Tài liệu Báo cáo khoa học: Chromatin under mechanical stress: from single 30 nm fibers to single nucleosomes pdf

These techniques have allowed direct mea-surements of several essential physical parameters of individual nucleo-somes and nucleosomal arrays, including the forces responsible for the ma

Chromatin under mechanical stress: from single 30 nm

fibers to single nucleosomes

Jan Bednar1,2,3and Stefan Dimitrov4

1 CNRS, Laboratoire de Spectrometrie Physique, St Martin d’Heres, France

2 Charles University in Prague, First Faculty of Medicine, Institute of Cellular Biology and Pathology, Prague, Czech Republic

3 Department of Cell Biology, Institute of Physiology, Academy of Science, Prague, Czech Republic

4 Institut Albert Bonniot, Grenoble, France

Introduction

Since the pioneering use of micromechanical and single

molecule manipulation approaches to probe biological

systems back in the late 1980s and 1990s (e.g [1–5]),

their use has continuously expanded In this review we

will focus mainly on the approaches using optical and

magnetic tweezers for studying the structure and

con-formational transitions of chromatin

The basic repeating unit of chromatin, the

nucleo-some, represents the first level of the chromatin

organi-zation [6] The major part of the nucleosome (termed

the chromatosome [7]) is composed of an octamer of

core histones (two each of H2A, H2B, H3 and H4), a

linker histone and  166 bp ( 56 nm) of DNA [6]

The histone octamer alone associates with 146 bp of DNA ( 50 nm) wrapped round in 1.65 left-handed superhelical turns (Fig 1) to form the nucleosome core particle (NCP), the structure of which has been solved

to 1.9 A˚ resolution by X-ray crystallography [8] The neighboring chromatosomes are connected by linker DNA

The linear array of nucleosomes folds into 30 nm fiber, the second level of chromatin organization The linker histones and the core histone NH2 tails and their post-translational modifications are essential for both the folding process and the maintenance of the chromatin fiber [9–11] as well as for the maintenance

Keywords

chromatin, micro-manipulation, nucleosome,

optical tweezers

Correspondence

J Bednar, CNRS, Laboratoire de

Spectrometrie Physique, UMR 5588, BP87,

140 Av de la Physique, 38402 St Martin

d’Heres Cedex, France

Fax: +33 476 51 45 44

Tel: +33 476 51 47 61

E-mail: jbedn@lf1.cuni.cz

(Received 22 November 2010, revised 7

April 2011, accepted 28 April 2011)

doi:10.1111/j.1742-4658.2011.08153.x

About a decade ago, the elastic properties of a single chromatin fiber and, subsequently, those of a single nucleosome started to be explored using optical and magnetic tweezers These techniques have allowed direct mea-surements of several essential physical parameters of individual nucleo-somes and nucleosomal arrays, including the forces responsible for the maintenance of the structure of both the chromatin fiber and the individual nucleosomes, as well as the mechanism of their unwinding under mechani-cal stress Experiments on the assembly of individual chromatin fibers have illustrated the complexity of the process and the key role of certain specific components Nevertheless a substantial disparity exists in the data reported from various experiments Chromatin, unlike naked DNA, is a system which is extremely sensitive to environmental conditions, and studies car-ried out under even slightly different conditions are difficult to compare directly In this review we summarize the available data and their impact

on our knowledge of both nucleosomal structure and the dynamics of nucleosome and chromatin fiber assembly and organization

Abbreviations

ACF, ATP-dependent chromatin assembly and remodeling factor; HMG, high-mobility group; NAP-1, nucleosome assembly protein 1; NCP, nucleosome core particle.

of mitotic chromosomes [12,13] The globular domain

of the linker histone is internally located in the 30 -nm

chromatin fiber [14], although how it interacts with

both the NCP and the linker DNA remains a subject

of debate [15,16]

The conformation of the 30 nm chromatin fiber is

sensitive to ionic conditions [9] The fiber adopts a

relaxed zigzag structure at low ionic strength and

undergoes compaction with increasing salt

concentra-tion, reaching a very compact form under

physiologi-cal conditions The linker DNA arrangement in the

most compact form of the chromatin fiber continues to

be a controversial issue [15–19]

Micromechanical approaches were used to study

three different aspects of chromatin organization: the

mechanical properties of (a) mitotic chromosomes and

(b) an individual nucleosome or a single 30 nm

chro-matin fiber, and (c) the rheology of chrochro-matin in vivo

(e.g [20–22]) Mitotic chromosomes have been the

sub-ject of several ‘mechanical’ studies [23–29] and some of

the stretching experiments were performed long before

the invention of optical tweezers [30–33] These studies

have recently been thoroughly reviewed [34] and this

review will thus concentrate on reviewing single

mole-cule studies of individual nucleosomes, nucleosomal

arrays and 30 nm chromatin fibers

Chromatin samples ‘eligible’ for single

molecule experiments

All micromechanical experiments applied to a

nucleo-some or chromatin fiber require an adaptation of the

substrate in order to make it suitable for attachment

to a ‘micro-handle’ In the case of experiments with

optical tweezers, micro-beads of dielectric material

(sil-ica, polystyrene) are the most frequently used type of

‘handle’ A typical configuration of the optical

twee-zers stretching experiment is depicted in Fig 2 The

chromatin substrate is tethered between the two beads

by means of a very tight interaction, typically using biotin⁄ streptavidin or digoxigenin⁄ anti-digoxigenin coupling between the fiber ends and beads

Four distinct types of chromatin substrates have been used for stretching experiments: (a) native chro-matin, isolated from nuclei after microccocal nuclease digestion, (b) chromatin reconstituted in vitro by salt

Fig 1 Chromatin organization and scheme of chromatin array stretching under different force regimes A nucleosomal core particle, formed

by 147 bp of DNA and a histone octamer, is complemented with linker histone (H1) and an additional 20 DNA bp to form the chromato-some Linker DNA completes and links consecutive nucleosomes which fold into the 30-nm chromatin fiber During stretching the nucleoso-mal array is first stretched to its contour length Additional stretching leads to the rupture of inter-nucleosonucleoso-mal interactions and the array is stretched to the beads-on-a-string configuration Further force increase results in progressive eviction of histone octamers The force values are approximate (see text) (adapted from [69,80]).

Fig 2 Optical tweezers experimental setup The laser beam (LB)

is conducted via a dichroic mirror (DM) to the back aperture of the objective lens (OL) which focuses the beam and creates the optical trap (OT) at the focal point The filament (F) (DNA, chromatin fiber etc.) is tethered between a trapped bead (TB) and a bead (FB) held

by suction onto a micropipette (MP) The micropipette is coupled to

a high precision micro-positioning system (typically a piezoelectric

XY plate) The image of the bead is projected onto a position sen-sor (PS) As the fiber is stretched beyond its curvilinear length, the bead in the trap will start to displace from the center of the trap and the force which is trying to bring the bead back is linearly pro-portional to this displacement Thus, the change of the fiber length

as a function of the force can be measured, resulting in a so-called force–extension curve.

dialysis, (c) nucleosome assembly protein 1 (NAP-1)

and ATP-dependent chromatin assembly and

remodel-ing factor (ACF) assembled chromatin and (d)

chro-matin assembled in nuclear extracts These distinct

substrates have different properties and

advanta-ges⁄ disadvantages for the experiments Native

chro-matin fibers, isolated after light micrococcal nuclease

digestion from nuclei, exhibit heterogeneous lengths

(different numbers of nucleosomes per individual

fiber) and both their protein composition and state of

histone modifications are poorly defined The

nucleos-omal arrays reconstituted by salt dialysis on tandem

repeats of positioning DNA sequences (601 [35] or 5S

[36]) have defined length, and the nature and

modifi-cation state of histones can be controlled, but the

proper association of linker histones in vitro is

diffi-cult Therefore, this material is mostly studied in their

absence The use of NAP-1⁄ ACF systems has allowed

the reconstitution of chromatin using any DNA

sequence, but it does not solve the issue of linker

his-tone assembly The preparation of chromatin

frag-ments in nuclear extracts does not require a DNA

substrate bearing positioning sequences and the

num-ber of nucleosomes will depend only on the length of

DNA used The linker histone will be present,

although its type will vary depending on the type of

extract used Unfortunately, in addition to the

chro-matin assembly proteins, the extracts contain a large

number of other proteins which can eventually form

distinct DNA–protein complexes These could affect

the physical properties of the assembled chromatin

fiber and consequently the interpretation of the

mea-sured elastic parameters

Mechanical properties of native 30 nm

fiber

The first ever single molecule micromanipulation

experiment on chromatin focused mostly on the elastic

behavior of the 30-nm chromatin fiber as a function of

its environmental conditions [37] In the low ionic

strength (5 mm NaCl) and low force regime

(< 10 pN), the measured stretching curve exhibited a

rather extended plateau, which was interpreted as fiber

accordion-like extension and disruption of

inter-nucle-osomal interactions The energy of these interactions

was estimated to be around 3.4 kBT per nucleosome

The authors observed the onset of a hysteresis in

repeated stretch⁄ relaxation cycle curves at a force of

about 20 pN Its origin was attributed to eviction of

some histone octamers from the fiber by mechanical

stress These experiments allowed the determination of

several physical parameters The persistence length of

the fiber and its stretch modulus at low salt conditions were determined to be 30 nm and 5 pN, respectively The chromatin fiber showed similar elastic behavior when the experiments were performed in 40 mm NaCl However, the forces necessary to achieve the same extension of the fiber were significantly higher This was attributed to the more compact initial conforma-tion of the fiber Although the compacconforma-tion level of native chromatin fibers (containing the linker histone)

is significantly higher in 150 mm NaCl than in 40 mm [9], quite surprisingly the experiments in 150 mm did not show significant differences in the fiber elastic characteristics compared with those at 40 mm

As mentioned earlier, about 166 bp of DNA is asso-ciated with the chromatosome and 145 bp with the NCP The nucleosome structure can thus be consid-ered as a ‘DNA length’ buffer When the stretching forces applied on the chain of nucleosomes exceeds the mechanical resistance of the DNA⁄ histone contacts, a mechanical unwrapping of DNA from the histone oct-amer will occur If this release is discontinuous (i.e a certain characteristic length of DNA is released in an all-or-none event) this will lead to a drop of instant stretching force and a sawtooth profile of the force⁄ extension curve will appear This is referred to as

a ‘disruption’ event As the nucleosome will not reas-semble, the length of the fiber will remain increased and in the next stretch⁄ relaxation cycle a different extension curve will be observed In this study [37], the sawtooth pattern could not be directly observed as the stretching was effected in discrete steps of about

50 nm, a value similar to the total length of nucleoso-mal DNA

Mechanical properties of chromatin reconstituted in egg extract

Another work used chromatin fibers reconstituted in Xenopus laevis egg extract [38] Stretching these fibers

at a continuous speed of 1 lmÆs)1revealed a sawtooth profile, which started to appear at forces above 20 pN and continued until about 40 pN The analysis revealed three distinct characteristic DNA release lengths: 65, 130 and 195 nm The 65 nm was attributed

to single nucleosome disruption and the two others were attributed to the simultaneous dissociation of two and three nucleosomes, respectively

The direct attribution of the observed released lengths to a single nucleosomal DNA unwrapping, however, appeared not to be straightforward Upon eviction of the histone octamer and histone H1 (i.e the disruption of the chromatosome) 166 bp of DNA

is expected to be released, i.e 56.4 nm and not 65 nm

To explain this, it was suggested [38] that other

non-histone proteins, namely high-mobility group (HMG)

family members, abundant in the X laevis egg extract,

were associated with the nucleosome This would result

in reinforcing the nucleosome mechanical resistance

and in locking of additional DNA into the complex

These suggestions were not experimentally addressed,

however

These experiments allowed also calculation of the

assembly rate of the nucleosomes, which was found to

be about three nucleosomes per second For the length

of k DNA (48 kbp) and the nucleosomal repeat length

(200 bp), the assembly of chromatin would thus be

complete in about 80 s under these experimental

condi-tions This is far shorter than the chromatin assembly

time (typically a few hours) in bulk in vitro

reconstitu-tion in egg extracts [39] When a force countering the

DNA shortening (due to nucleosome formation) was

applied, the rate of DNA shortening gradually

decreased and was finally halted at forces above 10

pN A similar fast rate of nucleosome assembly was

also observed in experiments where DNA was

stretched by hydrodynamic shear forces and incubated

in nuclear extracts [40] This apparent contradiction

between the rates of assembly of single molecules and

bulk chromatin could be explained by the very high

histone : DNA ratio in the single molecule experiment

compared with those in the bulk experiments When

competitive DNA (in amounts needed to reach the

DNA : histone ratio typical for bulk experiments) is

added to such a system, the nucleosome assembly rate

dramatically decreases (Claudet, Bednar and Dimitrov,

unpublished results)

Unwrapping individual nucleosomes in

reconstituted nucleosomal arrays

A detailed study of the mechanical behavior of in vitro

(by salt dialysis) reconstituted nucleosomal arrays (on

DNA templates containing 17 tandem repeats of the

5S positioning sequence from sea urchin) was

accom-plished by Brower-Toland and colleagues [41] In their

experiments, the arrays did not contain linker histones

and the stretching was performed in 100 mm NaCl,

1.5 mm MgCl2 The stretching profiles were recorded

with either constant stretching speeds or at constant

force The force–extension curves showed characteristic

sawtooth patterns at forces starting at about 20 pN

with 17 nominal peaks and a regular length of

sub-strate elongation steps of about 27 nm Very similar

values were observed in the constant force regime

Interestingly, the authors also observed a continuous

non-DNA stretching profile under a low force regime

(< 15 pN) This part of the curve was interpreted as a continuous unwinding of nucleosomal DNA from the histone octamer, mainly from contacts with histones H2A⁄ H2B where the DNA–histone interactions are supposed to be weak [42] The total amount of DNA released was calculated to be 158 bp per nucleosome, a value slightly higher than the 147 bp expected It was concluded that 76 bp of DNA per nucleosome is unwound continuously in the low force regime, and

82 bp dissociates under stresses higher than 20 pN in

an all-or-none fashion The calculated energy (using dynamic force spectroscopy theory [43]) necessary to dissociate the DNA from the histone octamer was 21–

22 kcalÆmol)1

In the multiple stretching cycle experiments, the reappearance of peaks was observed when the time gap between successive cycles was sufficiently long (at least 10 s) and the stretching force in the preceding cycle did not exceed 50 pN It was suggested that forces below this value did not cause a complete evic-tion of all histone octamers Some of the octamers may have remained attached to the DNA (probably at the dyad region, where the DNA–histone interactions are the strongest), and upon DNA relaxation nucleo-some reassembly could occur This phenomenon was not observed in the case of stretching experiments using chromatin reconstituted in X laevis egg extracts [38]

The part of the stretching profile interpreted as ‘con-tinuous release of the outer DNA turn’ [41] is very similar to the initial plateau in the experiments with native chromatin under similar ionic conditions [37], interpreted as chromatin fiber accordion-like extension and reflecting the disruption of the nucleosome–nucle-osome interactions Unfortunately, in [41] there is no comparison with stretching profiles in low salt condi-tions, which would help to clarify the contribution of inter-nucleosomal interactions or elastic contributions

of chromatin fiber compaction Note that the direct comparison of results reported in these two studies [37,41] is rather difficult as the fibers used in [37] were about 15-fold longer and contained linker histones In addition, the experiments were carried out under dif-ferent ionic conditions

The experiments were further refined with arrays reconstituted with tail-less histones or histones with modified NH2-termini [44] The removal of N-termini

of all core histones had a strong impact on both the length of the outer DNA turn and the peak force, which dropped by nearly 40 bp (from 65 bp in intact

to 28 bp in tail-less octamer nucleosomes) and to 3

pN, respectively Also the released DNA lengths in the case of the nucleosome with intact tails were revised

and found to be 65 bp (instead of 76 bp in [41]) for

continuous release of the outer DNA turn and 72 bp

(instead of 82 bp) for disruption of the inner turn In

all studied cases the removal or modification of histone

tails influenced the stretching profile and the effect

concerned mainly the outer DNA turn while the inner

turn was only minimally affected A similar

phenome-non was also observed for nucleosomal arrays

reconsti-tuted with the H2A.Bbd histone variant octamer In

this case a 2 pN drop of threshold disruption forces

(from 19 pN for conventional nucleosomes to 17 pN

for H2A.Bbd nucleosomes) was measured [45] The

last result is in agreement with the data obtained from

other methods showing a weaker association of the

variant H2A.Bbd octamer with DNA [45,46]

Obviously, similar experiments performed on

nucle-osomal arrays prepared by salt dialysis and by

assem-bly in egg extracts (see above) gave quite divergent

results While the threshold force values were very

sim-ilar (about 20 pN), the lengths of DNA released upon

mechanical disruption of nucleosomes were quite

dif-ferent The values of 65 nm or 130 nm measured in

the case of egg extract assembled fibers [38] were never

observed for chromatin reconstituted by salt dialysis

Gemmen et al [47] performed analogous

experi-ments on nucleosomal arrays prepared in vitro by

using the histone chaperone NAP-1 and ACF which

forms nucleosomal arrays on random DNA sequences

with nucleosomal repeat of about 168 bp [48,49]

Although the features of the measured stretching

pro-files were generally comparable with the results of [41]

(including the DNA re-wrapping in repeated stretching

cycles), some important differences were observed The

disruption length varied from 55 bp to 95 bp and the

threshold forces ranged from 5 to 65 pN In addition,

the authors found a clear dependence of the average

threshold force on ionic conditions, ranging from 24

pN in 100 mm NaCl to 31 pN in 5 mm NaCl The

wide range of measured threshold forces was attributed

to the variation of histone octamer affinities to the

given underlying DNA sequence

We have studied the elastic properties of both native

chromatin samples (isolated from chicken erythrocytes

and containing linker histones) and nucleosomal arrays

reconstituted by salt dialysis [50] We found the same

values of basic characteristics of the majority of

dis-ruption events, i.e the peak force and the released

DNA length, as reported in [42] (20 pN and 25 nm)

However, a minor population of events exhibited a

dis-ruption length centered at 50 nm, thus corresponding

very closely to the 147 bp of DNA released upon

dis-ruption How can we explain this finding? It was

previ-ously reported that the integrity of the nucleosomal

structure depends on two factors: (a) the ionic condi-tions and (b) the concentration of the chromatin itself [51] At very low concentrations of chromatin, the structure is destabilized and a progressive dissociation

of the linker histone and H2A–H2B dimers from the nucleosome is observed [50,51] When the stretching experiments with native chromatin fibers were repeated under conditions favoring histone octamer stability (presence of exogenous chromatin or low ionic strength) a significant increase in the number of 50-nm events was observed This was interpreted as an effect

of histone octamer stabilization and the release of all the DNA associated with a histone octamer in an all-or-none event [50] A similar effect was observed with arrays containing 12 nucleosomes reconstituted on 5s positioning sequences Further analysis showed that indeed under conditions typical for single molecule experiments (where the chromatin concentration is usually extremely low) H2A–H2B dimers as well as lin-ker histones readily dissociated from the nucleosomes even at moderate ionic concentrations [50] The remaining (H3–H4)2 tetramers associate with only one superhelical turn of DNA and consequently, upon stretching, the release of only 25 nm in a single disrup-tion event will be observed

Why then were the peaks with 25-nm release length not observed in experiments with egg extract reconsti-tuted chromatin? One of the possible explanations is the association of non-histone proteins (e.g HMG proteins) with chromatin, leading to an additional sta-bilization, mainly of the outer turn The study of Pope and colleagues [52] showed that the situation might be even more complex In their work they focused mainly

on the elastic response of chromatin fibers assembled

in X laevis egg extract to different loading rates (i.e the force increase per time unit) They detected three typical disruption lengths: 30 nm, 59 nm and 117 nm These data differed from the results of Bennink et al [38], where the 30-nm disruption length was not detected, and revealed two distinct energy barriers hav-ing values of 25 and 28 kBT (14.5 kcalÆmol)1 and

16 kcalÆmol)1) With high loading rates the value of the first barrier dropped to 20 kBT (12 kcalÆmol)1) The individual lengths were attributed to a disruption

of the entire nucleosome in one event (60 nm), simulta-neous release of the DNA from two nucleosomes (117 nm) or the partial unraveling of one DNA turn (30 nm) In addition to the explanation of Brower-To-land and Wang [53], Pope et al [52] also considered the possibility of disruption of an incomplete nucleo-some – missing either one or both H2A–H2B dimers The individual energy barriers were attributed

to nucleosomes with and without linker histone B4

(the embryonic linker histone variant present in the

egg extract) Based on the analysis, the linker histone

contribution to nucleosomal ‘stability’ was estimated

to be rather low, about 3 kBT, which would reflect the

fact that no significant difference in threshold force

was observed between nucleosomal arrays with and

without linker histones [50] In repeated stretching

experiments the number of events with high energy

barriers (28 kBT) rapidly decreased suggesting the

per-manent removal of B4 from the nucleosomes during

the initial stretching No correlation between the

dis-ruption length and the energy barrier was found The

value of the barrier was significantly lower than that

reported in [41] (16 kcalÆmol)1 versus 20–22

kcalÆ-mol)1) but again the experiments were carried out in

different ionic conditions (10 mm Tris⁄ HCl, pH 7.5,

1 mm EDTA, 150 mm NaCl, 0.05% BSA and 0.01%

NaN3) and the chromatin samples were assembled by

different techniques

The mechanical properties of nucleosomal arrays

reconstituted on African green monkey alpha-satellite

DNA were studied by Bussiek et al [54] They found

disruption of alpha-satellite nucleosomes to occur at a

higher force on average – 26.4 pN versus 21.7 pN for

random DNA nucleosomes The authors hypothesized

that the increased bending flexibility of alpha-satellite

DNA (due to the presence of clustered CA⁄ TG steps)

would result in the formation of more stable

nucleo-somes as less energy is needed for DNA bending

Zooming in on the stretching of a

single nucleosome

Analysis of the experiments with nucleosomal arrays is

always complicated by the elastic contribution of the

inter-nucleosomal interactions at different ionic

con-centrations This could be overcome by analyzing the

properties of a single mononucleosomal template

Mihardja et al [55] prepared a mononucleosomal

tem-plate on a 2582-bp long DNA construct containing a

single 601 positioning sequence [35] Stretching profiles

of these particles showed several features not

previ-ously observed Pulling the template at very low

load-ing rates, the first discontinuity in the stretchload-ing curve

was observed at forces centered at  3 pN and the

length of the released DNA was determined to be

21 nm A second peak occurred at forces around

8–9 pN with a similar length, 22 nm These events

were interpreted as a successive release of the outer

and the inner wrap of the nucleosomal DNA The first

unwrapping was reversible, provided the stretching

curve did not reach the second discontinuity

Experi-ments conducted under a constant force regime

ranging between 2 and 3 pN revealed a bistable char-acter of the first event with a dwell time in the unwrapped state depending on the force value, increas-ing with increased force From these measurements the free energy of the outer turn unwrapping was calcu-lated to be  6 kcalÆmol)1 The unwrapping of the sec-ond, inner turn represented by the second peak at about 8 pN was not reversible Its analysis with load-ing rates in the range 2.4–11 pNÆs)1 revealed that the dependence of the probability of unwrapping on the force was not linear Therefore, the unwrapping of the inner turn cannot be considered as a simple two-state process but will involve some intermediate states as well The same experiments were also performed at high salt concentrations (200 mm potassium acetate) Under these conditions, the first low force transition was transformed into a nearly continuous plateau rather than a sharp peak and the high force transition was shifted to lower forces

These experiments identified at least two novel fea-tures of the nucleosome elasticity behavior First, the value of the disruption force was lowered to about half of that originally reported (9 pN versus 20 pN) and, second, the experiments clearly showed that unwrapping of the outer turn was not continuous as reported previously [41] The differences in these experimental data from those obtained with nucleoso-mal arrays could reflect both the differences in the experimental conditions and the nature of the starting material The single nucleosome experiments avoid all contributions coming from the fiber-like behavior of the nucleosomal arrays, which is strongly dependent

on the ionic conditions The forces needed to stretch the fibers containing native linker histone without dis-rupting the nucleosomes (5 pN [37]) are roughly equal

to or greater than the threshold forces for unwrapping

of the outer turn (3 pN [55]) It is thus likely that at forces up to 5 pN two events are happening simulta-neously – an unwrapping of the outer DNA turn and stretching of the folded nucleosomal array The result-ing elastic profile would reflect a superposition of these two events This would in turn result in a smeared, plateau-like characteristic of the stretching curve at low forces rather than resolved peaks The different composition of buffers used in the experi-ments make the comparison even more difficult As the nucleosome stability strongly depends on ionic conditions, in order to directly compare the data the experiments have to be carried out under exactly the same ionic conditions Rather high concentrations of

Mg2+ (10 mm magnesium acetate) together with

 50 mm potassium acetate, however, were used in the single nucleosome experiments [55], while 100 mm

NaCl and 1.5 mm MgCl2 were used for nucleosomal

array stretching [41]

An interesting approach to investigate the stability

of a single nucleosome was used by Shundrovsky et al

[56] Instead of pulling tethered nucleosomal templates,

they ‘unzipped’ the DNA of a reconstituted template

containing a single 601-positioned nucleosome The

nucleosome was flanked by free DNA arms and, upon

stretching, the first 220 bp of naked DNA were

unzipped before the histone octamer was reached The

unzipping of DNA associated with the histone octamer

was affected by histone–DNA contacts within the

nucleosome and reflected the strength of the histone–

DNA interactions The unzipping profile of the

nucleo-some showed three distinct high force regions

(contrary to the first two, the third region was not

reg-ularly observed) Within these regions, forces up to 45

pN had to be applied in order to overcome the barrier

The first peak was observed at about 50 bp from the

dyad upon applying an average force of 31 pN, while

the second one was observed in the vicinity of the

nucleosome dyad and at 37 pN average force These

peaks were attributed to the disruptions of the strong

interactions between H2A–H2B dimers and H3–H4

tetramers, respectively The attribution of the first peak

to the disruption of the H2A⁄ H2B–DNA interaction

was confirmed by stretching a particle reconstituted

with the (H3–H4)2 tetramer only The unzipping

pro-file of this tetrameric particle exhibited only the

sec-ond, high force peak According to the authors, the

third peak was associated with the instability of the

nucleosome when most of the nucleosomal DNA was

unzipped

These experiments were further refined [57], allowing

analysis of the DNA–histone interactions with near

base-pair resolution The unzipping was carried out

under a constant force regime using a 28-pN trapping

force The strength of the interaction was found to be

proportional to the time needed for its disruption This

allowed mapping of the interaction strength of the

dif-ferent regions with a resolution of about 1.5 bp The

recorded data again revealed three regions of strong

interactions (longer dwell times): one was located close

to the dyad, while the other two were symmetrically

located at positions ±40 bp from the dyad All three

exhibited a 5-bp periodicity The data demonstrated

that the unzipping of the first 20 bp of nucleosomal

DNA had the same characteristics as those of naked

DNA, indicating a loose interaction of the histones

with DNA at the entry⁄ exit points of the NCP Very

similar results were obtained in continuous stretching

regime measurements with loading rates of 8 pNÆs)1,

as well as when random DNA sequences instead of

positioning sequences were used for nucleosome recon-stitution

Magnetic tweezer experiments

Several experiments with magnetic tweezers have also been reported (for the principles of magnetic tweezers see for example [58,59]) Magnetic tweezers can mea-sure forces about 1–2 orders smaller than optical twee-zers and, unlike optical tweetwee-zers, they can also control the torsion of the fiber

Leuba et al [60] studied NAP-1 mediated assembly

of chromatin fibers on k DNA using magnetic twee-zers They observed an inhibition of the fiber assembly

at forces of  10 pN, but they also registered disas-sembly events (in an otherwise progressive asdisas-sembly process) at forces of about 5 to 7.5 pN This suggested that the equilibrium forces were in this range

Experiments using a similar strategy, but in X laevis egg extracts, were realized by Yan et al [61] The experiments were carried out either in ATP-depleted extract or in extract containing a defined concentration

of ATP or non-hydrolyzable ATP They found that in ATP-depleted extract forces of only 4 pN resulted in inhibition of nucleosome assembly At forces below 3.5 pN, the extract was able to accomplish the assem-bly although the number of assembled nucleosomes was significantly lower relative to the nucleosomal array reconstituted under optimal conditions (the mea-sured nucleosomal repeat was only 280 bp in contrast

to the 180–160 bp repeat reported for fully extract-assembled chromatin [39]) The 3.5 pN value was determined as an equilibrium force of ATP-indepen-dent nucleosome assembly giving straightforwardly the free energy of DNA–histone octamer association as

27 kcalÆmol)1 Once the assembly was completed, the fibers were stretched with different loading rates Dur-ing this process, a step-wise fiber lengthenDur-ing was observed with a predominating step value of 50 nm, attributed to an unwrapping of one complete nucleo-some The presence of 30- and 100-nm steps was also detected Interesting changes were induced by addition

of ATP to the extract In this case, the disassembly threshold force decreased to  1 pN Non-hydrolyz-able ATP did not affect the nucleosome assembly⁄ disassembly equilibrium force determined in ATP-depleted extract

Why did these two very similar experiments give rise

to such different results? First, the egg extract contains

a poorly defined composition of proteins compared with the purified NAP-1 assembly system It is quite possible that some ATP-independent protein com-plexes present in the egg extract can associate with the

nucleosomes and modify their mechanical stability.

The steep drop to 1 pN in the stall force in the

pres-ence of ATP, however, is quite surprising The events

observed in the stretching profile under these

condi-tions did not correspond to an assembly of individual

nucleosomes, but rather to formation and release of

rather long ‘loops’ (200–400 nm) The fact that the

energy provided by the added ATP in the system was

not even partly used for assisted nucleosome assembly

is also surprising However, the authors have observed

nucleosome-like disassembly steps of 50 and 100 nm

when the force was increased to over 5 pN

Impor-tantly, no reverse (i.e assembly) events were detected

even at low forces

Kruithof et al [62] carried out experiments on

strongly subsaturated oligonucleosomal arrays (one to

four nucleosomes present on 17 tandem repeats of 5s

DNA) using magnetic tweezers with sub picoNewton

resolution This experiment is directly comparable with

the work of Mihardja et al [55] Although both groups

used very similar conditions, Kruithof et al did not

observe any DNA unwrapping from the nucleosome

below forces of 6 pN, even though they used a

posi-tioning sequence with lower affinity for the histone

octamer (5s versus 601)

The data obtained by force spectroscopy of

chroma-tin are not always easy to interpret unambiguously

and to explain in terms of changes in nucleosome and

fiber structure and dynamics While in lower salt

con-centrations (50 mm) and in the absence of bivalent

ions the inter-nucleosomal interactions can be

neglected, the situation becomes more complex when

the fiber is studied in its compact form, where the

pres-ence⁄ absence of linker histones, the higher

concentra-tion of monovalent ions, and the presence of bivalent

or polyvalent ions contribute significantly to the fiber

properties Kruithof et al [63] used improved

tech-niques of linker histone association [64,65] to prepare

defined chromatin arrays of 25 nucleosomes with two

different nucleosomal repeat lengths (197 and 167 bp)

and used magnetic tweezers to study their elastic

behavior The stretching curves of these samples

exhib-ited four major regions The first was attributed to the

extension of the DNA segments flanking the array of

25 nucleosomes which serve as a handle for tethering

The second region (at forces up to 4 pN) represented

the extension of the chromatin fiber The third region

(a plateau observed at 4–4.5 pN) was attributed to the

disruption of inter-nucleosomal interactions The last

region was interpreted to reflect extension of the

beads-on-a-string fiber The incorporation of the linker

histone had only a minor effect on the overall form of

the stretching profile Upon H5 association, the third

region (plateau) was shifted to a higher force value –

7 pN – suggesting that linker histone stabilizes nucleosomal stacking However, its absence did not compromise chromatin folding when Mg2+ was pres-ent (1.5 mm MgCl2) When Mg2+ ions were depleted from the solution, the behavior of the fibers without linker histones changed A disruption of the inter-nu-cleosomal interactions at forces of about 3.5 pN and

an increasing irreversibility upon repeated stretching cycles (in the presence of 100 mm NaCl) were observed Reintroduction of Mg2+ resulted in a com-plete recovery of the original folding pattern, suggest-ing that, at least under these conditions, the linker histone might not be required for proper chromatin folding The analysis of fiber stretching profiles, their Hookian behavior, their length and transition to extended beads-on-a-string structures in the third and fourth regions of the stretching curve led the authors

to conclude that in its compact form the fiber is orga-nized in a one-start solenoidal topology The data obtained on fibers with 167 bp nucleosomal repeat were significantly different [63] Surprisingly, their con-tour length at 0.5 pN stretching force was longer than for fibers with 197 bp nucleosomal repeat and their measured stiffness was found to be 2.7-fold higher (0.052 versus 0.019 pNÆnm)1) This was interpreted as

a consequence of their different topological organiza-tion and a two-start helix topology was suggested as best fitting the observed data

However, the story of chromatin fiber folding is apparently more complex Other studies have demon-strated that for longer nucleosomal repeats both linker histone and Mg2 +ions are required in order to reach maximal packing levels of the chromatin [66] This is not valid for short nucleosomal repeats where, even in the absence of linker histone, the fiber can maximally pack in a regular manner [66] It would therefore be interesting to see whether the same elastic behavior (i.e linker-histone-independent compaction in the pres-ence of bivalent ions) would be observed for longer nucleosomal repeats The presence of Mg2+ also resulted in a substantial increase of the inter-nucleoso-mal stacking energy to about 17 kBT, compared with the value of 3.4 kBT observed in [37] for native chro-matin fibers in the absence of bivalent ions This clearly demonstrates an important role for bivalent ions in chromatin fiber stabilization It should also be mentioned that the presence of bivalent ions not only influences chromatin stability [10,67,68] but may also direct the topology of its folding Indeed, analysis of the data obtained on native chromatin fibers with rather long nucleosomal repeat and in the absence

of bivalent ions [37], using metropolis–Monte Carlo

DNA–histone octamer

continuously unwrapped,

simulation [69], proposed the zigzag organization of

the fiber as the best fitting to measured elastic profiles

Therefore, the organization of the chromatin fiber in

its compact state remains an open issue and it is very

likely that variable topologies can be adopted

depend-ing on the given conditions [18]

Chromatin arrays under twist

The group of Viovy used magnetic tweezers to study

the behavior of a 36 nucleosome long array

reconsti-tuted on the tandem repeat of 5s DNA under torsional

stress [70] The acquired data allowed the

determina-tion of several elastic parameters of the fiber, namely

the persistence length (28 nm) and the stretch modulus

(8 pN), which are quite close to the values obtained

for native chromatin fibers (30 nm persistence length

and 8 pN stretch modulus) determined in [37]

How-ever, the determined torsional persistence length

(5 nm) differed markedly from the value of 35 nm

obtained by WLC (worm-like chain) modeling of

simi-lar arrays, using canonical nucleosomes [71] A new

model of the fiber was therefore proposed, where the

nucleosomes could exist in three different

configura-tions according to the crossing of the entry⁄ exit DNA

segments: negatively crossed, open and positively

crossed Transitions between the different

configura-tions are possible and energies of 0.4 kcalÆmol)1 and

1.2 kcalÆmol)1 from negative to open and positive to

open nucleosome states, respectively, fitted the

experi-mental data very well As linker histone was not

pres-ent in the system, transitions between individual

configurations (crossings) of nucleosomes could be

facilitated

Further experiments have revealed that the behavior

of the fiber differed significantly during stress

relaxa-tion [70] While in the case of negative twist the

pro-cess was essentially reversible, in the case of positive

twist a very significant hysteresis was observed, as if

the stress (and the resulting shortening) was released in

time by an internal structural rearrangement of the

fiber When the H2A–H2B histone dimers were

selec-tively removed from the nucleosomes, the hysteresis

disappeared Based on previous detailed studies on

nucleosomal polymorphism [72–76], the authors

pro-posed a specific mechanism for this rearrangement,

which required a flip of the nucleosomal chirality from

left-handed to right-handed

Concluding remarks

In this review, we have summarized available data on

the mechanical properties of nuclesomes and

chroma-tin We did not include single molecule experiments in which functional aspects of nucleosomal interactions with other complexes were examined (e.g [77,78]) or experiments where single molecule techniques other than micromechanical manipulation were used (e.g [79]) Still, the situation appears to be rather complex,

as documented in Table 1 where data obtained in selected studies are compared As can be seen, in some cases the data from very similar experiments are quite divergent This reflects the high sensitivity of the stud-ied chromatin samples to a number of parameters Obviously, the traction parameters, i.e the loading rate, turns out to be particularly important It is there-fore not surprising that data from early experiments, using in general quite high loads, are quite similar (e.g

a disruption force around 20 pN), but very different from the latest data (3–11 pN) The ionic conditions and the buffer composition are also very important factors, as they can influence the octamer stability or the DNA–octamer association strength It is also clear that the choice of DNA substrate has an impact on the results [57] The question of the effect of the linker histone association still remains an open issue as most

of the array stretching experiments were carried out in the absence of linker histone Although substantial progress has been made in the micromanipulation of chromatin substrates, many additional experiments will certainly be needed in order to evaluate the effects of individual factors that potentially influence the mechanical properties of chromatin substrates

Acknowledgement

This work was supported by grants from INSERM and CNRS S.D acknowledges ANR-09-BLAN-NT09-485720 ‘CHROREMBER’ J.B acknowledges the support of the Ministry of Education, Youth and Sports (MSM0021620806 and LC535) and the Acad-emy of Sciences of the Czech Republic (Grant

#AV0Z50110509)

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