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In Vitro Reconstitution of In Vivo-Like Nucleosome Positioning on Yeast DNA

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 833)

Abstract

Genome-wide nucleosome mapping in vivo highlighted the extensive degree of well-defined nucleosome positioning. Such positioned nucleosomes, especially in promoter regions, control access to DNA and constitute an important level of genome regulation. However, the molecular mechanisms that lead to nucleosome positioning are far from understood. In order to dissect this mechanism in detail with biochemical tools, an in vitro system is necessary that can generate proper nucleosome positioning de novo. We present a protocol that allows the assembly of nucleosomes with very much in vivo-like positioning on budding yeast DNA, either of single loci or of the whole-genome. Our method combines salt gradient dialysis and incubation with yeast extract in the presence of ATP. It provides an invaluable tool for the study of nucleosome positioning mechanisms, and can be used to assess the relative stability of properly positioned nucleosomes. It may also generate more physiological templates for in vitro studies of, e.g., nucleosome remodeling or transcription through chromatin.

Key words

Nucleosome positioning In vitro reconstitution Saccharomyces cerevisiae Yeast extract Salt gradient dialysis 
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Notes

Acknowledgments

Work in our laboratory is funded by the German Research Community (DFG, grant within the SFB/Transregio 5) and through the 6th Framework Programme of the European Community (NET grant within the Network of Excellence The Epigenome). We thank Nils Krietenstein for critical reading of the manuscript. This paper is dedicated to the memory of Eduard Buchner, who founded biochemistry by demonstrating the power of yeast extracts.

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© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Molecular Biology Unit, Adolf-Butenandt-InstitutUniversity of MunichMunichGermany

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