Meiosis pp 253-266 | Cite as

Analysis of Chromatin Structure at Meiotic DSB Sites in Yeasts

  • Kouji Hirota
  • Tomoyuki Fukuda
  • Takatomi Yamada
  • Kunihiro Ohta
Part of the Methods in Molecular Biology book series (MIMB, volume 557)


One of the major features of meiosis is a high frequency of homologous recombination that not only confers genetic diversity to a successive generation but also ensures proper segregation of chromosomes. Meiotic recombination is initiated by DNA double-strand breaks that require many proteins including the catalytic core, Spo11. In this regard, like transcription and repair, etc., recombination is hindered by a compacted chromatin structure because trans-acting factors cannot easily access the DNA. Such inhibitory effects must be alleviated prior to recombination initiation. Indeed, a number of groups showed that chromatin around recombination hotspots is less condensed, by using nucleases as a probe to assess local DNA accessibility. Here we describe a method to analyze chromatin structure of a recombination hotspot in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This method, combining micrococcal nuclease (MNase) digestion of chromatin DNA and subsequent Southern blotting, is expected to provide information as to chromatin context around a hotspot. Moreover, by virtue of MNase preferentially targeting linker DNA, positions of several nucleosomes surrounding a hotspot can also be determined. Our protocol is a very powerful way to analyze several-kb regions of interest and can be applied to other purposes.

Key words

Meiotic recombination recombination hotspot chromatin structure nucleosome linker DNA micrococcal nuclease (MNase) yeast indirect end labeling 



We thank Dr. Hajime Murakami for the original image of Fig. 16.1 .


  1. 1.
    Wolffe, A. (1997) in: Chromatin: Structure and function, 3rd edn, Academic Press, San Diego, USA.Google Scholar
  2. 2.
    Nightingale, K.P., O’Neill, L.P. and Turner, B.M. (2006) Histone modifications: signalling receptors and potential elements of a heritable epigenetic code. Curr. Opin. Genet. Dev. 16, 125–36.PubMedCrossRefGoogle Scholar
  3. 3.
    Eberharter, A. and Becker, P.B. (2004) ATP-dependent nucleosome remodelling: factors and functions. J. Cell Sci. 117, 3707–11.PubMedCrossRefGoogle Scholar
  4. 4.
    Felsenfeld, G., Boyes, J., Chung, J., Clark, D. and Studitsky, V. (1996) Chromatin structure and gene expression. Proc. Natl. Acad. Sci. U. S. A. 93, 9384–8.PubMedCrossRefGoogle Scholar
  5. 5.
    Keeney, S. and Neale, M.J. (2006) Initiation of meiotic recombination by formation of DNA double-strand breaks: mechanism and regulation. Biochem. Soc. Trans. 34, 523–5.PubMedCrossRefGoogle Scholar
  6. 6.
    Wu, T.C. and Lichten, M. (1994) Meiosis-induced double-strand break sites determined by yeast chromatin structure. Science 263, 515–8.PubMedCrossRefGoogle Scholar
  7. 7.
    Ohta, K., Shibata, T. and Nicolas, A. (1994) Changes in chromatin structure at recombination initiation sites during yeast meiosis. EMBO J. 13, 5754–63.PubMedGoogle Scholar
  8. 8.
    Mizuno, K., Emura, Y., Baur, M., Kohli, J., Ohta, K. and Shibata, T. (1997) The meiotic recombination hot spot created by the single-base substitution ade6-M26 results in remodeling of chromatin structure in fission yeast. Genes Dev. 11, 876–86.PubMedCrossRefGoogle Scholar
  9. 9.
    Moreno, S., Klar, A. and Nurse, P. (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823.PubMedCrossRefGoogle Scholar
  10. 10.
    Isshiki, T., Mochizuki, N., Maeda, T. and Yamamoto, M. (1992) Characterization of a fission yeast gene, gpa2, that encodes a G alpha subunit involved in the monitoring of nutrition. Genes Dev. 6, 2455–62.PubMedCrossRefGoogle Scholar
  11. 11.
    Hirota, K., Steiner, W.W., Shibata, T. and Ohta, K. (2007) Multiple modes of chromatin configuration at natural meiotic recombination hotspots in fission yeast. Eukaryot. Cell 6, 2072–80.PubMedCrossRefGoogle Scholar
  12. 12.
    Hirota, K., Hasemi, T., Yamada, T., Mizuno, K.I., Hoffman, C.S., Shibata, T. and Ohta, K. (2004) Fission yeast global repressors regulate the specificity of chromatin alteration in response to distinct environmental stresses. Nucleic Acids Res. 32, 855–62.PubMedCrossRefGoogle Scholar
  13. 13.
    Hirota, K., Hoffman, C.S. and Ohta, K. (2006) Reciprocal nuclear shuttling of two antagonizing Zn finger proteins modulates Tup family corepressor function to repress chromatin remodeling. Eukaryot Cell. 5, 1980–9.PubMedCrossRefGoogle Scholar
  14. 14.
    Hirota, K., Hoffman, C.S., Shibata, T. and Ohta, K. (2003) Fission yeast tup1-like repressors repress chromatin remodeling at the fbp1(+) promoter and the ade6-M26 recombination hotspot. Genetics 165, 505–15.PubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Kouji Hirota
    • 1
  • Tomoyuki Fukuda
    • 1
  • Takatomi Yamada
    • 1
  • Kunihiro Ohta
    • 1
  1. 1.Department of Life Sciences, Graduate School of Arts and SciencesThe University of Tokyo, and Shibata Distinguished Senior Scientist Laboratory, RIKEN Discovery Research InstituteJapan

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