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Structure and Function of the Core Nucleosome Particle:
The Molecular Basis of Chromatin Organization
Cassidy Crook
Chromatin and Genome Organization
All cells face the considerable challenge of compacting the genome to a
small fraction of its original volume such that it can exist within the space of a cell.
Prokaryotes as well as eukaryotes accomplish this feat through the combined
effects of protein interactions and DNA supercoiling. However, dissimilar to
eukaryotes, the genetic material of prokaryotes is maintained in an irregular
entanglement known as the bacterial nucleoid. The fluid nature of the nucleoid is
governed principally by enzyme-mediated (as well as spontaneous) topological
modifications, with the association of structural proteins serving largely to stabilize
supercoiled DNA states. In contrast, DNA compaction in eukaryotes is achieved
through an extensively ordered genome architecture for which an organizational
hierarchy is defined. The nucleosome represents the fundamental unit of
eukaryotic genome organization (Bates & Maxwell, 2006). Roughly 200 bp
segments of relaxed eukaryotic DNA are regularly coiled around octameric histone
proteins to form an assembly of nucleosome particles with a DNA packing ratio of
around seven (Lewin, 2004). Nucleosomes arranged along a span of duplex DNA
constitute a 10 nm chromatin fiber and have the canonical appearance of beads-on-
a-string (Olins & Olins, 1974). Subsequently, nucleosomes aggregate to form a 30
nm second-order chromatin fiber with a helical super-structure and a packing ratio
of around forty. The 30 nm fiber is arranged into 20-100 kbp loop domains which
associate with nuclear matrix proteins and scaffold proteins, including
topoisomerases and RNA polymerases, to achieve a packing ratio exceeding 1000.
Surprisingly, little is known about chromatin organization beyond the level of the
30 nm filament. Higher order structures appear to include looped rosette
configurations (or chromomeres), 100-130 nm chomomena, a 200-250 nm fiber,
and, ultimately, the eukaryotic chromosome (Nelson & Cox, 2008). Based on this
model of DNA compaction in eukaryotes, it has been suggested that the
composition of the bacterial nucleoid exemplifies a rudimentary chromosome. Still,
a fundamental difference between chromosome organization and nucleoid
composition is the primary role of DNA-histone interactions in the manifestation of
DNA supercoiling in eukaryotes as opposed to topological regulation achieved
cheifly by the competition of topoisomerase enzymes within the nucleoid.
Specifically, prokaryotes rely on the essential bacterial enzyme DNA gyrase, a type
II topoisomerase (topo), to introduce negative supercoils into DNA through an ATP-
dependent mechanism , while negative supercoiling in eukaryotic DNA is achieved
exclusively through the winding of DNA around nucleosomal histone octamers.
Interestingly, eukaryotic type II topos are not able to induce negative supercoiling
in DNA, yet they carry out ATP hydrolysis through a mechanism reminiscent of
bacterial gyrase. The free energy of ATP hydrolysis in eukaryotic topoII is utilized
in what has been described as a highly inefficient manner, to needlessly drive sub-
equilibrium topological simplification of DNA (Bates & Maxwell, 2006). Thus, one
might speculate that ATP-hydrolysis in eukaryotic topoII is an evolutionary relic of
an ancestral prokaryotic topoII once essential for the maintenance of genome
compaction through negative supercoiling.
A Historical Perspective of Chromatin and the Nucleosome
The term ‘chromatin’ was first introduced by the highly-renowned German
cytogeneticist Walther Flemming in the early 1880’s and was so-named to convey
its refractive properties and its affinity for dyes. In 1884 Albrecht Kossel described
‘histon’, a ‘proteoid’ isolated from the phosphrous-rich acidic ‘nuclein’ extracts of
avian erythrocyte nuclei. Jointly, these preliminary discoveries established the
basis for our current understanding of eukaryotic DNA organization. Yet,
approximately 80 years after histones were first described, the discovery of a basic
subunit of chromatin structure in 1973-1974, coined the ‘nucleosome’ shortly
thereafter, revolutionized the field of chromosome organization and sparked what
has been recognized as the ‘nucleosome era’ (Olins & Olins, 2003).
Prior to the earliest results indicating a discrete sub-structure repeated in
chromatin, the most widely accepted model for chromatin structure was the
Pardon-Wilkins super-helical model. Based on the results of low-angle x-ray
diffraction, the super-helical model of chromatin compaction essentially described
a nucleohistone coiled-coil in which the histone protein structure was that of a long
fibrillous coil around which DNA was wrapped (Olins & Olins, 1972, 1974, 1997;
Pardon & Wilkins, 1972). In the early 1970’s the work of Olins and Olins (1974),
Woodcock (1973), and Kornberg and Thomas (1974) independently utilized
electron scattering to visualize chromatin filaments at low-resolution. Consistently,
chromatin appeared to form irregularly distributed spherical bodies, designated
‘nu-bodies’, which exhibited a tendency to arrange in clusters (Olins & Olins, 1974;
Woodcock, 1973; Kornberg & Thomas, 1974). Micrograph images of nucleosomes
were paralleled by experimental evidence demonstrating a repeating structure in
condensed DNA which facilitated protection of contiguous 200 bp segments in
DNase digest assays (Hewish & Burgoyne, 1973). Furthermore, studies
characterizing the properties of histone proteins made vital contributions to a
unified theory of chromatin structure. Notably, such studies demonstrated the
presence of one of each class of histone per 100 bp of DNA (excluding H1), the in
vitro assembly of an (H3-H4)2 tetramer, and the vital role of histones H2A, H2B,
H3, and H4 in the formation of chromatin ‘beads’ (Kornberg, 1974). Thus, by the
mid-1970’s the sum of experimental data relating to chromatin was sufficient to
formulate a cohesive theory for chromatin structure – and so the nucleosome
model was conceived. In a basic sense, the nucleosome model identifies the
nucleosome unit as the basic repeating sub-structure of chromatin. Specifically,
this model recognized, in considerable detail, the structure and organization of the
nucleosome as comprised of a 200 bp segment of DNA oriented around a tripartite
histone octamer composed of a (H3-H4)2 tetramer and two H2A-H2B dimers (see
figs. 2, 3, & 4) (Kornberg, 1974).
The Core Histone Architecture
Fundamentally, the histone octamer is composed of the four primary histone
monomers, H2A, H2B, H3, and H4, each of which is structurally similar to the rest.
In particular, the core histones are distinguished by a unique secondary structure
known as the histone-fold. The histone-fold is a three-helix motif composed of an N-
terminal 11-residue alpha-helix (αI), a long central 27-residue helix (αII), and a C-
terminal 11-residue helix (αIII) consecutively joined by two short flexible linking
domains each containing a single beta-sheet. The three helices of the histone-fold
are arranged with respect to one another such that they create an overall ‘Z’-like
form.The N and C terminal regions are described as helix-sheet-helix regions
(HSH) and are designated HSH1 and HSH2 respectively (see fig. 1). Additionally,
the amino acid residues of the histone-fold have been categorized into four classes:
surface, self, pair, and interface residues. Of relevance, surface residues are
positioned on the outside of the histones in the context of the nucleosome to
facilitate histone-DNA interactions, pair residues are involved in histone
dimerization, and interface residues contribute to the contacts formed between
two dimers – either between the H2A-H2B dimers or in the formation of the (H3-
H4)2 tetramer. Surface residues demonstrate sequence conservation of the basic
amino acids Arg and Lys, which are critical in the binding of DNA to the histone
octamer through ionic interactions. The high degree of structural similarity
between the core histones is thought to be essential to the nature of DNA binding
and chromatin organization in general. It has been suggested that transitions in
chromatin are achieved through discrete changes in dimer-dimer and dimer-
tetramer interactions. One mode of structural regulation may be an inherent
susceptibility of the histones to subtle environmental changes at the interface
regions between the three histone multimers. This idea is supported by the
observation that interface residues are generally found to be less hydrophobic and
more diversified than pair residues (Arents & Moudrianakis, 1995).
The stability of the core histone octamer is DNA-dependent at physiological
conditions; however, biochemical studies have demonstrated that the octamer is
also stable in vitro under highly ionic conditions, particularly at high NaCl
concentrations. This property of the octamer ultimately enabled the elucidation of
high-resolution structures in the absence of DNA (Ramakrishnan, 1997). The first
accurate crystal structure of the core histone complex was not solved until 1991
(Arents et al, 1991). The 1991 structure, solved at 3.1 Ǻ, demonstrated that a
previous structure, resolved at 3.3 Ǻ, was critically flawed – the unfortunate
consequence of a subtle error in the coordinates for heavy atoms along the
octamer’s two-fold axis of symmetry, resulting in an erroneous phase estimate
(Ramakrishnan, 1997). Nonetheless, the 3.1 Ǻ resolution structure of the
nucleosome core histone complex represents a major leap in our understanding of
the nucleosome and its role in chromatin structure and regulation.
The nucleosomal core histone octamer is arranged in a tripartite left-handed
superhelix made up of two H2A-H2B dimers and a central (H3-H4)2 tetramer. Core
complex assembly is preceded by the binding of individual histone monomers in an
antiparellel head-to-tail arrangement, forming a characteristic “handshake” motif.
This motif describes the unique way in which the long helices of paired histone
monomers intersect such that the HSH domains converge to create a structure
that is distinctly similar to two hands clasped together. In contrast to localized
contacts formed when most proteins associate, histones form extensive
interactions spanning the entire chains (Arents et al. 1991). Following H3-H4
dimerization, formation of the (H3-H4)2 tetramer occurs through the binding of a
pair of H3-H4 heterodimers at the HSH2 domains of the H3 subunits.
Tetramerization represents the initial step of nucleosome assembly. The H3-H3 C-
terminal interaction is arranged in a 4-helical bundle and is facilitated in part by an
essential His113 residue, which forms a hydrogen bond with Asp123 buried within the
two helices of the adjacent chain (Luger et al. 1997). Docking of HSH2 domains to
form the (H3-H4)2 complex results in a twisted crescent structure with a central
hinge at the H3-H3 interface through which a two-fold axis of symmetry lies (see
fig. 3) (Ramakrishnan, 1997). Moreover, each half-crescent of the tetramer is
rotated approximately 15° away from the two-fold axis (Arents et al. 1991).
Assembly of the core histone complex is completed through a final octamerization
step in which H2A-H2B heterodimers bind to the (H3-H4)2 tetramer at its terminal
regions. The H2B histone interacts with the H4 histone at their respective HSH2
domains. Due to the conserved structure of the HSH2 domain among the four
histones the H2B-H4 interaction is remarkably similar to the association of the H3
histones during tetramerization. HSH2 domains at the H2B-H4 interface constitute
the 4-helical bundle in which His74 of H4 inserts between the two adjacent helices
to form a hydrogen bond with Glu90 on the H2B chain (Luger et al. 1997).
Importantly, the H2B-H4 interaction is weaker than the tetrameric H3-H3
interaction, despite a more extensive contact interface between H2B and H4; thus,
the association of H2B and H4 is more susceptible to changes in the surrounding
solvent (Ramakrishnan, 1997). Importantly, the fully assembled histone octamer
assumes the form of a left-handed protein superhelix. The radial axis of symmetry
for the superhelical conformation of the octamer runs perpendicular to its two-fold
axis of symmetry. When the complex is viewed down its radial axis it assumes the
appearance of a disk with a diameter of around 65 Ǻ; this would be akin to viewing
a vertically oriented cylinder from above or below. The outer surface of the protein
complex is characterized by regularly arranged ridges and grooves that constitute
an unambiguous path with a pitch of 28 Ǻ that follows the left-handed superhelical
form of the protein. This path creates an ideal binding site for DNA to wrap around
the surface of the histone octamer like thread on a spool (see fig. 4; Arents et al.
1997).
DNA Binding on the Histone Octamer
Structural studies of the nucleosome have been largely facilitated by
the use of micrococcal nuclease (MNase). Digestion of unraveled chromatin using
MNase is initiated at the nucleosomal DNA linker regions to produces a size
distribution of 10 nm chromatin fragments, described as oligonucleosomes.
However, oligonucleosomes tend to exhibit poor stability in solution. Extended
digests with MNase result in structures of progressively increased stability, such
that particle stability is directly proportional to the length of the free DNA fixed to
the nucleosome core. Of interest, particles consisting of a 165 bp DNA component
can be obtained from digests and are unique in that they are often associated with
the H1 histone. This More extensive nuclease digestion results in the isolation of
the highly stable core nucleosomal particle. This discrete unit is defined by a DNA
element of around 146 bp wound approximately 1.65 turns around the histone
octamer in a left-handed superhelix (Bates & Maxwell, 2006). Due to its stability,
the nucleosome core particle has provided the basis for x-ray diffraction studies
and is, thus, central to our present understanding of nucleosomal DNA-histone
surface interactions.
Similar to the subunit organization of the histone octamer, binding of a
continuous DNA segment on the surface of the histone complex is facilitated
predominantly through the conserved intrinsic properties of the histone-fold motif.
Yet, DNA-protein interactions are coordinated largely on the scale of the
handshake motif – that is, through surface contacts formed with the four histone
pairs and irrespective of the tripartite nature of the octamer. Each dimer is directly
associated with approximately 27-28 bp of DNA, while unbound 4-bp segments of
duplex join the discrete regions of DNA-histone contacts. Furthermore, an
invariable Lys residue occupying the second position of the C-terminal loop II
doman (L2) of each histone monomer (see fig. 1) forms a salt bridge with a distal
phosphate group of the DNA backbone such that Lys residues of adjacent dimers
traverse one another and effectively form a dimer-DNA-dimer cross-linkage (see
fig. 5; Ramakrishnan, 1997; Luger et al. 1997). Extruding L2 Lys residues of the
H2A-H2B dimers at the superhelical ends of the histone octamer facilitate docking
of the remainder of DNA bases in the formation of the 165 bp core particle. The
histone-DNA contacts occur predominantly through basic amino acid residues
bonding with the negatively charged phosphate groups of the DNA duplex to
establish a sequence-independent association. This general mode of ionic
interaction is a recurrent theme in nucleoprotein complexes. However, the helical
structure of DNA in the nucleosome is such that only two adjacent phosphate
groups per DNA strand are within direct hydrogen bonding range of the histone-
fold dimers. Despite this, it is thought that solvation of the individual
macromolecules mediates nucleosome assembly through the formation of water-
bridges which transiently stabilize indirect hydrogen bonding between the two
structures (Davey et al. 2006). The primary DNA binding site for each histone
dimer is positioned parallel to the central junction of the αII helices, such that it is
localized amid the N-termini of the α1 helices, which form arm-like protrusions
converging on the adjacent DNA backbone phosphates. Thus, the positive charges
of the two αI dipoles, as well as main chain amide groups, and variable side chain
interactions act cooperatively to create a stable interaction between the DNA and
histone dimer (Luger et al. 1997).
Nucleosome Phasing and Gene Expression
Histone-DNA interactions within the nucleosome are achieved
predominantly through non-specific histone contacts with the DNA phosphodiester
backbone. Nevertheless, a preferential specificity for histone binding at certain
DNA sequences has been observed. Early descriptions of nucleosome specificity
include work by Simpson and coworkers (1983) in which nucleosome
reconstitution was carried out in vitro using a 260 bp DNA fragment encoding the
5S rRNA from the sea urchin L. variegatus. A strong preference was identified for
nucleosome localization within the rRNA coding sequence such that the
transcription start site was positioned precisely at the center of the nucleosomal
DNA superhelix (i.e. proximal to the H3-H3 junction) (Simpson & Stafford, 1983).
In addition to preferential nucleosome binding in L. variegatus, this intrinsic
property of nucleosomes has been distinguished in various other contexts,
including the 5S rRNA locus of Drosophila and Xenopus, as well as at discrete sites
in the SV40 genome (Ramakrishnan, 1997). The phenomenon of sequence-
dependent nucleosome positioning is broadly referred to as ‘nucleosome phasing’.
In terms of function, it has been suggested that nucleosome phasing is integral to
the formation of higher-order chromatin structures by directly impacting linker
DNA length and, thus, the flexibility of the 10 nm fiber. Moreover, sequence-
dependent phasing suggests a unique role for the nucleosome in the suppression of
transcription by physically obstructing RNA polymerases during initiation and
elongation of RNA synthesis; thus, nucleosomes (and perhaps higher order
chromatin structures as well) possess crucial gene-regulatory significance (Blank
& Becker, 1996). However, prior to considering the myriad cellular consequences
of nucleosome phasing, it is critical to resolve the molecular underpinnings of
nucleosomal sequence specificity.
Nucleosome phasing principally implies that electrostatic interactions
cannot be the sole factor governing the way in which DNA is wrapped around a
histone octamer. Rather, the process of nucleosome formation is inherently
multipartite and, accordingly, far more dynamic than first suggested by structural
studies. Demonstrating remarkable intuition, studies by Drew and Travers (1985)
provided early insights to the molecular basis of sequence specificity in
nucleosome positioning. Such work united two distinct emerging fields,
nucleosome phasing and intrinsic DNA bending, to consider the former in terms of
the latter. Electrophoretic fractionation studies of tightly compacted DNA
minicircles in the trypanosome L. tarentolae led to the discovery of DNA bending
at phased adenine/thymine (AT) tracts due to intrinsic deformation of the DNA
helix, characterized by minor groove compression (Marini et al. 1982; Koo et al.
1986). It was found that intrinsically bent DNA is configured such that the minor
groove at AT tracts is positioned on the inner face of the DNA bend, whereas
intermittent GC clusters appear to be distinguished by a minor groove oriented
outwards. Nucleosome reconstitution experiments using DNA oligonucleotides
with sequence-directed curvature demonstrated that the helical conformation of
intrinsic bending was conserved in the arrangement of DNA in the nucleosome (i.e.
the ‘rotational setting’ of the nucleosome). Consequently, it was suggested that
intrinsically bent DNA may favor histone binding, while inherently rigid DNA
sequences may prohibit nucleosome assembly (Drew & Travers, 1985). Further
studies comparing the structure of DNA in the nucleosome to that of free DNA
have bolstered the idea that the inherent flexibility of a DNA sequence conveys
preference in nucleosome positioning (Hayes et al. 1990). Importantly, histone
octamerization is accomplished in a DNA-dependent manner, wherein the (H3-H4)2
histone tetramer binds to DNA before the two H2A-H2B dimers are incorporated in
the core histone complex. If one considers the general curved architecture of the
histone tetramer (see Fig. x) it is apparent that this structure provides a suitable
scaffold for preferential DNA binding on the basis of intrinsic flexibility (Dong &
Holde, 1991). Although the bent shape of the histone tetramer may underlie
nucleosome phasing, it is critical to conceptualize phasing in terms of the kinetic
and thermodynamic challenges to nucleosome formation. Namely, DNA bending by
the (H3-H4)2 tetramer is a thermodynamically unfavorable process due to the
structural distortions imposed on native B-form DNA. However, AT rich sequences
are relatively more flexible than GC rich sequences due to the fact that A-T base
pairing forms two hydrogen bonds, whereas G-C base pairing involves three. Thus,
the free energy required to induce deformation of an AT rich sequence is
comparatively lesser than that needed to deform GC rich DNA. Likewise, DNA that
possesses intrinsic curvature due to the presence of A/T tracts in phase with the
helical periodicity presents an optimally suited binding site for the (H3-H4)2
tetramer. Such sequences with highly favorable rotational settings minimize the
energetic requirements of nucleosome formation and, consequently, facilitate
preferential nucleosome binding (Ramakrishnan, 1997). Therefore, while
nucleosome positioning is certainly sequence-directed, it is not precisely sequence-
specific; rather, a nucleosome will bind over a contiguous range of sites about a
preferred sequence due to the comparable rotational settings of adjacent DNA
regions. Consistent with this idea, nucleosome positioning has been found to be
characterized by major and minor sites at a given locus (,). This property of
nucleosome phasing reflects a limited role for DNA sequence in nucleosome
binding. Major and minor nucleosome binding sites are thought to reflect local
fluctuations in nucleosome position, wherein the complex transiently dissociates
and reassembles at an adjacent site. This kinetic hallmark of the nucleosome is
referred to as ‘nucleosome mobility’ and is dependent on the degree of stability
that a given complex possesses. Accordingly, nucleosomes are found to occupy
major and minor sites at an equilibrium distribution (,).
Mobility is a fundamental characteristic of nucleosomes because complex
stability directly establishes the ease with which RNA polymerase (RNAP) can
transcribe through nucleosomal DNA. It is thought that many RNAPs, including
phage RNAP, bacterial RNAP and eukaryotic RNA polymerases II and III (Pol II
and PolIII), adopt a paused conformation upstream of a nucleosome, such that the
histone complex may effectively ‘step around’ the transiently immobilized RNAP by
means of a DNA loop (Workman & Kingston, 1998; Hodges et al. 2009; Workman,
2006). Of primary significance, nucleosome displacement and histone transfer
during transcription-elongation are explicitly dependent on the mobile capacity of
a given nucleosome complex. A seeming quandary then arises: while AT rich DNA
sequences favor nucleosome formation, nucleosomes with an AT rich DNA moiety
(including intrinsically bent DNA) are so-favored because they possess optimal
thermodynamic stabilization and, thus, preclude transcription by RNAP. This
problematic relationship between nucleosome positioning and nucleosome mobility
during transcription-elongation appears to be resolved through nucleosome
(de)stabilization factors and epigenetic histone modifications that regulate the
strength with which a histone octamer binds DNA (Henikoff, 2008). Post-
translational [histone] modifications (PTMs) are localized to the C-terminal
domains (CTDs), which are characteristically short ‘tails’, less than 40 amino acids,
extending from the globular body of the histone complex (Campos & Reinberg,
2009). In this way, histone CTD tails are thought to act as platforms for covalent
modifications, including ubiquitination, phosphorylation, methylation, acetylation,
and SUMOylation (i.e. the addition of small ubiquitin-like modifier proteins).
Collectively, histone modifications comprise a highly orchestrated ‘epigenetic’
histone code, which appears to specifically regulate the strength and efficiency of
intra-nucleosomal contacts, as well as extra-nucleosomal interactions (e.g.
nucleosome-nucleosome contacts in the 30 nm fiber) by inducing conformational
changes in the nucleosome complex. In addition to directly altering intra- and
extra-nucleosomal histone contacts, PTMs can be ‘read’ by effector proteins
through particular domains, such as chromodomains, which recognize methylated
sites, and bromodomains, which distinguish acetylated residues (Campos &
Reinberg, 2009). The tremendous complexity of the histone code is established
through the diverse classes of PTMs, the various residues to which PTMs can be
made, and the fact that a single amino acid can acquire several modifications
(Berger, 2007). Ongoing research aims to further decipher the histone code and
improve our current understanding of nucleosome remodeling. Nevertheless, it is
apparent that such phenomena contribute significantly to global chromatin
organization and the large-scale regulation of gene expression.
In the past forty years our understanding of chromatin organization and its
functional implications has expanded exponentially. Since the nucleosome model of
chromatin organization was first described by Roger Kornberg in 1974, our
perception of chromatin and its fundamental structure, the nucleosome, has
undergone an ideological metamorphosis. No longer is the nucleosome simply the
primary unit of chromatin by which genome compaction is achieved in eukaryotes.
Rather, we now recognize the nucleosome, as well as chromatin in general, in
terms of the paradigms of gene expression, and thus cellular diversity, which they
mutually establish. Most remarkable, this monumental function of chromatin
organization is communicated through discrete molecular modifications to the
primary structure of the nucleosomal histone core; thereby, specifying nucleosome
structure as central to the vast epigenetic ‘language’ of our cells.
I
Figure 1. Histone H2A of X. laevis resolved at 2.8 Å resolution – the histone fold. N and C terminal helix-sheet-helix motifs are displayed at helices I and II. Also, note the overall inverted Z-form characteristic of the fold.
Figure 2. The H2A-H2B heterodimer of X. laevis resolved at 2.8 Å resolution.
HSH2
III
N term.
HSH2
HSH1
HSH2
HSH1
Loop I
Figure 3. H3-H4 tetramer of X. laevis resolved at 2.8 Å resolution. The general form of the tetramer is that of a crescent. The tetramer is formed through contacts between the H3 histones of the two dimers via binding at the HSH1 domains to form a four
Figure 4. Complete nucleosome complex (top- side view, bottom-birds-eye.). The DNA follows the left-handed superhelical organization of the histone complex (above). H2A-H2B dimers are displayed in yellow and cyan, while H3-H4 dimers are shown is blue and magenta. The structure was obtained from X. laevis and
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