Advancing Epigenetics Towards Systems Biology

Nucleosome mobilization assay (Prot 24)

Karim Bouazoune

Introduction

The organization of eukaryotic genomes into nucleosome arrays restricts DNA sequence accessibility to many nuclear factors. Thus most DNA-based processes require opening (or "re-closing") of these arrays. One major class of enzymes, the "chromatin/nucleosome remodeling" factors, uses ATP hydrolysis to alter the canonical histone-DNA contacts. The term "nucleosome remodeling" can be defined and monitored in different ways (Flaus and Owen-Hughes, 2004). The simplest configuration to study one aspect of nucleosome remodeling is to use a purely reconstituted system consisting of mononucleosomes and an ATP-dependent nucleosome remodeler in the so-called "nucleosome sliding" or "nucleosome mobilization" assay. This technique was initially developed by Carl Wu and Peter Becker laboratories (Hamiche et al., 1999; Langst et al., 1999) by taking advantage of two nucleosome properties: Nucleosomal histones can moderately move on DNA under rather mild temperature and salt conditions (Beard, 1978; Meersseman et al., 1991; Pennings et al., 1991) and nucleosomes reconstituted on a short DNA fragment can adopt multiple positions that can be separated by native gel electrophoresis (Linxweiler and Horz, 1984; Pennings et al., 1991).

In fact, the sliding assay monitors alterations in nucleosomes electrophoretic mobility in native gel that are caused by remodeling factors in an ATP-dependent manner. Repositioning of the histone octamer along a DNA fragment usually accounts for these mobility shifts. However, changes in electrophoretic mobility can also result from an altered (non-canonical) nucleosomal DNA path (Kassabov et al., 2003; Narlikar et al., 2001). Hence, actual repositioning of the histone octamer may need to be confirmed by mapping of the new positions for uncharacterized remodelers. It is also noteworthy that mononucleosomes do not recapitulate
all chromatin properties (Hansen, 2002). Consequently, all conclusions derived from using this substrate may not always apply to nucleosome arrays (even not to other mononucleosomes using a different DNA template). Despite these caveats, the sliding assay is still a powerful tool that has greatly contributed to our understanding of how chromatin remodeling factors work.

Acknowledgements: I am grateful to Gernot Längst and Anton Eberharter for introducing me to this great technique.

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Karim Bouazoune

Massachusetts General Hospital - Department of Molecular Biology - Harvard Medical School, Department of Genetics - 185 Cambridge Street - Boston, MA 02114, USA

Karim Bouazoune