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Yeast Nucleoplasmic Extracts and an Application to Visualize Chromatin Assembly on Single Molecules of DNA

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Yeast Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2196))

Abstract

In eukaryotic cells, the genomic DNA is packaged into chromatin, the basic unit of which is the nucleosome. Studying the mechanism of chromatin formation under physiological conditions is inherently difficult due to the limitations of research approaches. Here we describe how to prepare a biochemical system called yeast nucleoplasmic extracts (YNPE). YNPE is derived from yeast nuclei, and the in vitro system can mimic the physiological conditions of the yeast nucleus in vivo. In YNPE, the dynamic process of chromatin assembly has been observed in real time at the single-molecule level by total internal reflection fluorescence microscopy. YNPE provides a novel tool to investigate many aspects of chromatin assembly under physiological conditions and is competent for single-molecule approaches.

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References

  1. Olins AL, Carlson RD, Olins DE (1975) Visualization of chromatin substructure: upsilon bodies. J Cell Biol 64(3):528–537

    Article  CAS  Google Scholar 

  2. Olins AL, Senior MB, Olins DE (1976) Ultrastructural features of chromatin nu bodies. J Cell Biol 68(3):787–793

    Article  CAS  Google Scholar 

  3. Richmond TJ, Davey CA (2003) The structure of DNA in the nucleosome core. Nature 423(6936):145–150. https://doi.org/10.1038/nature01595

    Article  PubMed  CAS  Google Scholar 

  4. Woodcock CL, Skoultchi AI, Fan Y (2006) Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length. Chromosom Res 14(1):17–25. https://doi.org/10.1007/s10577-005-1024-3

    Article  CAS  Google Scholar 

  5. Sedat J, Manuelidis L (1978) A direct approach to the structure of eukaryotic chromosomes. Cold Spring Harb Symp Quant Biol 1:331–350

    Article  Google Scholar 

  6. Rattner JB, Lin CC (1985) Radial loops and helical coils coexist in metaphase chromosomes. Cell 42(1):291–296

    Article  CAS  Google Scholar 

  7. Belmont AS, Sedat JW, Agard DA (1987) A three-dimensional approach to mitotic chromosome structure: evidence for a complex hierarchical organization. J Cell Biol 105(1):77–92

    Article  CAS  Google Scholar 

  8. Kireeva N, Lakonishok M, Kireev I, Hirano T, Belmont AS (2004) Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure. J Cell Biol 166(6):775–785. https://doi.org/10.1083/jcb.200406049

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Dehghani H, Dellaire G, Bazett-Jones DP (2005) Organization of chromatin in the interphase mammalian cell. Micron 36(2):95–108. https://doi.org/10.1016/j.micron.2004.10.003

    Article  PubMed  Google Scholar 

  10. Schalch T, Duda S, Sargent DF, Richmond TJ (2005) X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Nature 436(7047):138–141. https://doi.org/10.1038/nature03686

    Article  PubMed  CAS  Google Scholar 

  11. Robinson PJ, Fairall L, Huynh VA, Rhodes D (2006) EM measurements define the dimensions of the "30-nm" chromatin fiber: evidence for a compact, interdigitated structure. Proc Natl Acad Sci U S A 103(17):6506–6511. https://doi.org/10.1073/pnas.0601212103

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Song F, Chen P, Sun D, Wang M, Dong L, Liang D, Xu RM, Zhu P, Li G (2014) Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344(6182):376–380. https://doi.org/10.1126/science.1251413

    Article  PubMed  CAS  Google Scholar 

  13. Guerra RF, Imperadori L, Mantovani R, Dunlap DD, Finzi L (2007) DNA compaction by the nuclear factor-Y. Biophys J 93(1):176–182. https://doi.org/10.1529/biophysj.106.099929

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Bhaskara S, Knutson SK, Jiang G, Chandrasekharan MB, Wilson AJ, Zheng S, Yenamandra A, Locke K, Yuan JL, Bonine-Summers AR, Wells CE, Kaiser JF, Washington MK, Zhao Z, Wagner FF, Sun ZW, Xia F, Holson EB, Khabele D, Hiebert SW (2010) Hdac3 is essential for the maintenance of chromatin structure and genome stability. Cancer Cell 18(5):436–447. https://doi.org/10.1016/j.ccr.2010.10.022

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Campagna-Slater V, Mok MW, Nguyen KT, Feher M, Najmanovich R, Schapira M (2011) Structural chemistry of the histone methyltransferases cofactor binding site. J Chem Inf Model 51(3):612–623. https://doi.org/10.1021/ci100479z

    Article  PubMed  CAS  Google Scholar 

  16. Novo CL, Tang C, Ahmed K, Djuric U, Fussner E, Mullin NP, Morgan NP, Hayre J, Sienerth AR, Elderkin S, Nishinakamura R, Chambers I, Ellis J, Bazett-Jones DP, Rugg-Gunn PJ (2016) The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells. Genes Dev 30(9):1101–1115. https://doi.org/10.1101/gad.275685.115

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Lebofsky R, Takahashi T, Walter JC (2009) DNA replication in nucleus-free Xenopus egg extracts. Methods Mol Biol 521:229–252. https://doi.org/10.1007/978-1-60327-815-7_13

    Article  PubMed  CAS  Google Scholar 

  18. Sparks J, Walter JC (2018) Extracts for analysis of DNA replication in a nucleus-free system. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot097154

  19. Banaszynski LA, Allis CD, Shechter D (2010) Analysis of histones and chromatin in Xenopus laevis egg and oocyte extracts. Methods 51(1):3–10. https://doi.org/10.1016/j.ymeth.2009.12.014

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Liao S, Toczylowski T, Yan H (2008) Identification of the Xenopus DNA2 protein as a major nuclease for the 5′ → 3′ strand-specific processing of DNA ends. Nucleic Acids Res 36(19):6091–6100. https://doi.org/10.1093/nar/gkn616

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Sukhanova MV, D'Herin C, Boiteux S, Lavrik OI (2014) Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: proteolytic degradation of the large subunit of replication protein a in ddc1Delta strains. DNA Repair (Amst) 22:30–40. https://doi.org/10.1016/j.dnarep.2014.07.002

    Article  CAS  Google Scholar 

  22. Damodaren N, Van Eeuwen T, Zamel J, Lin-Shiao E, Kalisman N, Murakami K (2017) Def1 interacts with TFIIH and modulates RNA polymerase II transcription. Proc Natl Acad Sci U S A 114(50):13230–13235. https://doi.org/10.1073/pnas.1707955114

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Rodriguez-Campos A, Koop R, Faraudo S, Beato M (2004) Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract. Nucleic Acids Res 32(13):e111. https://doi.org/10.1093/nar/gnh107

    Article  PubMed  PubMed Central  Google Scholar 

  24. Zhang Z, Reese JC (2006) Isolation of yeast nuclei and micrococcal nuclease mapping of nucleosome positioning. Methods Mol Biol 313:245–255. https://doi.org/10.1385/1-59259-958-3:245

    Article  PubMed  CAS  Google Scholar 

  25. Estruch F, Perez-Ortin JE, Franco L (1986) Fractionation of yeast chromatin by micrococcal nuclease digestion. Cell Mol Biol 32(2):195–199

    PubMed  CAS  Google Scholar 

  26. Germond JE, Hirt B, Oudet P, Gross-Bellark M, Chambon P (1975) Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc Natl Acad Sci U S A 72(5):1843–1847

    Article  CAS  Google Scholar 

  27. Greene EC, Wind S, Fazio T, Gorman J, Visnapuu ML (2010) DNA curtains for high-throughput single-molecule optical imaging. Methods Enzymol 472:293–315. https://doi.org/10.1016/S0076-6879(10)72006-1

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Ladoux B, Quivy JP, Doyle P, du Roure O, Almouzni G, Viovy JL (2000) Fast kinetics of chromatin assembly revealed by single-molecule videomicroscopy and scanning force microscopy. Proc Natl Acad Sci U S A 97(26):14251–14256. https://doi.org/10.1073/pnas.250471597

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Wagner G, Bancaud A, Quivy JP, Clapier C, Almouzni G, Viovy JL (2005) Compaction kinetics on single DNAs: purified nucleosome reconstitution systems versus crude extract. Biophys J 89(5):3647–3659. https://doi.org/10.1529/biophysj.105.062786

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Chen X, Zhao E, Fu YV (2017) Using single-molecule approach to visualize the nucleosome assembly in yeast nucleoplasmic extracts. Sci Bull 62(6):399–404. https://doi.org/10.1016/j.scib.2017.02.011

    Article  CAS  Google Scholar 

  31. Xue H, Zhan Z, Zhang K, Fu YV (2018) Visualizing the interaction between the Qdot-labeled protein and site-specifically modified λDNA at the single molecule level. J Vis Exp 137:e57967. https://doi.org/10.3791/57967

    Article  CAS  Google Scholar 

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Acknowledgments

We are grateful to Dr. Yujie Sun of Peking University and Dr. Chunlai Chen of Tsinghua University for useful discussion. We thank that Dr. Hasan Yardimci and Dr.Sevim Yardimci of the Francis Crick Institute for their kind help in the single-molecule experiments. This study was supported by the National Natural Science Foundation of China (31970553, 31571288), CAS Interdisciplinary Innovation Team, and the Newton Advanced Fellowship (NA140085) from the Royal Society.

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Authors and Affiliations

  1. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Yong Wang & Yu V. Fu

  2. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China

    Yong Wang

  3. Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China

    Yu V. Fu

Authors
  1. Yong Wang
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  2. Yu V. Fu
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Correspondence to Yu V. Fu .

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Editors and Affiliations

  1. Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, College of Life Sciences, Capital Normal University Beijing, China, Saskatoon, SK, Canada

    Wei Xiao

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Wang, Y., Fu, Y.V. (2021). Yeast Nucleoplasmic Extracts and an Application to Visualize Chromatin Assembly on Single Molecules of DNA. In: Xiao, W. (eds) Yeast Protocols. Methods in Molecular Biology, vol 2196. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0868-5_15

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  • DOI: https://doi.org/10.1007/978-1-0716-0868-5_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0867-8

  • Online ISBN: 978-1-0716-0868-5

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