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The location of the linker histone
on the nucleosome
To understand the mechanism of condensation of transcriptionally inactive
chromatin we must identify the details
of interactions between its components.
The fundamental structural unit of the
condensed 300-Å fibre is the chromatosome (see Box 1); this contains a histone
octamer and one molecule of linker histone, which are associated with 168 bp
of DNA. The chromatosomes are separated by varying lengths of linker DNA,
and the folding of the fibre is facilitated
by linker histone H1 and its variants,
which include H5. The linker histones
themselves contain basic N- and Cterminal tails, which flank a central
globular domain. This domain is sufficient for chromatosome formation but
its position in the particle has remained
controversial1. One recent model2 proposes that the globular domain bridges
two superhelical turns between one terminus and the dyad (see Box 1), while a
second places the domain on the inside
of one turn away from the dyad3,4. New
evidence5 indicates that, at least in bulk
chromatin, the first model is correct.
Location and orientation of the globular
domain of histone H5 in the nucleosome
An early model of the 300-Å fibre proposed that it has a solenoidal structure
in which a helical array of nucleosomes
is linked by histone H1 and that the
globular domain of this histone binds to
the dyad region located on the interior
of the solenoid6. In this model, adjacent
4
nucleosomes in the helix are connected
by a single linker DNA. Since this proposal, various alternative structures that
differ in the path of the linker DNA (e.g.
the zig-zag model) or in the location of
linker histone on the chromatosome
have been proposed.
Ramakrishnan and co-workers7 experimentally confirmed the interior location
of H1 by neutron diffraction, but evidence
for its binding site in the chromatosome
remained elusive1. The three-dimensional
structures of the globular domains of
both H1 and H5 (GH1 and GH5, respectively) showed that the globular domain
is a three-helix bundle that shares structural homology with helix-turn-helix
DNA-binding proteins. These structures
also revealed the existence of a secondary putative DNA-binding site, which is
separated from the recognition helix by
25 Å8,9 – an observation that is consistent
with the demonstration that this domain can bind two DNA duplexes simultaneously10,11. This structure suggested
a model in which the globular domain
bridges two adjacent DNA gyres (see
Box 1 and Fig. 1).
Both these predictions have now been
confirmed. Goytisolo et al.12 have demonstrated that mutations in the secondary
binding site abolish the ability of GH5 to
bind two duplexes and to form chromatosomes. In addition, the groups of
Muyldermans and Ramakrishnan, in
collaboration with our group5, have
mapped the binding site of the globular
and Kumar, S. (1998) J. Biol. Chem. 273,
26566–26570
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1243–1253
23 Colussi, P. A., Harvey, N. L. and Kumar, S.
(1998) J. Biol. Chem. 273, 24535–24542
24 Koseki, T., Inohara, N., Chen, S. and Nunez, G.
(1998) Proc. Natl. Acad. Sci. U. S. A. 95,
5156–5160
SHARAD KUMAR AND PAUL A. COLUSSI
Hanson Centre for Cancer Research, Institute
of Medical and Veterinary Science, Frome Rd,
Adelaide, Australia.
Email: sharad.kumar@imvs.sa.gov.au
domain of linker histone H5 on mixedsequence chicken chromatosomes
by conjugating a protein–DNA crosslinking reagent to specific Ser→Cys substitutions. These experiments show
that helix III binds in the major groove
of the first helical turn of the chromatosomal DNA (i.e. close to the end) while
the secondary DNA-binding site on the
opposite face of the globular domain of
H5 contacts the chromatosomal DNA
close to its midpoint. By exploiting the
ability of some Ser→Cys mutants to
self-dimerize, we5 inferred that helix I
and helix II of the globular domain of H5
face the solvent and the nucleosome,
respectively. In bulk chromatin, the
globular domain of the linker histone
thus forms a bridge between one terminus of chromatosomal DNA and the midpoint. This position of GH5 on the chromatosome would place its C-terminus
on the outside of the particle, between
one end of chromatosomal DNA and the
central gyre, and its N-terminus on the
inner surface of the entering DNA. The
orientation of the globular domain relative to the surface of the octamer is also
consistent with the protection of lysine
residues in helix II from reductive
methylation in chromatin containing H5
(Ref. 13).
Both the original model for GH5 positioning proposed by Allan et al.14 and
our more recent variant5 argue that GH5
binds at, or close to, the chromatosome
dyad (Fig. 1b,c). However, a second radically different model was recently proposed by Wolffe and Hayes and their
colleagues3,4. This model is based on
crosslinking3, site-directed DNA cleavage4 and micrococcal-nuclease (an enzyme that cuts linker DNA) mapping15
on a chromatosome formed on Xenopus
borealis somatic 5S rDNA, and proposes
that the globular domain of linker histone
0968–0004/99/$ – See front matter © 1999, Elsevier Science. All rights reserved.
PII: S0968-0004(98)01339-5
FRONTLINES
TIBS 24 – JANUARY 1999
H5 binds on the inside of one DNA gyre
at a single internal site 65 bp from the
dyad. They report that the single DNA
contact site is about two double-helical
turns from the proximal terminus of the
chromatosomal DNA (see Fig. 1d). No
contact is made with either the DNA termini or the dyad region but instead the
putative secondary DNA-binding site is
positioned on the upper surface of the
octamer close to the H2A–H2B dimer1.
The Wolffe–Hayes and bridging models
accordingly differ significantly in both
the position and the number of contacts
GH5 makes with nucleosomal DNA. Not
only do the two models differ fundamentally in the positioning of GH5 on the nucleosome but they also differ in another
significant respect. In the Wolffe–Hayes
model, the C-terminus of GH5 is directed
along the upper DNA gyre towards the
dyad, whereas in the bridging model
the C-terminus is directed towards the
linker DNA. Because the C-terminal domain of H5 binds to linker DNA16, the
Wolffe–Hayes model invokes a U-turn in
the C-terminal domain17, whereas this is
unnecessary in the bridging model.
Studies on a chromatosome reconstituted on a unique DNA sequence provide additional support for the terminal
binding of GH1/GH5 (i.e. the latter’s binding close to one end of the chromatosomal DNA) proposed in the bridging
model. Guschin et al.18 show that a variant of GH1, GH10, crosslinks to DNA
less than one turn from one end of a
chromatosome containing a thyroidhormone-response element. Nevertheless, the chromatosomes formed on the
Xenopus borealis somatic 5S rDNA could
have a different organization from that
of those formed on most other DNA sequences. In principle, crosslinking and
site-directed DNA cleavage should accurately identify DNA sequence(s) in close
proximity to GH5, but the mapping of
these contacts onto the nucleosome
structure requires an independent reference point, of which the most appropriate in this case is the nucleosomal
pseudodyad. The derivation of the
Wolffe–Hayes model assumed that the
somatic 5S-rDNA chromatosome is
uniquely positioned on two different
5S-rDNA fragments and that, in each
case, there is a single highly preferred
dyad position (i.e. a single translational
position or reference point), although
the actual inferred dominant dyad position differed by one double-helical turn
in the two sets of experiments3,4. However, recent studies have shown that
chromatosomes can occupy more than
Box 1. Levels of chromatin organization
(a)
(c)
(b)
The core nucleosome (a) contains a histone octamer associated with 145 bp of DNA
wrapped in 1.65 left-handed superhelical turns. Each superhelical turn corresponds to one
gyre. The dyad (X) corresponds to the crystallographic axis of symmetry. The chromatosome
(b) contains a histone octamer and one molecule of linker histone, which are associated
with 168 bp of DNA. The 300-Å fibre (c) is a compact structure containing chromatosomes
that are separated by varying lengths (0–80 bp) of linker DNA and might be wrapped as a
flat spiral or solenoid.
On-line see Fig. I.
one position on the same X. borealis
somatic 5S-rDNA sequence. Using
low-resolution mapping, An et al.19 find
at least two core-particle positions,
whereas Panetta et al.20, by mapping
dyad positions directly (using the technique introduced by Flaus et al.21), show
that the population of core particles and
chromatosomes formed on this DNA is a
mixture of several different translational
settings (i.e. contains chromatosomes
in which the position of the dyad varies)
present in roughly equal proportions.
An et al.19 also raised the possibility
that the original detailed micrococcalnuclease mapping15 of the somatic 5SrDNA chromatosome was itself intrinsically unreliable. Although the precise
number of translational settings (i.e.
dyad positions) available to a chromatosome will depend on the length of the
DNA fragment used, even with only two
equally likely settings, the contacts deduced from crosslinking and site-directed
DNA cleavage cannot be assigned unambiguously to any particular translational position. Indeed, the data of
Wolffe, Hayes and their colleagues3,4
could be entirely consistent with the
bridging model if at least some of their
chromatosomes occupied the positions
observed by Panetta et al.20
Structural asymmetry of the chromatosome
In mixed-sequence chromatosomes,
GH5 binds asymmetrically with respect
to the DNA sequence. Initial studies on
the structure of the chromatosome proposed that the binding of the linker histone symmetrically extends the amount
of DNA that is protected from nuclease
digestion by approximately one doublehelical turn at both ends of the nucleosome core1. This conclusion was based
on the preservation of a symmetric
DNase-I-cleavage profile within the core
section of chromatosomes. However,
more-recent studies on positioned chromatosomes have indicated that such an
extension of protection might be asymmetric and that there is no increase at one
terminus of the core DNA but an ~20-bp
increase at the other22,23. This observation is consistent with the finding that
short DNA sequences corresponding to
those found preferentially at the dyad of
core particles are located ~93 bp from one
terminus of chromatosomal DNA24, where
the half-length of core DNA is just 72.5 bp.
The available data do not resolve the
fundamental structural issue of the location of the two binding sites for GH5.
We know that these two sites are separated by one superhelical turn, but the
precise location of the helix-III-binding
5
FRONTLINES
TIBS 24 – JANUARY 1999
(b)
(a)
Allan et al. 1980
Core particle
(c)
(d)
Bridging model
Wolffe–Hayes model
Figure 1
Models for the placement of the globular domain of linker histone on the nucleosome.
(a) The core particle. (b) Allan et al.14 (c) Bridging model2. (d) Wolffe–Hayes model3,4.
(b)
(a)
The structure of the 300-Å fibre
Symmetric extension
Core particle
(c)
Asymmetric extension
Figure 2
The core particle (a), and possible locations of the globular domain in the chromatosome
for symmetric (b) and asymmetric (c) extension of nuclease protection by linker histone.
The extra DNA that is protected is shown in a darker shade. X, histone-octamer dyad.
6
site relative to the location of the histone octamer is not clear. In the cases
where asymmetric extension is observed,
the data do not exclude the possibility
that the dyad of the core particle has
moved by one turn on binding linker histone. If extension is asymmetric, does
the recognition helix of GH5 bind to the
extended terminus or to the terminus
that defines one border of the core particle (Fig. 2)? One additional feature of
chromatosomal DNA from chicken erythrocytes is the frequent occurrence of sequences related to the tetranucleotide
AGGA within half a double-helical turn
of one terminus25,26. Although the position of this signal sequence is similar to
that of the contact point with helix III of
GH5, it could also constitute a preferred
binding site for a core-histone tail that
dislocates on GH5 binding.
A further unresolved question is that
of how the linker histone selects one of
two possible binding sites on an apparently symmetric core particle. The core
particle itself could adopt, however
transiently, an asymmetric configuration
– in which case, the binding of the linker
histone would be determined by the
properties of the core particle and, ultimately, by the DNA sequence.
Whatever the precise details of the
organizational symmetry of the chromatosome, the orientation and location
of the globular domain are such that the
position of the basic C-terminal tail would
allow the latter to bind the entering
and/or exiting (proximal or distal) linker
DNA. If both are bound, the resulting nucleosomal particles would then assume
the form observed by electron microscopy16. The N-terminal tail would also be
in close proximity to linker DNA; however, reconstitution studies show that
this domain is slightly inhibitory to monochromatosome formation16. Alternatively,
the N-terminal tail might contact a
neighbouring chromatosome or a distant portion of linker DNA.
The crystal structure of the core particle27 indicates that the N-terminal tail
of one of the two copies of histone H4 is
constrained by a contact between amino
acid residues that are 16–25 residues immediately distal to the acetylatable lysines
and the face of an H2A–H2B dimer on a
neighbouring nucleosome core. This, or
a similar, disposition in native chromatin
would direct the N-terminal tail of H4 towards the outer surface of the condensed
(solenoid) structure. The sequence of
this region, and hence the potential to
FRONTLINES
TIBS 24 – JANUARY 1999
form nucleosomal contacts, is very highly
conserved. Contacts of this type, if truly
representative of chromatin in vivo,
have significant biological implications.
First, their existence suggests that, without histone modification, nucleosomes
in an array are capable of mutual interactions; consequently, condensation, to
a greater or lesser extent, should be the
default state of chromatin. Second, any
remodelling (i.e. decondensation of this
state) must require the disruption of internucleosomal contacts, whatever their
nature. The observation that the product of the SWI/SNF and RSC remodelling
activities probably contains two altered
nucleosomal particles28–30 suggests that
the substrate of these activities is two
spatially adjacent nucleosomes whose
histone octamers contact each other.
Given that a single turn of the chromatin solenoid contains, on average, six nucleosomes, the effects of remodelling activities will depend on the connectivity
between spatially adjacent nucleosomes. If a pair of such nucleosomes are
also topologically adjacent (i.e. joined
by linker DNA without any intervening
nucleosomes), remodelling should result in highly localized decondensation.
However, if the topological connectivity
does not define spatial proximity, as in
some zig-zag models of condensed chromatin structure31, remodelling should
result in more-extensive disruption. In
this context, the observation that the
remodelling induced by the yeast protein Adr1p is limited to two connected
nucleosomes32 argues strongly that, in
an ordered array, nucleosomes connected
by linker DNA also make histone–histone
contacts with each other. However, if
these nucleosomes are not spatially adjacent in the condensed fibre, this pattern of disruption should necessitate a
prior, more extensive, decondensation.
A further common feature of zig-zag models of condensed chromatin is straight
linker DNA. The observation that nucleosome dimers, which in these models
would contain a single straight linker of
invariant length, compact with increasing
ionic strength is more consistent with
solenoid-type models in which the linker
DNA is bent or kinked33,34. The overall
picture that emerges is thus more a detailed refinement of the original proposals
for the structure of condensed chromatin
than any significant radical departure.
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ANDREW TRAVERS
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, UK CB2 2QH.
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