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

tel-00413908, version 1 - 7 Sep 2009
showed a shift in ΔL distribution with the peak value at ~ 50 bp indicative of octamer
movement to the end of the DNA (Figure III.3 D).
We conclude that prior to mobilization, SWI/SNF generates particles associated with
additional ~30 bp DNA and this results in strong perturbations of the histone DNA-
interactions. For simplicity we will refer to these particles, further in the text, as remosomes
(remodeled nucleosomes). The remosomes were stable since we have observed them after gel
elution and gel eluted remosomes exhibited the same morphology as the remosomes observed
directly in the reaction mixture without gel purification (Montel et al., 2007).
A B
Probability density
Nucleosome Remodeling
SWI - + ++
1 2
3
PAGE fractionation
0.014
0.012
0.01
0.008
0.006
0.004
0.002
Elution of nucleosome
particles from gel
AFM
Band 1 (α)
Band 2 (α+β)
Band 3 (γ)
0
0 50 100 150 200 250 300
DNA complexed length (bp)
Band 1 (α)
Band 2 (α+β)
Band 3 (γ)
C D
Probability density
Figure III.3. SWI/SNF generates non-mobilized nucleosomes particles associated with ~180 bp of
DNA. (A) Schematics of the experiment. Centrally positioned nucleosomes were reconstituted on 255
bp 601 DNA sequence. The histone octamer is localized close to the center of the fragment, leaving
two free DNA arms with legths of 56 bp (L+) and 52 bp (L-), respectively. The nucleosomes were
incubated with increasing amounts of SWI/SNF for 45 minutes at 29°C and after arresting the reaction
with apyrase, they were run on a 5% PAGE. The bands corresponding to either the non-slided (upper
band) or slided (lower band) nucleosomes were cut, the nucleosome particles were eluted from the gel
slices and analyzed with AFM. (B) AFM visualization of the gel-eluted nucleosomes. First row, gel
eluted control nucleosomes (incubated in the absence of SWI/SNF); 2 nd row, nucleosomes from upper
electrophoretic band incubated with SWI/SNF in the presence of ATP; 3 rd row, nucleosomes eluted
from the lower gel band. (C) Lc distribution of the gel eluted nucleosomes from the non-slided and
slided nucleosome fractions, Lc is the length of the DNA associated with the histone octamer [Lc= Lt-
(L+-L-)]. (D) ΔL distribution of gel eluted nucleosomes to measure the position of octamer with respect
to DNA arms. For unremodeled (α) (n=5806), remodeled (α+β) (n=4448) and slided (γ) (n= 6410)
nucleosome Lc and ΔL distributions (C and D) are represented in blue, red and green color
respectively.
122
0.01
0.008
0.006
0.004
0.002
100 nm
Band 1 (α)
Band 2 (α+β)
Band 3 (γ)
0
0 50 100
Position ΔL (bp)
150