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Detection and Quantitation of Acetylated Histones on Replicating DNA Using In Situ Proximity Ligation Assay and Click-It Chemistry

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Protein Acetylation

Part of the book series: Methods in Molecular Biology ((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.

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References

  1. Alabert C, Groth A (2012) Chromatin replication and epigenome maintenance. Nat Rev Mol Cell Biol 13:153–167

    Article  CAS  Google Scholar 

  2. Gong F, Miller KM (2013) Mammalian DNA repair: HATs and HDACs make their mark through histone acetylation. Mutat Res 750:23–30

    Article  CAS  Google Scholar 

  3. Galvani A, Thiriet C (2015) Nucleosome dancing at the tempo of histone tail acetylation. Genes (Basel) 6:607–621

    Article  CAS  Google Scholar 

  4. Gong F, Chiu LY, Miller KM (2016) Acetylation reader proteins: linking acetylation signaling to genome maintenance and cancer. PLoS Genet 12:e1006272

    Article  Google Scholar 

  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 311–333

    Chapter  Google Scholar 

  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:e1003518

    Article  CAS  Google Scholar 

  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 33:3286–3298

    Article  CAS  Google Scholar 

  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 6:27

    Article  CAS  Google Scholar 

  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 291:24487–24503

    Article  CAS  Google Scholar 

  10. Annunziato AT, Seale RL (1983) Histone deacetylation is required for the maturation of newly replicated chromatin. J Biol Chem 258:12675–12684

    CAS  PubMed  Google Scholar 

  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 281:9287–9296

    Article  CAS  Google Scholar 

  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 25:1320–1327

    Article  CAS  Google 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 16:281–293

    Article  CAS  Google Scholar 

  14. Filippakopoulos P, Knapp S (2014) Targeting bromodomains: epigenetic readers of lysine acetylation. Nat Rev Drug Discov 13:337–356

    Article  CAS  Google Scholar 

  15. Chiu LY, Gong F, Miller KM (2017) Bromodomain proteins: repairing DNA damage within chromatin. Philos Trans R Soc Lond Ser B Biol Sci 372:20160286

    Article  Google Scholar 

  16. Fujisawa T, Filippakopoulos P (2017) Functions of bromodomain-containing proteins and their roles in homeostasis and cancer. Nat Rev Mol Cell Biol 18:246–262

    Article  CAS  Google 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 288:31458–31467

    Article  CAS  Google Scholar 

  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 7:594–605

    Article  CAS  Google Scholar 

  19. Roy S, Luzwick JW, Schlacher K (2018) SIRF: quantitative in situ analysis of protein interactions at DNA replication forks. J Cell Biol 217:1521–1536

    Article  CAS  Google Scholar 

  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 150:922–933

    Article  CAS  Google 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.e245

    Article  CAS  Google Scholar 

  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 3:995–1000

    Article  Google Scholar 

  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 20:473–477

    Article  CAS  Google Scholar 

  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 43:9788–9803

    CAS  PubMed  PubMed Central  Google Scholar 

  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 29:589–598

    Article  CAS  Google Scholar 

  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) 22:1785–1792

    Article  CAS  Google Scholar 

  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.e418

    Article  CAS  Google Scholar 

  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:e31723

    Article  Google Scholar 

  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 35:193–207

    Article  CAS  Google Scholar 

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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.

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Authors and Affiliations

  1. Department of Pathology, University of Washington, Seattle, WA, USA

    Pavlo Lazarchuk & Julia Sidorova

  2. Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Sunetra Roy & Katharina Schlacher

Authors
  1. Pavlo Lazarchuk
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  2. Sunetra Roy
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  3. Katharina Schlacher
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  4. Julia Sidorova
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Corresponding author

Correspondence to Julia Sidorova .

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Editors and Affiliations

  1. NIH Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA

    Robert M. Brosh, Jr.

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Lazarchuk, P., Roy, S., Schlacher, K., Sidorova, J. (2019). Detection and Quantitation of Acetylated Histones on Replicating DNA Using In Situ Proximity Ligation Assay and Click-It Chemistry. In: Brosh, Jr., R. (eds) Protein Acetylation. Methods in Molecular Biology, vol 1983. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9434-2_3

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  • DOI: https://doi.org/10.1007/978-1-4939-9434-2_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9433-5

  • Online ISBN: 978-1-4939-9434-2

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