scispace - formally typeset
Search or ask a question
Journal ArticleDOI

Exploring the Free Energy Landscape of Nucleosomes

27 Jun 2016-Journal of the American Chemical Society (American Chemical Society)-Vol. 138, Iss: 26, pp 8126-8133
TL;DR: This work investigates the disassembly of single nucleosomes using a predictive coarse-grained protein DNA model with transferable force fields and quantitatively describes the thermodynamic stability of both the histone core complex and the nucleosome and predicts rates of transientucleosome opening that match experimental measurements.
Abstract: The nucleosome is the fundamental unit for packaging the genome. A detailed molecular picture for its conformational dynamics is crucial for understanding transcription and gene regulation. We investigate the disassembly of single nucleosomes using a predictive coarse-grained protein DNA model with transferable force fields. This model quantitatively describes the thermodynamic stability of both the histone core complex and the nucleosome and predicts rates of transient nucleosome opening that match experimental measurements. Quantitative characterization of the free-energy landscapes reveals the mechanism of nucleosome unfolding in which DNA unwinding and histone protein disassembly are coupled. The interfaces between H2A-H2B dimers and the (H3-H4)2 tetramer are first lost when the nucleosome opens releasing a large fraction but not all of its bound DNA. For the short strands studied in single molecule experiments, the DNA unwinds asymmetrically from the histone proteins, with only one of its two ends pr...

Summary (2 min read)

■ INTRODUCTION

  • The genome, the blueprint of life, contains nearly all the information needed to build and maintain an entire organism.
  • 15, 16 A detailed characterization of the molecular mechanism for nucleosome assembly will not only improve their understanding of genome packaging but will also shed light on the various pathways that regulate gene expression kinetically.
  • These experiments used an assay that merges single molecule FRET together with optical tweezers.
  • Though the octamer structure is stable when the DNA is bound, a similar intermediate state which has lost the (H3-H4) 2 tetramer/H2A-H2B dimer interface is observed as the DNA unwinds, supporting the idea that DNA unwrapping and histone core complex unfolding are coupled processes.
  • Finally, the freeenergy landscape of nucleosome disassembly supports asymmetric conformations of DNA unwrapping that predominantly expose only one of the two ends.

■ METHODS

  • Computational modeling promises to provide a detailed molecular characterization for the assembly of a single nucleosome as well as the higher order structures formed by oligonucleosomes.
  • 10, 11 Each amino acid in AWSEM is modeled with three atoms, C α , C β , and O, and the transferable interactions among amino acids are parametrized following the energy landscape theory prescription to maximize the ratio of folding temperature over glass transition temperature for a set of training proteins.
  • Using this nonspecific simplification of the protein−DNA interactions is not unreasonable for the nucleosome assembly problem because the X-ray structure indicates the lack of base-specific interactions between histone proteins and the DNA as well as the predominance of water molecules at the interface of the two.
  • Unfortunately, such a simple treatment of electrostatics, though useful in capturing long-range interactions, may give rise to some double counting at short-range.
  • All simulations were performed using the software Large-Scale Atomic/Molecular Massively Parallel Simulator .

■ RESULTS AND DISCUSSION

  • Free-Energy Landscape of the Assembly of the Histone Protein Core.
  • The coupling between histone disassembly and DNA unwinding revealed in Figure 4 suggests that histone proteins will remain bound to the DNA by disrupting protein− protein interfaces at large R DNA , thus explaining the plateau of average bound DNA base pairs shown in Figure 3 .
  • Figure 5 further consolidates this observation with a free-energy landscape as a function of the R DNA for each of the two equally divided DNA segments separated at the nucleosome dyad.
  • As shown in Figure 6B , the two H2A-H2B dimer/H3-H4 tetramer interfaces are lost at large R DNA while the tetramer itself remains stable.
  • The significant drop in the energetic cost of unwinding the DNA when comparing the free-energy landscapes shown in Figure 6 and Figure 3 predicts a faster DNA unwrapping rate for tailless nucleosomes.

■ CONCLUSIONS

  • The authors have introduced a chemically accurate and computationally efficient coarse-grained protein−DNA model to investigate quantitatively the molecular mechanism of nucleosome disassembly.
  • Proteins in this model are described using the AWSEM force field, the parameters of which were determined from statistical optimization algorithms that sculpt a funnelled energy landscape for natural proteins in the known structural database.
  • Intermediate states observed along these simulated pathways, in which interfacial contacts between H2A-H2B dimers and the (H3-H4) 2 tetramer are only partially formed, have indeed been indicated in prior experimental studies.
  • 54 When combined with the 3SPN.2C DNA model, AWSEM not only quantitatively reproduces the thermodynamics and kinetics of nucleosome unfolding but also provides a detailed molecular picture for the nucleosome assembly pathway.
  • The coupling between DNA unwinding and histone core disassembly observed in the simulations has been suggested previously by single molecule FRET experiments.

Did you find this useful? Give us your feedback

Figures (6)

Content maybe subject to copyright    Report

Exploring the Free Energy Landscape of Nucleosomes
Bin Zhang,
Weihua Zheng,
Garegin A. Papoian,
and Peter G. Wolynes*
,,§
Department of Chemistry and Center for Theoretical Biological Physics and
§
Department of Physics and Astronomy, Rice
University, Houston, Texas 77005, United States
Department of Chemistry and Biochemistry and Institute for Physical Science and Technology, University of Maryland, College
Park, Maryland 20742, United States
*
S
Supporting Information
ABSTRACT: The nucleosome is the fundamental unit for
packaging the genome. A detailed molecular picture for its
conformational dynamics is crucial for understanding tran-
scription and gene regulation. We investigate the disassembly
of single nucleosomes using a predictive coarse-grained protein
DNA model with transferable force elds. This model
quantitatively describes the thermodynamic stability of both
the histone core complex and the nucleosome and predicts
rates of transient nucleosome opening that match experimental
measurements. Quantitative characterization of the free-energy
landscapes reveals the mechanism of nucleosome unfolding in
which DNA unwinding and histone protein disassembly are
coupled. The interfaces between H2A-H2B dimers and the
(H3-H4)
2
tetramer are rst lost when the nucleosome opens releasing a large fraction but not all of its bound DNA. For the short
strands studied in single molecule experiments, the DNA unwinds asymmetrically from the histone proteins, with only one of its
two ends preferentially exposed. The detailed molecular mechanism revealed in this work provides a structural basis for
interpreting experimental studies of nucleosome unfolding.
INTRODUCTION
The genome, the blueprint of life, contains nearly all the
information needed to build and maintain an entire organism.
In higher organisms, at the chromosomal level, the three-
dimensional structural organization of the genome is crucial for
its function.
13
Large scale chromosome folding can bring into
proximity regulatory elements, i.e., enhancers and promoters,
that are separated far away in sequence in order to control gene
expression.
4,5
At a ner nanometer scale, the packaging of the
genome plays an important role in gene regulation as well.
6
For
eukaryotic cells, the fundamental unit of DNA organization is
the so-called nucleosome, in whose crystal structure the DNA
wraps approximately 1.7 times around a core made of histone
proteins.
7
We investigate the stability and conformational
dynamics of single nucleosomes by computing the free-energy
landscapes for nucleosome disassembly using a coarse-grained
model that includes a transferable protein force eld suitable for
structure prediction
8,9
and a DNA force eld that successfully
predicts its elastic properties.
10,11
The nucleosome structure itself presents a steric barrier for
gene transcription.
6
Approximately 147 base pairs of duplex
DNA wrap around each histone octamer, which is formed from
two copies of the histone heterodimers (H2A-H2B)
α,β
and
(H3-H4)
α,β
. The linker length between neighboring nucleo-
somes ranges from 20 to 90 base pairs long,
12
and
approximately 75% of the DNA is sterically occluded by
being bound in nucleosomes.
13,14
In order for other proteins,
including transcription factors and RNA polymerases, to access
their binding sites, the tightly bound DNA must at least
partially unwind from the histone core. Numerous mechanisms
inside the cell regulate the stability of the nucleosome and
thereby ne-tune the amount of accessible DNA. These
mechanisms range from passive histone modications to active
remodeling that uses ATP.
15,16
A detailed characterization of
the molecular mechanism for nucleosome assembly will not
only improve our understanding of genome packaging but will
also shed light on the various pathways that regulate gene
expression kinetically.
Many experimental studies already have provided insight into
how the nucleosome assembles. Single molecule stretching
experiments using optical traps suggest that the DNA may
unwrap from the histone core following a three step process
that includes rst (i) the release of the outer turn, next (ii) the
release of the inner turn, and nally (iii) irrev ersible
dissociation of the histone core.
17,18
When the three stage
picture was proposed, the histone core was assumed to be
rather rigid retaining a stable octamer conformation. Recently,
however single molecule Fo
rster resonance energy transfer
(FRET) experiments have revealed that the protein core is
rather exible and disassembles with a loss of the (H3-H4)
2
tetramer/(H2A-H2B) dimer interface as the DNA unwraps.
7,19
Received: March 19, 2016
Article
pubs.acs.org/JACS
© XXXX American Chemical Society A DOI: 10.1021/jacs.6b02893
J. Am. Chem. Soc. XXXX, XXX, XXXXXX

Furthermore, in contrast to the symmetric unwinding of the
two DNA ends initially proposed, recent experiments on short
DNA suggest that the DNA unwraps asymmetrically with one
end being predominantly exposed. These experiments used an
assay that merges single molecule FRET together with optical
tweezers.
20
We show here that computational modeling of
nucleosome disassembly further elucidates the molecular
mechanism and provides a quantitative theoretical foundation
that is needed to unify these experiments.
Using computer simulations of a coarse-grained protein
DNA model, we provide a comprehensive characterization of
free-energy landscapes for the histone complex and nucleosome
disassembly. We nd that the histone core complex without the
DNA is unstable at physiologic al conditions. Instead an
intermediate stat e in which the (H3-H4)
2
tetramer is
sandwiched between the two H2A-H2B dimers with non-
specic interactions is favored thermodynamically. Though the
octamer structure is stable when the DNA is bound, a similar
intermediate state which has lost the (H3-H4)
2
tetramer/H2A-
H2B dimer interface is observed as the DNA unwinds,
supporting the idea that DNA unwrapping and histone core
complex unfolding are coupled processes. Finally, the free-
energy landscape of nucleosome disassembly supports asym-
metric conformations of DNA unwrapping that predominantly
expose only one of the two ends. Comparing the free-energy
landscapes of intact and tailless nucleosomes, we show that this
asymmetric unwrapping mainly arises from electrostatic
interactions between histone tails and the DNA, and we nd
that the tails of histone H3 have the most profound eect. The
combined chemical accuracy and computational eciency of
the coarse-grained model thus enables a rigorous energy
landscape analysis for a single nucleosome and paves the way
for further investigation of higher order structures formed by
oligonucleosomes.
METHODS
Coarse-Grained ProteinDNA Model. Computational modeling
promises to provide a detailed molecular characterization for the
assembly of a single nucleosome as well as the higher order structures
formed by oligonucleosomes. In fact, atomistic simulations have
already provided structural insight into the transient DNA unwrapping
near its entry/exit sites
2123
and revealed the eect of post-
translational modic ations and di erent histone variants on
nucleosomal dynamics.
22,24
The minimal systems are large in size
and involve a complex ensemble of molecular players having intricate
physicochemical interactions. These features make the modeling of
nucleosomes a challenge
25
and limit the time scale currently accessible
from fully atomistic simulations to microseconds.
22,23,26,27
This
limitation constrains the application of all atom models currently
providing a comprehensive landscape characterization. Instead, we
adopt a coarse-grained modeling approach, which has already proven
fruitful in investigating a wide range of biological systems.
2830
To investigate proteinDNA interactions in the nucleosome, we
combine the associative memory, water-mediated, structure and energy
model (AWSEM) for protein
9
with an improved version of the three
site per nucleotide model (3SPN.2C) for DNA.
10,11
Each amino acid
in AWSEM is modeled with three atoms, C
α
,C
β
, and O, and the
transferable interactions among amino acids are parametrized
following the energy landscape theory prescription to maximize the
ratio of folding temperature over glass transition temperature for a set
of training proteins.
3134
AWSEM has been shown to predict
monomer structures reasonably well from sequence alone and to
predict proteinprotein interfaces in dimers with remarkable accuracy
when monomer structures are known.
8,35
The coarse-grained DNA
model developed by de Pablo and co-workers quantitatively
reproduces the persistence length of double-stranded DNA at varying
ionic concentrations and for dierent DNA sequences.
10,11
Thus, it
encodes DNAs elastic properties in a predictive fashion. When we
combine the protein and DNA models, we preserve the original ne-
tuned force elds for proteinprotein and DNADNA interactions by
themselves. In the present model, we introduce additional protein
DNA interactions at a nonspecic level using a screened Debye
Hu
ckel potential for the electrostatics along with a Lennard-Jones
potential for excluded volume (see Supporting Information (SI) for
details). A typical dielectric constant of 78.0 for water and an ionic
concentration of 100 mM at the physiological condition are used for
the electrostatic interactions in this study. Using this nonspecic
simplication of the proteinDNA interactions is not unreasonable for
the nucleosome assembly problem because the X-ray structure
indicates the lack of base-specic interactions between histone
proteins and the DNA as well as the predominance of water molecules
at the interface of the two.
7,36
We note this kind of simple treatment of
the direct proteinDNA interaction has already been applied
successfully to study proteinDNA interactions in a wide range of
biological systems,
3739
including some studies of nucleosomes.
40,41
We introduce two modications to the original AWSEM force eld
presented in ref 8 in order to improve modeling the chemical
complexity of histone proteins. First, to better characterize long-range
electrostatic interaction among histone proteins, we explicitly include
DebyeHu
kcel potential among charged amino acid residues
following ref 42 . Unfortunately, such a simple treatment of
electrostatics, though useful in capturing long-range interactions,
may give rise to some double counting at short-range. This double
counting arises since AWSEM, in its original form, already includes
short-range direct contact potentials between charged residues that
implicitly involve electrostatics. Another modication we employ
remedies to some extent the double counting issue. Additional weak
nonadditive Go
-potentials derived from the octamer conformation in
the nucleosome crystal structure were introduced for the histone
protein core. These partially counteract the repulsion among positively
charged residues in short range. Nonadditive Go
-potentials are further
helpful in quantitatively reproducing energetic barriers and sucient
cooperativity while tuning an already funneled energy landscape more
completely toward native conformations.
43
It is important to note that
the strength of the nonadditive Go
-potential employed here is small
(<30% in the native state of the physically motivated AWSEM contact
potentials, i.e., λ
c
V
contact
in eq S1 of the SI), so the emergence of a basin
of attraction in calculated free-energy landscapes should be attributed
mostly to the original physical potentials. Most of the features seen in
the simulation also appear in simulations completely lacking the
nonadditive Go
term, albeit with somewhat less clarity (see Figure S1).
Details of the denition and the parametrization of the nonadditive
Go
-potential are provided in the SI.
Reaction Coordinates and Free-Energy Calculations. We
determine the free-energy proles using reaction coordinates Q and
R
DNA
to monitor the disassembly of the histone core and the global
DNA portion of the nucleosome, respectively. The fraction of native
contacts Q is dened as
σ
=
−−
<−
Q
NN
rr
2
(2)(3)
exp
()
2
ij
ij ij
N
2
2
2
(1)
with σ = 3 Å and N being the total number of nontail residues from all
eight histone proteins. The summation in eq 1 only includes C
α
atoms.
The native separation r
ij
N
is the distance between the two C
α
atoms
from amino acids i and j calculated using the coordinates from the
crystal structure.
36
Similar measures can be dened to study protein
protein interfaces when only intermolecular contacts are included in
the summation. Q ranges from 0 to 1, with a higher value
corresponding to greater similarity to the native structure. For
funnele d surfaces, Q has bee n shown to provide an excellent
characterization of the progression of folding for single proteins
44,45
and binding for proteinprotein complexes.
46
To study how the DNA
becomes unwrapped when the nucleosome unfolds, we use the radius
of gyration of the DNA R
DNA
dened as
Journal of the American Chemical Society Article
DOI: 10.1021/jacs.6b02893
J. Am. Chem. Soc. XXXX, XXX, XXXXXX
B

=−
=
R
N
rr
1
()
i
N
iDNA
1
com
2
(2)
where r
com
is the center of mass, and the summation is conducted over
all the coarse-grained sugar beads of the DNA.
We used umbrella sampling together with replica exchange
techniques to enhance conformation sampling for free-energy
calculations.
47,48
Harmonic potentials 1/2K
q
(Q Q
o
)
2
and 1/
2K
r
(R
DNA
R
DNA
o
)
2
were introduced to restrain constant temperature
molecular dynamics simulations toward reference values, with K
q
=
1000 kcal/mol and K
r
= 0.8 kcal/mol/Å
2
. The reference values for Q
o
are equally spaced from 0.2 to 0.8 with a step size 0.1. For R
DNA
o
,12
references were chosen from 45 to 72.5 Å with an increment of 2.5 Å.
Twelve replicas were used for each umbrella window with temperature
ranging from 260 to 370 K with a step of 10 K. Data from dierent
windows were stitched together with the weighted histogram analysis
method (WHAM) to construct free-energy landscapes.
49
Simulation Details. All simulations were performed using the
software Large-Scale Atomic/Molecular Massively Parallel Simulator
(LAMMPS). Initial congurations of the simulation and the DNA
sequence are obtained from the crystal structure with PDB ID: 1KX5
(see Figure S2). Molecular dynamics trajectories were performed at
constant temperatur e and volume without p eriodic boundary
conditions for 5 million steps with a time step of 20 fs, and exchanges
among dierent replicas were attempted at every 100 steps. Due to the
coarse graining, we note the simulation time scale cannot be converted
precisely into real time units.
50
In any event, we checked the
convergence of the simulations by performing rigorous error analysis
of the calculated free-energy proles (see SI Section: Convergence of
the Simulation for Details). Since the simulations were performed
without periodic boundary conditions, we introduced a constraint on
the radius of gyration of the histone core complex when the protein
assembly is studied without the presence of the DNA in order to
prevent molecules from diusing too far away from each other
unproductively. De nition of this constraint and additional simulation
details are provided in the SI.
RESULTS AND DISCUSSION
Free-Energy Landscape of the Assembly of the
Histone Protein Core. Several coarse-grained models have
already been used for the investigation of nucleosome
dynamics.
40,41,51,52
In most of these studies, the histone core
complex was restrained to having the octamer conformation
found in the crystal structure because the models that were
employed lack a transferable force eld for protein molecules.
These structural restraints prohibit large-scale conformational
changes away from the crystal structure, whether they are
artifactual and unphysical or physical and mechanistically
required. In our view, allowing protein exibility is essential
because the histo ne octamer structure is unstable under
physiological conditions in the absence of the DNA.
5355
Partial disassembly of the histone core complex as the
nucleosome unfolds has also been observed in single molecule
FRET experi ment s.
19,56
Conformational exib ilit y of t he
histone core complex must therefore play a crucial role in
nucleosome dynamics. We rst investigate whether the histone
core would assemble in the absence of the DNA using free-
energy landscape analysis.
Figure 1 presents the free-energy prole as a function of the
fraction of native contacts Q, with representative structures of
the protein complex at various Q values shown at the top.
Ther e are three free-energy basins i n this landscape at
approximately Q = 0.35, 0.45, and 0.75, respectively. At Q =
0.35, the system has disassembled into a tetramer (H3-H4)
2
and two H2A-H2B dimers, and no specic contacts between
H2A-H2B and the tetramer are present. Throughout our
simulation, we have not observed complete dissociation of any
of the four dimers into monomer structures. The stability of
this low Q basin is sensitive to protein concentrations due to
the entropic contributions from the free diusion of proteins in
the solution.
54
At Q = 0.45, the two dimers H2A-H2B begin to
assemble around (H3-H4)
2
from two sides, and the tetramer is
seen to be sandwiched in between H2A-H2B dimers, as
illustrated in the top panel. Finally, specic contacts form
between the dimers and the tetramer at Q = 0.75, and the
histone core complex adopts the conformation captured in the
nucleosome crystal structure that contains DNA. Average
contact maps of protein structures at various Q values are
provided in Figure S3 .
Mechanistic insight about the assembly process can be
obtained by following the formation of interfacial contacts as a
function of Q . As shown in Figure 2A, the formation of contacts
between the two H3-H4 dimers (blue) precedes the formation
of contacts between H3-H4 and H2A-H2B (red and yellow).
The partially disassembled state at small Q values thus consists
of a (H3-H4)
2
tetramer and two H2A-H2B dimers. As the two
distinct interfaces between H3-H4 and H2A-H2B are chemi-
cally identical, it is reassuring that their average number of
contacts are found to be the same within numerical accuracy.
The attachment of the two H2A-H2B dimers to the (H3-
H4)
2
tetramer occurs largely sequentially, as shown in Figure
2B. The free-energy prole as a function of the two interfacial
contact numbers exhibits two parallel reaction channels from
the disassembled state to the octamer conformation, as
indicated by the arrows. Along either one of the reaction
pathways, the formation of each of the two interfaces is
decoupled from the formation of the other, suggesting two
parallel serial mechanisms.
The molecular picture for the assembly of histone proteins
revealed from our s imulation is consistent with prior
experimental observations. For example, in agreement with
Figure 2A, the histone proteins are known to stabilize into a
(H3-H4)
2
tetramer and two H2A-H2B dimers when
disassembled.
5355,57
Furthermore, in support of the sequential
Figure 1. Free energy prole as a function of the fraction of native
contacts Q for the folding of the histone protein core. Error bars
shown in gray represent the standard deviation of the mean. Example
congurations of the histone complex at various values of Q are shown
in the top panel, with the two H3-H4 dimers drawn in blue and green
and the two H2A-H2B dimers in red and orange. Histone tails are not
displayed for clarity.
Journal of the American Chemical Society Article
DOI: 10.1021/jacs.6b02893
J. Am. Chem. Soc. XXXX, XXX, XXXXXX
C

pathway shown in Figure 2B, an intermediate hexameric
structure consisting of the H3-H4 tetramer and one copy of the
H2A-H2B dimer has been observed in ref 54.
Free-Energy Landscape of Full Nucleosome Disas-
sembly. The free-energy landscape for the histone core
complex quanties the stability of the octamer state and
provides a detailed molecular pathway for the assembly process.
The remarkable agreement between the molecular mechanism
predicted from simulation and that proposed from experimental
observations on the histone complex by itself encourages the
application of AWSEM for studying full nucleosome
disassembly. We therefore now turn to investigate the coupling
between DNA unwrapping and the conformational changes of
the histone core complex as the nucleosome unfolds.
Figure 3 presents the free-energy prole (yellow) as a
function of the radius of gyration of the DNA (R
DNA
).
Unfolded nucleosome conformations with exposed DNA can
be seen from example snapshots shown in the top panel
together with Figures S5 and S6. The free-energy landscape
exhibits a single basin around the crystal structure at R
DNA
=45
Å, where the DNA is tightly bound to histone proteins. The
model thus reproduces the expected stability of the nucleosome
as a packaging unit for the genome. The free-energy cost of
unwrapping the outer layer of the DNA in intact nucleosomes
has been estimated to be around 7 to 10 kcal/mol.
17,18,58
As
detailed in the SI (Section: Thermodynamics and Kinetics of
DNA Unwrapping), by carefully dening the state in which the
outer layer DNA has been unwound, our simulation predicts
the free-energy cost for unwinding the DNA to be 8 kcal/mol.
Furthermore, using a diusion constant of D = 5500 bp
2
/s
estimated experimentally,
59
we nd the rate for unwrapping the
outer layer DNA for the intact nucleosome to be approximately
3.6 × 10
4
s
1
, which is in good agreement with reported rate
0.00038 s
1
from single molecule pulling experiments.
18
The average number of DNA base pairs bound to histone
proteins (see the SI for a rigorous denition) as a function of
R
DNA
is also shown in Figure 3 as a blue curve. As R
DNA
increases, we nd that the number of bound DNA base pairs
changes in a stepwise manner. For example, most of the DNA
base pairs remain bound over the range of extension 45 < R
DNA
< 47.5 Å, which is followed by a sudden drop of 10 bp,
followed again by another plateau region 48 < R
DNA
<5.
The stepwise unwinding is even clearer at lower temperature, as
shown in Figure S7. Step-wise DNA unwinding is expected
from the periodic contacts that form between histone proteins
and the DNA at a 1011 bp frequency;
36,57,60,61
see SI Section:
Periodicity of Histone DNA Contacts for a detailed discussion.
Figure 4A presents a two-dimensional free-energy landscape
as a function of the DNA radius of gyration R
DNA
and the
fraction of native contacts Q. At small R
DNA
when the DNA is
fully bound, the histone core complex is highly stable around
the octamer conformation that is captured in the crystal
structure with Q 0.8. As the DNA unwraps at large R
DNA
, the
free-energy of the low and high Q conformations become
comparable, and the histone proteins begin to fall away from
the core and begin to deviate from the octamer X-ray structure.
Figure 4B further characterizes in detail the unfolding of various
proteinprotein interfaces. The interface between the two
copies of H3-H4 f orming the tetramer remains stable
throughout the entire range of R
DNA
studied. On the other
hand, the two interfaces between H3-H4 and H2A-H2B
gradually disappear as the nucleosome unfolds. We note the
loss of the interface between H3-H4 and H2A-H2B is
consistent with the stability of dierent interfaces determined
from the free-energy landscape for histone core assembly
shown in Figure 2. The coupling between histone disassembly
Figure 2. Formation of dierent proteinprotein interfaces as the
histone protein core assembles. (A) Average fraction of native contacts
for various proteinprotein interfaces as a function of the global Q.
(B) Two-dimensional free-energy prole for the two chemically
identical interfaces formed between H3-H4 and H2A-H2B hetero-
dimers. The arrows indicate the two parallel reaction channels for
folding. Standard deviation is provided in Figure S4.
Figure 3. Free-energy prole as a function of the DNA radius of
gyration R
DNA
for the unfolding of the nucleosome (yellow). Error
bars shown in gray represent the standard deviation of the mean. The
blue line measures the average number of DNA base pairs bound to
histone proteins. Examples of nucleosome congurations at various
values of R
DNA
are shown in the top panel, with the DNA colored in
yellow and the same coloring scheme as in Figure 1 for proteins.
Journal of the American Chemical Society Article
DOI: 10.1021/jacs.6b02893
J. Am. Chem. Soc. XXXX, XXX, XXXXXX
D

and DNA unwinding revealed in Figure 4 suggests that histone
proteins will remain bound to the DNA by disrupting protein
protein interfaces at large R
DNA
, thus explaining the plateau of
average bound DNA base pairs shown in Figure 3.
As illustrated in the top panel of Figure 3B, the DNA
molecule unwraps asymmetrically, and only one of two ends is
preferentially exposed at large R
DNA
. Figure 5 further
consolidates this observation with a free-energy landscape as
a function of the R
DNA
for each of the two equally divided DNA
segments separated at the nucleosome dyad. This landscape
clearly illustrates that the energetic cost of opening the two
ends simultaneously, i.e., moving along the diagonal of the
landscape, is much higher than opening only one end following
the pathways highlighted with arrows. Two examples of
sampled half open nucleosome structures are shown in Figure
5A, with the two segments of the DNA colored in yellow and
purple, respectively. It is important to point out that the short
DNA sequence employed in our simulation is palindromic, and
thus the two segments of the DNA are identical chemically.
There is thus no preference for either one DNA end to unwrap
rst or the other, as reected in the symmetry of the two
reaction channels on the free-energy landscape.
The asymmetric DNA conformation having only one of its
two ends unwrapped allows the other end to interact more
favorably with histone proteins. This energetic preference can
be seen from Figure 5C, which plots the average proteinDNA
electrostatic interaction energy of various nucleosome con-
formations. The y-axis of this gure is a measure for DNA
asymmetry dened as ξ =(R
DNA
first
)/(R
DNA
first
+ R
DNA
second
). This
denition is motivated by the observation that the unwrapped
end has larger radius of gyration. From Figure 5C, we see that
as we pull the DNA apart, for R
DNA
between 58 and 62 Å, the
proteinDNA electrostatic interaction energy is indeed lower
either for ξ < 0.5 or for ξ > 0.5 compared with the symmetric
conguration with ξ 0.5.
Sampling becomes dicult at large distances, but we note
that at the R
DNA
= 62 Å, a new set of congurations, in which
the DNA folds back onto itself along with a completely
dissociated histone protein core occasionally appears in the
simulation (see Figure S8). These congurations also have a
low electrostatic energy. This multiplicity of observed structures
at large R
DNA
suggests that in vivo unwrapping is likely to be
mechanically coupled to large scale DNA motions that are
promoted by motor proteins.
62
The detailed molecular model for nucleosome disassembly
put forward by our simulation is well supported with
experimental single molecule studies. For example, the
predicted loss of the H3-H4 tetramer/H2A-H2B dimer
interface along with DNA unwinding was indeed observed in
single molecule FRET experiments.
19,56
Similarly, the sequen-
tial asymmetric unwrapping of the two DNA ends has been
detected in recent single molecule pulling experiments.
20
Figure 4. Coupling between DNA unwrapping and histone protein
core disassembling. (A) Two-dimensional free-energy prole as a
function of the DNA radius of gyration (R
DNA
) and the fraction of
native contacts for the histone protein core (Q). Energies in kcal/mol.
(B) Average fraction of native contacts for various proteinprotein
interfaces as a function of R
DNA
.
Figure 5. Asymmetric unwrapping of the two DNA ends as the
nucleosome unfolds. (A) Example asymmetric nucleosome conforma-
tions with the rst DNA segment colored in purple and the second in
yellow. The coloring scheme for proteins is identical to Figure 1. (B)
Two-dimensional free-energy prole for the radius of gyration of the
two chemically identical DNA segments separated at the nucleosome
dyad. (C) Average proteinDNA electrostatic interactions as a
function of the DNA radius of gyration R
DNA
and the asymmetry
measure ξ. Energy scales in both part (B) and (C) are kcal/mol.
Journal of the American Chemical Society Article
DOI: 10.1021/jacs.6b02893
J. Am. Chem. Soc. XXXX, XXX, XXXXXX
E

Citations
More filters
Journal ArticleDOI
TL;DR: This work introduces AWSEM-IDP, a new AWSEM branch for simulating intrinsically disordered proteins (IDPs), where the weights of the potentials determining secondary structure formation have been finely tuned, and a novel potential is introduced that helps to precisely control both the average extent of protein chain collapse and the chain's fluctuations in size.
Abstract: The associative memory, water-mediated, structure and energy model (AWSEM) has been successfully used to study protein folding, binding, and aggregation problems. In this work, we introduce AWSEM-IDP, a new AWSEM branch for simulating intrinsically disordered proteins (IDPs), where the weights of the potentials determining secondary structure formation have been finely tuned, and a novel potential is introduced that helps to precisely control both the average extent of protein chain collapse and the chain’s fluctuations in size. AWSEM-IDP can efficiently sample large conformational spaces, while retaining sufficient molecular accuracy to realistically model proteins. We applied this new model to two IDPs, demonstrating that AWSEM-IDP can reasonably well reproduce higher-resolution reference data, thus providing the foundation for a transferable IDP force field. Finally, we used thermodynamic perturbation theory to show that, in general, the conformational ensembles of IDPs are highly sensitive to fine-tun...

82 citations

Journal ArticleDOI
TL;DR: A coarse-grained active polymer model is developed where chromatin is represented as a confined flexible chain acted upon by molecular motors that drive fluid flows by exerting dipolar forces on the system, demonstrating that coherent motions emerge in systems involving extensile dipoles and are accompanied by large-scale chain reconfigurations and nematic ordering.
Abstract: The 3D spatiotemporal organization of the human genome inside the cell nucleus remains a major open question in cellular biology. In the time between two cell divisions, chromatin—the functional form of DNA in cells—fills the nucleus in its uncondensed polymeric form. Recent in vivo imaging experiments reveal that the chromatin moves coherently, having displacements with long-ranged correlations on the scale of micrometers and lasting for seconds. To elucidate the mechanism(s) behind these motions, we develop a coarse-grained active polymer model where chromatin is represented as a confined flexible chain acted upon by molecular motors that drive fluid flows by exerting dipolar forces on the system. Numerical simulations of this model account for steric and hydrodynamic interactions as well as internal chain mechanics. These demonstrate that coherent motions emerge in systems involving extensile dipoles and are accompanied by large-scale chain reconfigurations and nematic ordering. Comparisons with experiments show good qualitative agreement and support the hypothesis that self-organizing long-ranged hydrodynamic couplings between chromatin-associated active motor proteins are responsible for the observed coherent dynamics.

72 citations

Journal ArticleDOI
TL;DR: Analysis of the trajectories via Markov state modeling highlights how the sequence-dependence of the sliding dynamics is due to the different twist defect energy costs, and in particular how nucleosome regions where defects cannot easily form introduce the kinetic bottlenecks slowing down repositioning.
Abstract: While nucleosomes are highly stable structures as fundamental units of chromatin, they also slide along the DNA, either spontaneously or by active remodelers. Here, we investigate the microscopic mechanisms of nucleosome sliding by multiscale molecular simulations, characterizing how the screw-like motion of DNA proceeds via the formation and propagation of twist defects. Firstly, coarse-grained molecular simulations reveal that the sliding dynamics is highly dependent on DNA sequence. Depending on the sequence and the nucleosome super-helical location, we find two distinct types of twist defects: a locally under-twisted DNA region, previously observed in crystal structures, and a locally over-twisted DNA, an unprecedented feature. The stability of the over-twist defect was confirmed via all-atom simulations. Analysis of our trajectories via Markov state modeling highlights how the sequence-dependence of the sliding dynamics is due to the different twist defect energy costs, and in particular how nucleosome regions where defects cannot easily form introduce the kinetic bottlenecks slowing down repositioning. Twist defects can also mediate sliding of nucleosomes made with strong positioning sequences, albeit at a much lower diffusion coefficient, due to a high-energy intermediate state. Finally, we discuss how chromatin remodelers may exploit these spontaneous fluctuations to induce unidirectional sliding of nucleosomes.

67 citations

Journal ArticleDOI
TL;DR: A physics-based framework is proposed that predicts the effect of charge-altering PTMs in the histone core, quantitatively for several types of lysine charge-neutralizing PTMs including acetylation, and qualitatively for all phosphorylations, on the nucleosome stability and subsequent changes in DNA accessibility, making a connection to resulting biological phenotypes.
Abstract: Controlled modulation of nucleosomal DNA accessibility via post-translational modifications (PTM) is a critical component to many cellular functions. Charge-altering PTMs in the globular histone core—including acetylation, phosphorylation, crotonylation, propionylation, butyrylation, formylation, and citrullination—can alter the strong electrostatic interactions between the oppositely charged nucleosomal DNA and the histone proteins and thus modulate accessibility of the nucleosomal DNA, affecting processes that depend on access to the genetic information, such as transcription. However, direct experimental investigation of the effects of these PTMs is very difficult. Theoretical models can rationalize existing observations, suggest working hypotheses for future experiments, and provide a unifying framework for connecting PTMs with the observed effects. A physics-based framework is proposed that predicts the effect of charge-altering PTMs in the histone core, quantitatively for several types of lysine charge-neutralizing PTMs including acetylation, and qualitatively for all phosphorylations, on the nucleosome stability and subsequent changes in DNA accessibility, making a connection to resulting biological phenotypes. The framework takes into account multiple partially assembled states of the nucleosome at the atomic resolution. The framework is validated against experimentally known nucleosome stability changes due to the acetylation of specific lysines, and their effect on transcription. The predicted effect of charge-altering PTMs on DNA accessibility can vary dramatically, from virtually none to a strong, region-dependent increase in accessibility of the nucleosomal DNA; in some cases, e.g., H4K44, H2AK75, and H2BK57, the effect is significantly stronger than that of the extensively studied acetylation sites such H3K56, H3K115 or H3K122. Proximity to the DNA is suggestive of the strength of the PTM effect, but there are many exceptions. For the vast majority of charge-altering PTMs, the predicted increase in the DNA accessibility should be large enough to result in a measurable modulation of transcription. However, a few possible PTMs, such as acetylation of H4K77, counterintuitively decrease the DNA accessibility, suggestive of the repressed chromatin. A structural explanation for the phenomenon is provided. For the majority of charge-altering PTMs, the effect on DNA accessibility is simply additive (noncooperative), but there are exceptions, e.g., simultaneous acetylation of H4K79 and H3K122, where the combined effect is amplified. The amplification is a direct consequence of the nucleosome–DNA complex having more than two structural states. The effect of individual PTMs is classified based on changes in the accessibility of various regions throughout the nucleosomal DNA. The PTM’s resulting imprint on the DNA accessibility, “PTMprint,” is used to predict effects of many yet unexplored PTMs. For example, acetylation of H4K44 yields a PTMprint similar to the PTMprint of H3K56, and thus acetylation of H4K44 is predicted to lead to a wide range of strong biological effects. Charge-altering post-translational modifications in the relatively unexplored globular histone core may provide a precision mechanism for controlling accessibility to the nucleosomal DNA.

61 citations


Cites background from "Exploring the Free Energy Landscape..."

  • ...For example, the free energy, ΔG, of stripping the DNA completely from the histone core is a measure for the overall stability of the histone–DNA complex and is estimated to be over 20 kcal/mol [25, 56], which is much larger than the stability of a typical protein [57, 58]....

    [...]

Journal ArticleDOI
TL;DR: The CG content of nucleosomal DNA is found to anticorrelate with the extent of unwrapping, supporting the possibility that AT-rich segments may signal the start of transcription by forming less stable nucleosomes.

56 citations

References
More filters
Journal ArticleDOI
18 Sep 1997-Nature
TL;DR: The X-ray crystal structure of the nucleosome core particle of chromatin shows in atomic detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it.
Abstract: The X-ray crystal structure of the nucleosome core particle of chromatin shows in atomic detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it. Both histone/histone and histone/DNA interactions depend on the histone fold domains and additional, well ordered structure elements extending from this motif. Histone amino-terminal tails pass over and between the gyres of the DNA superhelix to contact neighbouring particles. The lack of uniformity between multiple histone/DNA-binding sites causes the DNA to deviate from ideal superhelix geometry.

7,841 citations

Journal ArticleDOI
18 Dec 2014-Cell
TL;DR: In situ Hi-C is used to probe the 3D architecture of genomes, constructing haploid and diploid maps of nine cell types, identifying ∼10,000 loops that frequently link promoters and enhancers, correlate with gene activation, and show conservation across cell types and species.

5,945 citations

Journal ArticleDOI
TL;DR: The Weighted Histogram Analysis Method (WHAM) as mentioned in this paper is an extension of Ferrenberg and Swendsen's multiple histogram technique for complex biomolecular Hamiltonians.
Abstract: The Weighted Histogram Analysis Method (WHAM), an extension of Ferrenberg and Swendsen's Multiple Histogram Technique, has been applied for the first time on complex biomolecular Hamiltonians. The method is presented here as an extension of the Umbrella Sampling method for free-energy and Potential of Mean Force calculations. This algorithm possesses the following advantages over methods that are currently employed: (1) It provides a built-in estimate of sampling errors thereby yielding objective estimates of the optimal location and length of additional simulations needed to achieve a desired level of precision; (2) it yields the “best” value of free energies by taking into account all the simulations so as to minimize the statistical errors; (3) in addition to optimizing the links between simulations, it also allows multiple overlaps of probability distributions for obtaining better estimates of the free-energy differences. By recasting the Ferrenberg–Swendsen Multiple Histogram equations in a form suitable for molecular mechanics type Hamiltonians, we have demonstrated the feasibility and robustness of this method by applying it to a test problem of the generation of the Potential of Mean Force profile of the pseudorotation phase angle of the sugar ring in deoxyadenosine. © 1992 by John Wiley & Sons, Inc.

5,784 citations

Journal ArticleDOI
TL;DR: In this article, a replica-exchange method was proposed to overcome the multiple-minima problem by exchanging non-interacting replicas of the system at several temperatures, which allows the calculation of any thermodynamic quantity as a function of temperature in that range.

4,135 citations

Journal ArticleDOI
06 Sep 2012-Nature
TL;DR: In this paper, the authors applied chromosome conformation capture carbon copy (5C) to interrogate comprehensively interactions between transcription start sites (TSSs) and distal elements in 1% of the human genome representing the ENCODE pilot project regions.
Abstract: The vast non-coding portion of the human genome is full of functional elements and disease-causing regulatory variants. The principles defining the relationships between these elements and distal target genes remain unknown. Promoters and distal elements can engage in looping interactions that have been implicated in gene regulation. Here we have applied chromosome conformation capture carbon copy (5C) to interrogate comprehensively interactions between transcription start sites (TSSs) and distal elements in 1% of the human genome representing the ENCODE pilot project regions. 5C maps were generated for GM12878, K562 and HeLa-S3 cells and results were integrated with data from the ENCODE consortium. In each cell line we discovered >1,000 long-range interactions between promoters and distal sites that include elements resembling enhancers, promoters and CTCF-bound sites. We observed significant correlations between gene expression, promoter-enhancer interactions and the presence of enhancer RNAs. Long-range interactions show marked asymmetry with a bias for interactions with elements located ∼120 kilobases upstream of the TSS. Long-range interactions are often not blocked by sites bound by CTCF and cohesin, indicating that many of these sites do not demarcate physically insulated gene domains. Furthermore, only ∼7% of looping interactions are with the nearest gene, indicating that genomic proximity is not a simple predictor for long-range interactions. Finally, promoters and distal elements are engaged in multiple long-range interactions to form complex networks. Our results start to place genes and regulatory elements in three-dimensional context, revealing their functional relationships.

1,438 citations

Frequently Asked Questions (1)
Q1. Who is the author of this paper?

P.G.W. would like to dedicate this paper to the memory of his old friend, Jonathan Widom, who pioneered the biophysical study of nucleosomal dynamics.■