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

tel-00413908, version 1 - 7 Sep 2009
conditions described in (A) and gel eluted (fraction I, see (A). (C) and (D), 2D histograms for the
upper gel band eluted nucleosomes incubated with 30 fmol (fraction II, see (A), N=635 nucleosomes
and 60 fmol, (fraction III, see (A) N=255 nucleosomes, of RSC. (E) 2D histogram for the nucleosomes
eluted from the excised lower gel band after incubation for 30 minutes with RSC (N= 538
nucleosomes). The inserts show the distinct nucleosome species corresponding to the different regions
of the 2D histograms.
The clear visualization of the free DNA arms allows the precise measurement of the DNA
length of each arm (indicated as L+ and L- for the longer and the shorter arm, respectively). To
measure the length of each arm, we have excluded the octamer part and the trajectory of the
free DNA was determined by using morphological tools avoiding false skeletonization by
heuristic algorithm (Figure II.2A and Materials and Methods). The precise measurements of
the length of the arms allowed the calculation of both the length of the DNA complexed with
the histone octamer Lc (Lc = Ltot - L+ - L-, where Ltot= 255 bp is the length of the 601 fragment
used for reconstitution) and the position of the nucleosome relative DNA template center ∆L=
(L+ - L-)/2. The 2D histogram Lc/∆L for the control nucleosomes (treated with ATP in the
absence of RSC and eluted from the gel particles) is presented in Figure II.2B. The maximum
of the distribution peaked at ∼ 145 bp and ∆L is ∼5-8 bp, which is in a good qualitative
agreement with the determination of the nucleosome position by biochemical approaches.
Importantly, in the absence of ATP, RSC has no effect on the Lc/∆L map (data not shown)
The 2D histograms Lc/∆L for the nucleosomes incubated with RSC (in the presence of ATP)
and eluted from the gel slice nucleosomes were, however, quite different (Figure II.2 C-E).
The data show that both variables, Lc and ∆L, are significantly different in the distinct RSC
generated nucleosome populations. Indeed, at the lower amount (30 fmol) of RSC present in
the remodeling reaction the Lc/∆L map for the nucleosomes isolated from the upper
electrophoretic band was getting wider indicating that particles with overcomplexed DNA
(more than 150 bp in length) were generated (Figure II.2C). The presence of the higher
amount (60 fmol) of RSC resulted in the generation of mainly particles with short free DNA
arms (isolated from the upper band) and containing about 180-190 bp DNA in complex with
the histone octamer (Figure II.2D). Importantly, the nucleosome position ∆L relative to the
DNA ends in these particles remained essentially the same as in the control particles,
suggesting that the increased amount of DNA associated with the octamer is achieved through
pumping of about 15-20 bp of DNA from each free DNA arm without nucleosome
repositioning. For simplicity, further in the text we will call these particles remosomes
(remodeled nucleosomes).
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