Etudes sur le mécanisme de remodelage des nucléosomes par ...

tel-00413908, version 1 - 7 Sep 2009
alterations in the nucleosome structure, induced by the incorporation of some histone variants,
affect the capacity of chromatin remodelers to mobilize the histone variant nucleosomes
(Angelov et al., 2004; Doyen et al., 2006a; Gautier et al., 2004).
SWI/SNF was the first discovered chromatin remodeling complex (Peterson and Herskowitz,
1992). SWI/SNF in involved several processes, including transcription (Peterson and
Herskowitz, 1992), DNA repair (Chai et al., 2005), splicing (Batsche et al., 2006) and
telomeric and ribosomal DNA silencing (Dror and Winston, 2004). It consists of ∼11 subunits
and exhibits a central cavity. The dimensions of the cavity (∼15 nm in diameter and ∼5 nm in
depth) fit well with these of the nucleosome, suggesting that the cavity would be viewed as a
nucleosome-binding pocket (Smith et al., 2003). This indicates that SWI/SNF would interact
and remodel only one nucleosome at the time.
Despite numerous studies, the mechanism of action of the remodeling complexes is far from
being clear. Two different general classes of models were proposed (recently reviewed in
(Gangaraju and Bartholomew, 2007). According to the first class of models, DNA moves on
the surface of the histone octamer in 1 bp waves. This model is, however, inconsistent with
several recent reports (see for review Gangaraju and Bartholomew, 2007). According to the
second class of models, favored in the literature, the remodeler creates a bulge on the
nucleosomal surface, which is further directionally propagated (Gangaraju and Bartholomew,
2007). Since the dimensions of SWI/SNF are quite large and its contacts with DNA are
extensive (the nucleosome is supposed to “fill” the SWI/SNF cavity), a large fragment of
DNA could be involved in the SWI/SNF induced bulge formation and indeed, according to
the single-molecule experiments the average size of the bulge was found to be about 110 bp
(Zhang et al., 2006). Note that each one of the models described the mobilization of the
nucleosome as a continuing, non-interrupted process, which is achieved without dissociation
of the remodeler from the nucleosome.
In this manuscript we have studied the SWI/SNF nucleosome mobilization mechanism by
using a combination of high resolution microscopy techniques (Atomic Force Microscopy
(AFM) and Electron Cryo-Microscopy (EC-M)) and novel biochemistry approaches, which
allowed measurements with high precision of the DNA accessibility towards restriction
enzymes at 10 bp resolution all along the nucleosomal DNA length. We showed that
SWI/SNF uses a two-step mechanism to mobilize the nucleosome. The first step involves
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