From the Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse Cedex, France
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ABSTRACT |
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It has recently been proposed that the histone (H3-H4)2 tetramer undergoes structural changes, which allow the particle to accommodate both negatively and positively constrained DNA. To investigate this process, we modified histone H3 at the H3-H3 interface, within the histone (H2A-H2B-H3-H4)2 octamer or the histone (H3-H4)2 tetramer, by forming adducts on the single cysteine of duck histone H3. We used three sulfhydryl reagents, iodoacetamide, N-ethylmaleimide, and 5,5'-dithiobis(2-nitrobenzoic acid). Torsionally constrained DNA was assembled on the modified histones. The H3 adducts, which have no effect on the structure of the nucleosome, dramatically affected the structural transitions that the (H3-H4)2 tetrameric nucleoprotein particle can undergo. Iodoacetamide and N-ethylmaleimide treatment prevented the assembly of positively constrained DNA on the tetrameric particle, whereas 5,5'-dithiobis(2-nitrobenzoic acid) treatment strongly favored it. Determination of DNA topoisomer equilibrium after relaxation of the tetrameric nucleoprotein particles with topoisomerase I demonstrated that the structural transition occurs without histone dissociation. Incorporation of H2A-H2B dimers into the tetrameric particle containing modified or unmodified cysteines allowed nucleosomes to reform and blocked the structural transition of the particle. We demonstrate the importance of the histone H3-H3 contact region in the conformational changes of the histone tetramer nucleoprotein particle and the role of H2A-H2B in preventing a structural transition of the nucleosome.
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INTRODUCTION |
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The nucleosome, the repeated structural unit of chromatin, consists of two each of the core histones H2A, H2B, H3, and H4 (building up an "octamer"), around which is wrapped 146 bp1 of DNA in a left-handed superhelix (1). Several lines of evidence have suggested that the arginine-rich histones H3 and H4, which interact in solution to form a stable (H3-H4)2 tetramer, play an important role in the establishment of nucleosomal structure (2-5). The two H2A-H2B dimers complete the nucleosome by binding on both sides of the (H3-H4)2-DNA structure (6). The interaction of the histone H2A-H2B dimer with the histone (H3-H4)2 tetramer involves mostly hydrogen bonds and is significantly weaker than the interaction between H3 and H4 within the tetramer. This supports the model of a tripartite organization of the core histone octamer, the assembly of which is governed by positive cooperativity and reversibility (7, 8). In addition, histone H2A-H2B dimers are more easily released and interchanged from chromatin than (H3-H4)2 tetramers (9); the lability of the H2A-H2B dimer interaction within the nucleosome appears to additionally depend on RNA polymerase activity (10-12).
Posttranslational modifications of the core histones, such as
ubiquitination, methylation, phosphorylation, ADP-ribosylation, and
acetylation, probably result in subtle changes in nucleosome structure.
Among these modifications, the reversible acetylation of -amino
groups of lysine residues present in the amino-terminal domains, or
"tails," of the core histones, is the most strongly linked with
transcriptional activity. Certain transcriptional coactivators have
acetyltransferase activity, and they can be targeted to specific
promoters by transcription factors (13-15). More recently, it has been
demonstrated that histone deacetylase can also be targeted by
transcriptional repressors (reviewed in Ref. 16). Targeted histone
acetylation/deacetylation is likely to play a major role in inducing
nucleosome structural changes, thus allowing transcription complex
assembly/disassembly and/or RNA polymerase tracking.
The structural complexity of the core histones makes the nucleosome a master molecule that governs biochemical processes implicating chromatin. Mechanisms that modify the conformation of chromatin in living cells are of considerable interest because they are likely to determine the efficiencies of transcription, replication, recombination, and DNA repair. Gene activation is accompanied by alterations in the conformation of chromatin, which for highly transcribed genes are revealed by an increased sensitivity to cleavage by nucleases. The nature of these alterations at the level of nucleosomal structure is not known, but it is clear that both histone-DNA interactions and histone content are modified. One possible way to spread alterations along a chromatin fiber is through the torsional stress generated by RNA polymerase during transcription (17, 18). Garrard and co-workers (19, 20) have proposed that transient positive supercoils downstream of a tracking RNA polymerase result in nucleosome splitting. A similar conclusion was reached from an investigation of the influence of positive stress on nucleosome assembly (9, 21, 22). Recently, it was also shown that the histone (H3-H4)2 tetramer can associate with positively supercoiled DNA with a high affinity, probably as a result of a change in tetramer topology (23). Based on these observations, it has been proposed that a slight shift in the dimer-dimer interface may be all that is needed for the DNA to wrap the (H3-H4)2 tetramer in a right-handed helix (24).
To address the question of how the (H3-H4)2 tetramer can undergo a structural change allowing it to accommodate either negatively or positively supercoiled DNA, we modified the cysteine located at position 110 within the H3 molecule. X-ray crystallography studies have demonstrated that modifications of this cysteine do not change the histone structure (25, 26). Because the two cysteines face each other at a distance of 6.2 Å, this should create steric hindrances at the H3-H3 interface within the (H3-H4)2 tetramer. Adducts on the cysteine were formed by treating histone octamers and (H3-H4)2 tetramers with three different sulfhydryl reagents. We used these treated histones to reconstitute nucleoprotein particles on DNA mini-circles of negative and positive superhelicities. These experiments, together with an analysis of the topoisomerase I relaxation of the particles, demonstrate that the (H3-H4)2 tetramer can flip from the left-handed conformation it adopts within the nucleosome to a right-handed conformation. This flipping requires a reorientation of the H3-H3 interface, because adducts on cysteine 110 allow the freezing of the tetrameric particle in left- or right-handed conformations. The presence of histones H2A-H2B prevents this transition by blocking the tetramer in a left-handed structure. Such a conformational flexibility of the (H3-H4)2 tetramer could provide a fine-tuned mechanism for modulating the accessibility of DNA sequences within the nucleosome.
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MATERIALS AND METHODS |
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Preparation of Topologically Constrained Mini-circles
The murine mammary tumor virus-long terminal repeat 359-bp
fragment used to generate mini-circles spans the promoter from 280 to
+72. It was amplified by polymerase chain reaction using the following
primers, which contain an EcoRI site: primer 1, 5'-GCGAATTCTAGAACATTATTCTGCAAAAA-3', and primer virus
2,5'-GCGAATTCAGGATCCGTGACGAGCGGAGA-3'. The resulting DNA fragment
was digested with EcoRI and fractionated by
electrophoresis on polyacrylamide gel. The 359-bp band was eluted from
the gel. The DNA fragment was ligated to linearized dephosphorylated
pUC18 under conditions whereby two tandemly repeated fragments were
inserted into the vector (construct pUC359.2m). Negatively and
positively supercoiled DNA topoisomers were prepared from the above
fragment, after purification by gel electrophoresis and 32P
end-labeling by circularization in the presence of ethidium bromide and
netropsin, respectively. The different DNA topoisomers were separated
by electrophoresis on acrylamide gels, the bands were excised, and the
DNA was eluted (27).
Histone Purification
Duck erythrocyte nuclei were prepared according to the method
described in Ref. 28, and histone octamers (1-2 mg/ml) were prepared
as described previously (29). Long soluble chromatin obtained from
micrococcal nuclease digestion of nuclei was adsorbed onto a
hydroxyapatite column as described in Ref. 30. Non-histone proteins
histone H1 and histone H5, were eluted first, and the core histones
were eluted second by sequential washes with 0.65 M and 2 M NaCl (in 0.1 M potassium phosphate, pH 7, 0.25 mM phenylmethylsulfonyl fluoride, and 1 mM
2- mercaptoethanol), respectively. To isolate H2A-H2B and H3-H4
histones separately, after the 0.65 M NaCl wash, a stepwise
elution with 0.93 M NaCl allowed recovery of most of the
H2A-H2B histone dimers without any contamination with
(H3-H4)2 histone tetramers; 1.2 M NaCl eluted
the remaining H2A-H2B histone dimers and also some of
(H3-H4)2 histone tetramers; and 2 M NaCl elution allowed recovery of (H3-H4)2 histone tetramers,
free from H2A-H2B histone dimers. Samples were dialyzed against 2 M NaCl, 10 mM potassium phosphate (pH 7.6), and
0.25 mM phenylmethylsulfonyl fluoride. Histone H2A-H2B
dimers and histone (H3-H4)2 tetramers were concentrated by
centrifugation in Centricon-10 and Centricon-30 microconcentrators
(Amicon), respectively. Histone preparations were aliquoted and stored
at
80 °C until use.
Treatment of Histones with Sulfhydryl Reagents
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) Treatment--
DTNB
was prepared as a 10 mM stock solution in 100 mM potassium phosphate (pH 8.0). Histone octamers and
(H3-H4)2 tetramers in 10 mM potassium phosphate
(pH 7.6), 2 M NaCl (at a final thiol group concentration of
30 mM) were incubated with 0.25 mM DTNB under
the conditions described under "Results." The reaction was followed
by recording absorbance at 412 nm. Product yield was calculated from
its extinction coefficient, M = 13.6 mM
1 (Ellman's reagent; Ref. 31). Treated
histone octamers and tetramers were dialyzed to remove the excess of
reagent and stored at
80 °C.
N-Ethylmaleimide (NEM) Treatment--
NEM was prepared as a 1 M stock solution in methanol. Histones were treated at room
temperature with 1 mM NEM in 2 M NaCl and 10 mM phosphate buffer, pH 7.6. Histones were added directly to this solution in a 1-ml cuvette, and the reaction was followed by
measuring the decrease of the absorbance at 305 nm (32). The extinction
coefficient value M = 0.620 mM
1 was used for the calculations of reacted
NEM.
Iodoacetamide (IA) Treatment-- IA was prepared as a 1 M stock solution in water. Histones were treated with 1 mM IA for 2-3 h at room temperature.
Chromatin Reconstitution
To generate nucleoprotein particles, histone octamers or tetramers were assembled on topologically constrained DNA circles according to the "salt jump" method (33) as described in Ref. 29. The procedure involved the addition of 32P-labeled DNA topoisomer to the plasmid DNA (form I) from which the fragment originated (final DNA concentration, 200 mg/ml), followed by the suitable amount of histones (histone:DNA weight ratio = rw), in 2 M NaCl, 10 mM Tris-HCl (pH 7.5), 100 mg/ml bovine serum albumin. The mixture was first incubated for 10 min at 37 °C, diluted to 50 mg/ml DNA and 0.5 M NaCl, incubated at the same temperature for 30 min, and finally dialyzed at 4 °C against 10 mM Tris-HCl (pH 7.5), 1 mM EDTA for at least 1.5 h. For the experiments involving measurement of the DNA topoisomer:histone ratio (two-dimensional gel electrophoresis), nonradioactive topoisomer was substituted to the carrier plasmid DNA.
Assembly of histones H2A-H2B on tetrameric nucleoprotein particles was performed as follows: increasing amounts of purified H2A-H2B dimers were added to particle preparations; adjusted to 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 50 mM KCl, 5 mM MgCl2, 100 mg/ml bovine serum albumin; and incubated for 30 min at 37 °C.
For relaxation studies, nucleoprotein particle preparations were adjusted to 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 50 mM KCl, 5 mM MgCl2, 100 mg/ml bovine serum albumin, and the DNA was relaxed by incubation with 800-1000 units of calf thymus topoisomerase I (Life Technologies, Inc.) per ml at 37 °C for 1 h (34).
Gel Electrophoresis
Octameric and tetrameric nucleoprotein particles were
electrophoresed at room temperature in 4% polyacrylamide
(acrylamide/bisacrylamide, 29:1, w/w) slab gels (0.15 × 17 × 18 cm) in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). Gels were pre-electrophoresed for 1 h at
200-250 V and electrophoresed at the same voltage for 3-4 h with
extensive buffer recirculation. Gels were dried and autoradiographed at 80 °C. For the two-dimensional gel analysis, after the first dimension run in TE buffer (see above), the bands were excised an
loaded onto a second dimension 18% acrylamide denaturing gel (35). DNA
and histones were visualized by silver staining (36). In preparative
relaxation experiments, "chromatin" gels were dried without heating
to allow the reswelling of the excised gel slices and the elution of
the DNAs. Unless otherwise stated, naked DNA was electrophoresed at
room temperature in 4% polyacrylamide (acrylamide/bisacrylamide, 20:1,
w/w) slab mini-gels (0.15 × 10 × 8 cm) for 2 h at 100 V in 20 mM sodium acetate, 2 mM EDTA, 40 mM Tris acetate (pH 7.8). When required for the separation
of DNA topoisomers, chloroquine (125 µM) was included.
The radioactivity in the bands was quantitated in the dried gels using
a phosporimager (Fuji PC-Bas).
Topoisomer Identification, Linking Number Reduction Associated with Single Nucleoprotein Particle Formation; Topological Constraint in the Loop
Topoisomer linking difference relative to its most probable configuration is given by the equation,
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(Eq. 1) |
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(Eq. 2) |
Lk was used to identify the topoisomers, using the value
h0 = 10.56 bp/turn (measured under the DNA gel
electrophoresis conditions used here and for a migration at 25 °C
(27, 41)). This h0 value and the ring size
N = 359 bp were used to calculate Lk0 = 34.0 from equation (2) Topoisomers with Lk = 35, 34, 33, 32, and
31 have linking number differences
Lk = +1, 0,
1,
2, and
3, respectively (see Eq. 1).
The linking number increment associated with an octameric or tetrameric particle reconstitution is given by the following equation.
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(Eq. 3) |
Lkp0 is the linking number of the most probable configuration of the mini-circle partially wrapped around the histones. Lkp0 can be measured from the amounts of the different topoisomers in the equilibrium distribution obtained upon particle relaxation with topoisomerase I. If Lki and ri are the linking number and the amount of topoisomer i, respectively, then the following equation applies.
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(Eq. 4) |
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(Eq. 5) |
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RESULTS |
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Titration of Histone H3 Cysteine within Histone Tetramers or Octamers with Sulfhydryl Reagents-- We have used sulfhydryl reagents to investigate the structure and the dynamics of the nucleosome. Titration of the unique histone H3 cysteine by DTNB has been reported (43). The authors of that study concluded that cysteine accessibility is different in histone (H3-H4)2 tetramers (two cysteines, corresponding to the two H3 molecules, are titrated) than in histone (H2A-H2B-H3-H4)2 octamers (only one cysteine was titrated). To ensure that under our experimental conditions the two cysteines were reacted with DTNB in both histone tetramers and octamers, we treated them with DTNB at 25 °C for different periods of time in the presence of 2 M NaCl to preserve their oligomeric structure. Fig. 1 presents the result of such an experiment. The two histone H3 cysteines were rapidly titrated by DTNB within the histone tetramer. In contrast, within histone octamers, titration of one and two cysteines was achieved only after a treatment time of 25-30 min and 2-3 h, respectively. During that time, spontaneous hydrolysis of the DTNB was negligible. To confirm that the maximal absorbance reflected a complete reaction of the two cysteines with DTNB, we performed the following controls. After each reaction had reached its maximal value, an aliquot of the mixture was brought to a final concentration of 5 M urea to allow complete accessibility of the cysteines and incubated for 10 min at 37 °C with DTNB, and the absorbance was measured. There was no increase in the absorbance, compared with the samples untreated with urea. This allowed us to conclude that the two cysteines were titrated in both histone tetramers and octamers. In addition, samples containing untreated histone tetramers or octamers were brought to a final concentration of 5 M urea and then incubated for 15 min at 37 °C with DTNB, and the absorbance of the samples was measured. This absorbance was identical to that obtained in the previous experiment. Taken together, these results demonstrate that providing the reaction is left to proceed long enough, DTNB can react with the two histone H3 cysteines in both the histone tetramer and octamer. The same results were obtained for the titration of the cysteines with N-ethylmaleimide (not shown).
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Influence of IA, NEM, or DTNB Cysteine Adducts on Nucleoprotein
Particle Assembly--
We used torsionally constrained DNA
mini-circles to reconstitute nucleoprotein particles on histone
octamers or tetramers that were treated or not treated with sulfhydryl
reagents. Fig. 2 presents the results of
an experiment in which histone octamers were treated with either NEM,
IA, or DTNB. The DNA topoisomers used to reconstitute a nucleosome had
a linking number difference of Lk =
1 (Fig. 2, lanes
1-5) or +1 (lanes 6-10). Lanes 1 and 6 show the naked DNA. Lanes 2 and 3 demonstrate the assembly of the untreated histone octamer particle on
topoisomer
1, resulting mainly in the formation of mononucleosomes
(OP) but also in the formation of some dinucleosomes
( Di). The amount of dinucleosomes assembled with the
mini-circles increased with the increase of the histone:DNA ratio. The
different dinucleosome bands correspond to different positions of the
two nucleosomes on the DNA. Treatment of the histone octamers with the
sulfhydryl reagents (Fig. 2, lanes 4-5) did not prevent the
assembly of the nucleosome on DNA topoisomer
1. However, the
formation of dinucleosomes was completely abolished, independent of the
reagent used, even at the highest histone:DNA ratio. Lanes 7 and 8 show the assembly of the DNA topoisomer +1 on histone
octamers. The migration of the DNA-protein complex was slower than that
of the naked circle and corresponds to a histone tetrameric particle,
illustrating the previously described release of H2A-H2B from histone
octamers upon assembly on positively supercoiled DNA (23). In contrast,
histone octamers treated with the sulfhydryl reagents were unable to
assemble with positively supercoiled mini-circles. Reaction of the
cysteine with these reagents probably induces a conformational change
of the histone (H3-H4)2 tetramer that does not allow it to
accommodate positively supercoiled DNA.
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Topological Analysis of the DNA within Particles Containing
Histones Modified with Sulfhydryl Reagents--
The wrapping of closed
circular DNA on a histone surface, followed by relaxation of unbound
DNA with topoisomerase I and removal of proteins, produces a
characteristic DNA linking deficiency, Lk. The determination of this
value provides information on DNA structural changes due to its
association with a protein. Consequently, changes in the protein
conformation will provoke a modification of the torsional stress of the
DNA. We used this approach to elucidate the nature of the conformation
changes in octameric and tetrameric nucleoprotein particles
reconstituted in the presence of histone H3 treated or not treated with
sulfhydryl reagents.
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DISCUSSION |
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Previous studies have shown that the (H3-H4)2 tetramer can accommodate both negative and positive DNA supercoiling (23). This may be due to a change from the left- to the right-handed conformation in the horseshoe structure of the tetramer, resulting from changes in contacts at the H3-H3 interface (the contact area between the two halves of the horseshoe).
To investigate the equilibrium between the two forms of the tetramer,
sulfhydryl reagents were used to trap these different conformations.
Histone H3 was treated with three different reagents to form adducts on
the single cysteine located at position 110. We used it to reconstitute
a mononucleosome or a tetrameric particle on topologically constrained
DNA mini-circles. We monitored structural changes of these particles in
response to variations in DNA supercoiling. Modification of histone H3
by the sulfhydryl reagents had no effect on the nucleosomal structure
because the LkP value (
1.15) was the same as for the
control particle. In contrast, these modifications had a dramatic
effect on the flexibility of the (H3-H4)2 tetrameric
nucleoprotein particle. The DNA topoisomer equilibrium resulting from
the relaxation with topoisomerase I of untreated tetrameric particles
includes three adjacent DNA topoisomers (topoisomers
1, 0, and +1).
In contrast, the equilibrium obtained under similar conditions with
mononucleosomes and naked DNA includes only two topoisomers. This
demonstrates that histone tetramer interaction with DNA strongly
accentuates DNA thermal fluctuations. In this case, the DNA topoisomer
equilibrium deviates significantly from the Gaussian distribution
normally expected upon DNA relaxation (44, 45). It is especially
striking for the topoisomer distribution obtained after relaxation of
the tetrameric particle assembled with DNA topoisomer
2. At
equilibrium, topoisomer 0 was only a minor component; topoisomers
1
and +1 were the major ones (
LkP =
0.65). We interpret
this as the result of the tetrameric nucleoprotein particle oscillation
between two conformations, one stable left-handed conformation
(represented by topoisomer
1) and a metastable right-handed one
(represented by topoisomer +1). The predominance of negative topoisomers in the relaxation equilibrium indicates that the
left-handed conformation of the tetrameric nucleoprotein particle is
the most stable. At the midpoint between the two conformations, the
tetramer is expected to adopt, at least transiently, a flat
conformation with no writhing (revealed by a trace of the relaxed DNA
topoisomer 0). The same result was obtained using topoisomer +1 instead
of topoisomer
2 as a starting topoisomer. As expected, for untreated particles,
LkP did not depend on the starting
topoisomer.
In contrast, for tetrameric particles containing DTNB-treated histone
H3, after relaxation, the final DNA topoisomer was strongly dependent
on the starting topoisomer. Relaxation of tetrameric particles
containing DTNB-treated histones reconstituted on DNA topoisomer 2
generated DNA topoisomers +1 (61%) and
1 (37%), with a
LkP value of +0.15. Although this
LkP
value is close to zero, it does not indicate the absence of writhing in
such particles, because a flat conformation of the tetramer should be
represented by topoisomer 0. The absence of topoisomer 0 from the final
relaxation equilibrium is due to the lack of affinity of the
DTNB-treated tetramers for relaxed DNA (data not shown). This
LkP value reflects the slight prevalence of the
right-handed conformation over the left-handed one and the absence of
the flat one. In contrast, relaxation of tetrameric particles
containing DTNB-treated histones reconstituted on DNA
topoisomer +1 generated only DNA topoisomer +1, resulting in a higher
positive
LkP value (+0.85). This indicates that for
tetrameric nucleoprotein particles containing DTNB-treated histones
assembled on positively supercoiled DNA, the more stable conformation
is the right-handed one. This dependence of
LkP on the
starting topoisomer can be explained by different interactions of the
two nitrobenzoic adducts arising from changes in protein topology,
rather than by a change in histone H3 structure. This hypothesis is
supported by the fact that tetrameric nucleoprotein particles
containing only one DTNB adduct per histone tetramer are
indistinguishable from untreated ones. Similar constraints have been
described for histone (H3-H4)2 tetramers treated with N-pyrene maleimide (46). An increase in excimer fluorescence intensity of N-pyrene maleimide-treated tetramers was
observed when H2A-H2B dimers were added simultaneously to linear DNA to assemble nucleosomes. This reflects the interaction through stacking of
the two N-pyrene maleimide molecules. The excimer
fluorescence was reduced by one-half when H2A-H2B dimers were not
present, and a dead-end complex DNA-histone (H3-H4)2
tetramer was produced. Such a structure precluded the subsequent
incorporation of H2A-H2B, suggesting a change in histone tetramer
conformation. We propose that such particles are in a right-handed
conformation, as are DTNB-treated tetramers.
An extreme situation was seen with tetrameric particles containing NEM
or IA-treated histones that relaxed completely into DNA topoisomer 1
with a negative
LkP value (
1.09). This value reflects
the lack of affinity of such a tetramer not only for topoisomer +1 but
also for topoisomer 0, suggesting that it is locked in the left-handed
conformation. The possibility that in these particles, two
(H3-H4)2 tetramers were stacked, allowing them to wrap
twice as much DNA as unmodified particles, was excluded by determining
protein/DNA stochiometry and analyzing the sucrose sedimentation
profile. In addition, as described here for the DTNB modified
tetramers, NEM- and IA-treated histone tetramers were able to
incorporate H2A-H2B dimers and nucleate into a normal mononucleosomes
(data not shown).
Histone (H3-H4)2 tetramers assembled on negative or
positive DNA topoisomers show similar horseshoe shapes under electron microscopy (23). This supports the hypothesis that the conformational transition of the tetrameric particle might be achieved through a
reorientation of the two H3-H4 dimers, due to reshuffling at the H3-H3
interface. This hypothesis is supported by the existence in archaea of
a nucleosome-like particle that, although it wraps DNA in a
right-handed superhelix (47), displays a structure resembling the
histone (H3-H4)2 tetramer (48). Many proteins undergo
conformational changes, and the switch between specific conformational
isomers is part of the mechanism of their function. The intrinsic
flexibility of proteins results from the ability of different segments
of the protein to move in relation to one another with only small
expenditures of energy. Protein motion can take two forms: hinge
motions in strands, -sheets, and
-helices that are not
constrained by tertiary packing interactions, and shear motions between
close-packed segments of polypeptides (for a review, see Ref. 49).
Crystallographic data show that H3-H4 histone pairs form tetramers
through the interaction of the carboxyl-terminal halves of the
2-
and
3-helices of H3' and H3 across the dyad in addition to
hydrophobic interactions (26). Two mechanisms may lead to the
structural transition of the tetrameric nucleoprotein particle: a hinge
motion in
-helices (e.g. catabolic activator protein,
CAP) or small shifts and rotations of
-helices at the interface
(e.g. citrate synthase). At present, we cannot discriminate between these two possibilities. Simulation work on the 2.8 Å resolution crystal structure of the tetramer within the nucleosome (26)
might help provide an answer.
The nucleosome structural changes described here may be of importance for transcription. The induction of positive supercoiling downstream from a tracking RNA polymerase should result in the release of H2A-H2B dimers. This would allow the formation of a flexible tetrameric nucleoprotein particle. Transient histone modifications, such as tail acetylation, might also favor the release of H2A-H2B dimers from the nucleosome. The existence of tetrameric particles, able to easily flip from a left- to a right-handed structure, might result in transient changes in DNA/histone contacts, facilitating polymerase tracking and rapid chromatin reassembly after transcription has taken place.
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ACKNOWLEDGEMENTS |
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A. H. thanks A. Prunell for fruitful and stimulating discussions. We are grateful to E. Käs and L. Poljak for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported in part by the Association pour la Recherche contre le Cancer, La Ligue contre le Cancer, the Conseil de Région Midi-Pyrénées, and European Economic Community Biomed 2 contract PL95-0181.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 33-5-61-335940;
Fax: 33-5-61-335886; E-mail: hrfoy{at}ibcg.biotoul.fr.
1 The abbreviations used are: bp, base pair(s); DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); NEM, N-ethylmaleimide IA, iodoacetamide; Lk, linking number of the topoisomer; Lk0, linking number of the most probable configuration of the topoisomer.
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REFERENCES |
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