrossRefGoogle Scholar
  20. 20.
    Petruk S, Sedkov Y, Johnston DM, Hodgson JW, Black KL, Kovermann SK, Beck S, Canaani E, Brock HW, Mazo A (2012) TrxG and PcG proteins but not methylated histones remain associated with DNA through replication. Cell 150:922–933CrossRefGoogle Scholar
  21. 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
  22. 22.
    Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG et al (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3:995–1000CrossRefGoogle Scholar
  23. 23.
    Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gustafsdottir SM, Ostman A, Landegren U (2002) Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol 20:473–477CrossRefGoogle Scholar
  24. 24.
    Iannascoli C, Palermo V, Murfuni I, Franchitto A, Pichierri P (2015) The WRN exonuclease domain protects nascent strands from pathological MRE11/EXO1-dependent degradation. Nucleic Acids Res 43:9788–9803PubMedPubMedCentralGoogle Scholar
  25. 25.
    Weibrecht I, Gavrilovic M, Lindbom L, Landegren U, Wahlby C, Soderberg O (2012) Visualising individual sequence-specific protein-DNA interactions in situ. New Biotechnol 29:589–598CrossRefGoogle Scholar
  26. 26.
    Zhang W, Xie M, Shu MD, Steitz JA, DiMaio D (2016) A proximity-dependent assay for specific RNA-protein interactions in intact cells. RNA (New York, NY) 22:1785–1792CrossRefGoogle Scholar
  27. 27.
    Taglialatela A, Alvarez S, Leuzzi G, Sannino V, Ranjha L, Huang JW, Madubata C, Anand R, Levy B, Rabadan R et al (2017) Restoration of replication fork stability in BRCA1- and BRCA2-deficient cells by inactivation of SNF2-family fork remodelers. Mol Cell 68:414–430.e418CrossRefGoogle Scholar
  28. 28.
    Roy S, Tomaszowski KH, Luzwick JW, Park S, Li J, Murphy M, Schlacher K (2018) p53 orchestrates DNA replication restart homeostasis by suppressing mutagenic RAD52 and POLtheta pathways. Elife 7:e31723CrossRefGoogle Scholar
  29. 29.
    Tutton S, Azzam GA, Stong N, Vladimirova O, Wiedmer A, Monteith JA, Beishline K, Wang Z, Deng Z, Riethman H et al (2016) Subtelomeric p53 binding prevents accumulation of DNA damage at human telomeres. EMBO J 35:193–207CrossRefGoogle Scholar

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

Personalised recommendations