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

tel-00413908, version 1 - 7 Sep 2009
III.3 Discussion
In this work we have studied the type and structure of the products of the SWI/SNF
nucleosome remodeling reaction by using highly resolution microscopy methods combined
with novel biochemistry approaches. This has allowed a detailed structural characterization of
the SWI/SNF reaction products. In the microscopy study we have used centrally positioned
nucleosomes, reconstituted on a 255 bp 601 DNA sequence. These nucleosomes exhibited
two free ∼50 bp DNA arms, which permitted the visualization of the structural alterations in
nucleosomal DNA upon remodeling. We found that, in addition to the mobilized
nucleosomes, SWI/SNF generates a multitude of nucleosome-like particles that we called
remosomes and which are associated with ∼180 bp of DNA, instead of 147 bp of DNA as in
the non-remodeled control nucleosomes. Importantly, the AFM data demonstrated that the
position of the histone octamer relative to the center of the DNA remained unchanged,
indicating that the remosome is generated by SWI/SNF “pumping” of 15-20 bp of DNA from
each individual free DNA arm. The “in gel one pot assay” illustrates that the histone-DNA
interactions within the remosomes are markedly perturbed all along the remosome DNA.
Importantly, the accessibility of dyad 6 and 7 (located at the very end of the nucleosomal
DNA) to HaeIII, in contrast to those of all the remaining dyads, is decreased in the
remosomes, which could be viewed as an evidence for generation of a stronger histone-DNA
interactions in the vicinity of this location, i.e. the “pumped” DNA interacting with the
histone octamer.
The DNase I footprinting pattern of the remosomes is clearly different from that of the
nucleosomes and is similar to free DNA. Since the remosomes appeared to be generated
without mobilization of the histone octamer, this points that the remosomes are not a set of
well defined particles as the parental nucleosomes are, but instead represent an ensemble of
heterogenous structures. The EC-M visualization of the remosomes confirms that this is really
the case. A common feature of the remosomes is their larger size than that of nucleosomes.
Importantly, each remosome shows an irregular and distinct DNA path, the strongest
irregulatities being observed at different locations relative to the center of the particles. This
indicates that within each individual remosome, a distinctly localized region with very
strongly perturbed histone-DNA interaction should exist. The presence of HaeIII immediate
cleavage regions all along the remosomal DNA is in perfect agreement with this statement.
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