1
IMA Sept 15th-21st 2007
Nucleosome dynamics probed by
torsional manipulation of single
chromatin fibers
Prunell et al.
2
Scheme of the magnetic tweezers set-up
Force
(500 bp)
(600 bp)
(2x18 5S 208- or 190-bp)
(600 bp)
(500 bp)
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EM of typical 2x18 5S 190 bp chromatin
fibers with linkers in TE
Clusters of close-packed nucleosomes
~1100-bp nucleosome-free DNA spacers and stickers
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Part 1
A fiber of three-state
nucleosomes
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Force-vs.-extension of a 5S 2x18
208-bp chromatin fiber in TE
Unfolding
regime
Entropic
regime
Chromatin
Naked DNA
Smooth curves by the worm-like rope elasticity model
(Bouchiat et al. 1998, Phys. Rev. Lett. 80, 1556-1559):
Chromatin stretching modulus ~ 8 pN
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Extension-vs.-rotation of a 5S 2x18 208bp chromatin fiber and its corresponding
naked DNA in TE
Elastic regime
Plectonemic
regime
Strand
separation
Chromatin
Naked DNA
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Fiber parameters before and after partial
nucleosome depletion
NAP-1 + 50 mM NaCl
f = 7.7 pN
f = 7.7 pN
f = 0.7 pN
14 steps of 24±2 nm =
14 nucleosomes removed
Topological shifts
∆(length)/topological shift ~ 1.35 µm/24 turns = ~55 nm/turn
or ~160 bp/turn
~ 700 nm/13 turns
or 54 nm /turn
∆(Topological shift)/∆(nucleosome) ~ -11/14 = -0.8±0.1
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Regular and irregular fibers
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Slope ~ 55 nm/turn
Regular fibers contain mostly regularly spaced nucleosomes
and are on the straight line
Irregular fibers contain a significant number of close-packed
nucleosomes of smaller topological deformation
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Fibers are extremely flexible in torsion
compared to DNA of the same length
2
2
Renormalized
naked DNA
Smooth curve by the worm-like rope model:
Bending persistence length ~ 28 nm
Torsional persistence length ~ 5 nm against 80 nm for DNA
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Irregular fibers are less torsionally flexible
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6
2
Slope ~ 55 nm/turn
2
7
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The two-angle molecular model
of the canonical (all-negative) 2x18 208-bp fiber
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∆Lkm(turn)
-1.4
 between entry/exit DNAs
 between successive nucleosomes
∆l : rise per nucleosome
∆Lkt : theoretical topological
deformation per nucleosome
∆Lkm : experimental deformation for a
single nucleosome on a DNA minicircle
of the same 5S sequence
Elastic regime
Torsional persistence length ~ 35 nm (against 5 nm for the real fiber)
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Nucleosome three conformational states
Recent reviews of the DNA minicircle approach :
Prunell A. & Sivolob A. (2004) New Comprehensive Biochemistry, 39,
45-73
Sivolob A. & Prunell A. (2004) Phil. Trans. Roy. Soc. A. 362, 1519 1547
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All-open- and all-positive fibers
entry/exit
nucleos./nucleos.
rise
t for theoretical
m for minicircle
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The equilibrium three-state model of the
chromatin fiber
Fiber 2
Elastic regime
Plectonemic
regimes
The model uses the experimentally-determined number of
nucleosomes and, as adjustable parameters, the difference
in energy between the states (negative - open = 0.7 kT;
positive - open = 2 kT)
Slope of the plectonemic regimes: ~ 25 nm/turn
against 90 nm/turn for the naked DNA
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Details of the modelled fiber
Torsionally-relaxed fiber
(zero torque)
negative
Steady-state
number of
nucleosomes
(total = 31)
Torque
(pN.nM/rad)
open
positive
Models:
worm-like rope
molecular
Circles: onsets of plectoneme formation at constant torque
The maximal length maximizes the number of open
nucleosomes at ~ +5 turns from the relaxed state
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Part 2
The nucleosome chiral
transition
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The hysteretic torsional response
Naked DNA
Hysteretic shifts on the positive side (
) remain constant for a given
fiber over many cycleds of rotation, while it varies on the negative side, in
particular as a function of the acquisition time
The fiber maximal extension is approximately the same in forward
and backward curves
Conclusion: The backward curve as a whole tends to shift to larger
rotations relative to the forward curve as a consequence of positive
turn trapping by the fiber after excursion at high torsion
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Hysteretic shifts are proportional to the
number of regularly-spaced nucleosomes
Lkfibre  0.85nreg  0.5(ntotal  nreg )
(1)
1200
208
 6.5nreg  L DNA
(36 ntotal )
8680
8680
50
4(ntotal  nreg ) L DNA
(ntotal  nreg  1)
8680
L fibre  L DNA
(2)
²Lk fibre : topological shift

Lfiber : length
nreg : number of regularly-spaced nucleosomes
ntotal : number of all nucleosomes
8260 bp : total DNA length (36 x 208 + 2 x 600)
50 bp : addition to the naked DNA length made by each close-packed nucleosome
Slope = 1.3 ±0.1
turns/reg. nucl.
ntotal
nreg
Conclusion: close-packed nucleosomes contribute little to the hysteresis
Shift increases at a rate of 1.3 turns per regularly spaced nucleosome.
With a topological deformation of the positively-crossed nucleosome
in the positive plectonemic regime of ~ -0.4 , the topological
deformation of the altered nucleosome form is 1.3 -0.4 = 0.9 ±0.2
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The altered nucleosome form contains
the H2A-H2B dimers
2- removal of H2A-H2B dimers at 3.5 pN with 250 nM yNAP-1 dimers in TE + 50 mM NaCl
followed by rinsing in TE and return to 0.2 pN results in a hysteresis-free fiber
3- rescue of initial fiber length and hysteresis upon incubation with 40 nM H2A-H2B dimers
chaperoned with 40 nM NAP-1 dimers and rinsing in TE
4- all histones removed (with heparin)
Conclusion:
The altered nucleosome form must contain H2A-H2B dimers as they are required
for the hysteresis
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An elusive tetrasome fiber
Direct reconstitutions with tetramers lead to authentic hairpin-like tetrasomes
only at low histone/DNA ratios.
Ratios insuring a reasonable particle density along the DNA produce pseudonucleosomes made of two stacked (H3-H4)2 tetramers
Insets: particles obtained upon reconstitutions on a ~ 200 bp 5S DNA fragment
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The true tetrasome fiber
Dimers removed by
Dimers removed
Naked DNA
1 µg/ml heparin
+ 1 µg/ml core particles
as histone
acceptors
700 mM NaCl
An authentic tetrasome fiber (obtained by H2A-H2B removal and not by direct
reconstitution) shows :
1) an absence of hysteresis
2) a maximal extension intermediate between the initial chromatin fiber and naked DNA
consistent with tetrasome wrapping of less than a turn
3) an absence of topological shift relative to naked DNA.
This is consistent with the known ability of single tetrasomes on DNA minicircles to
fluctuate between pseudo-mirror-symmetrical left- and right-handed chiral conformations
of nearly equal and opposite ∆Lk (-0.7 and + 0.6 (±0.05) on 5S DNA). This in turn implies
that the tetrasome fiber at its center of rotation contains an equal steady state number of
particles of each chirality
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The altered nucleosome versus the right-handed
tetrasome
1 µg/ml heparin
+ 1 µg/ml core particles
as acceptors
700 mM NaCl
1) The tetrasome fiber and the nucleosome backward fiber have the same
center of rotation.
2) Their responses have the same breath.
3) The topological deformation of the altered nucleosome (+0.9) is similar
to that of the right-handed tetrasome on the same 5S sequence (+0.6)
The backward transition process must be similar for the altered nucleosome and
the tetrasome.
The difference in the forward responses may then solely reflect the activation
energy of the nucleosome transition linked to the presence of H2A-H2B dimers.
Whole nucleosomes also switch their conformation from lefthanded to right-handed, and the core of the altered form is the righthanded tetrasome
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The transition in real time: a test for the
metastability of the altered nucleosome
A- Fiber length monitored at constant force in the backward curve.
The proportions of the different states (inset) is used to estimate the energy
parameters of the transition (from the ground state of the open nucleosome)
according to standard calculation:
- Equilibrium energy difference = 10 ±2 kT
- Energy barrier = 30 ±5kT
B- Length monitored in the forward curve at constant force.
No extension is observed in 30 min, consistent with the >90 % straight
nucleosome proportion at steady state (measured from the inset in A at infinite
time), and the high energy barrier.
A conversion to the altered form is observed only when the equilibrium is
displaced toward the altered state by a force >2.3 pN.
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Salt-dependence of the torsional response
and the chiral transition
•
•
Fibers in higher salt (25 - 50 mM NaCl) tend to progressively
compact from one cycle of torsion to the next. This is due to saltinduced attractive interactions between the nucleosomes, which
are mediated by the tails. However, a force of a few pN applied at
the center of rotation breaks these interactions and rescues the
initial response which is close to that in low salt.
The hysteresis tends to disappear and forward and backward
curves tend to merge toward an intermediate curve. Real-time
monitoring shows a substantial decrease in the energies:
– Equilibrium energy difference = 6 ±2 kT, against 10 ±2 kT in
low salt
– Energy barrier = 25 ±5 kT, against 30 ±5 kT in low salt.
Because the transition rate depends exponentially on the
energy barrier, the equilibrium between the two states
becomes more dynamics relative to the time-scale of data
acquisition, explaining the merging
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Putative scenario for the transition
Similar fiber extension in forward and backward curves
implies a similar length component along the force of canonical
and altered nucleosome forms
Conclusion :
The two forms should wrap the same length of DNA with similar
compactions
Nucleosome
Reversome
∆Lk ~-1
positive rotation
∆Lk ~+1
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Physiological relevance of the data
Part 1
Nucleosome access to three conformational states (depending on the
crossing status of entry-exit DNAs: negative, null or positive) solves a number
of previous topological enigmas of DNA in chromatin.
The most famous is the linking number paradox, where two negative
superhelical turns around the nucleosome reduce (on average) the linking
number by only 1 (instead of 2). The key nucleosomes here are the positively
crossed ones, because they are almost topologically neutral and contribute
little to the overall linking number reduction.
Part 2
Nucleosome chiral transition into reversomes is likely to occur during
transcriptional elongation.
RNA polymerases exert a positive torque >1.25 kT/rd, i. e. 8 kT/turn,
sufficient in principle to trigger the transition (6 kT/turn in 50 mM salt). However,
it is the activation energy which specifies the conversion rate, and for this
reason it was important to know whether reversomes could be produced at a
distance in a time-scale consistent with the polymerase elongation speed.
We thus implemented a kinetic model in which a fiber is twisted at constant
angular velocity, the torsional constraint being relaxed by the nucleosomereversome transition in a steady-state manner. Given the speed of RNA
polymerase II (~20 nucleotides or 2 turns/sec), the above energy parameters
lead to the effective torque involved, ~1.5 kT/rad. This figure, close to the
above minimal torque value, suggests that the transition may indeed propagate
ahead of a transcribing polymerase.
If reversomes indeed form, what could their role be ?
H2A-H2B dimers exert an almost absolute block against transcription by RNA
polymerase II at physiological ionic strength. This has been observed on short
chromatin substrates, when positive supercoiling cannot accumulate due to free
rotation of the ends.
Reversomes, owing to their open character, have their dimers relatively
destabilized, suggesting they could behave as torsion-driven “activated”
nucleosomes poised for polymerase traversing.
The chiral-switching ability of the tetramer may then be viewed as the lever
used by the main RNA polymerase to break the docking of the dimers, via the
wave of positive supercoiling it pushes in its front.
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Work attributions
1) Jean-Louis Viovy, with Aurélien Bancaud and Gaudeline Wagner
(Institut Curie-Paris) provided and operated the magnetic tweezers
set-up
2) Jean-Marc Victor, with Julien Mozziconacci, Hua Wong and Maria
Barbi (LPTMC, Paris) modeled and simulated
3) Eric le Cam and Christophe Lavelle (Institut Gustave Roussy,
Villejuif) visualized the fibers
4) Liliane Mouawad (Institut Curie-Orsay) performed the Normal
mode analysis of tetrasome chiral transition
5) Andrei Sivolob (Taras Shevchenko National University, Kiev)
calculated the energies
6) Ariel Prunell and Natalia Conde e Silva (Institut Jacques Monod,
Paris) prepared the chromatin fibers
For more details, see:
- Bancaud et al. (2006) Nat. Struct. Mol. Biol. 13, 444-450.
- Bancaud et al. (2007) Mol. Cell 27, 135-147.
-and for a genealogy of the nucleosome superfamily, see:
- Lavelle & Prunell (2007) Cell cycle 6,2113-2119 (Review)