Download letters The essential histone variant H2A.Z regulates the

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The essential histone
variant H2A.Z regulates the
equilibrium between
different chromatin
conformational states
Jun Y. Fan1, Faye Gordon2, Karolin Luger3,
Jeffrey C. Hansen2 and David John Tremethick1
1
The John Curtin School of Medical Research, The Australian National
University, P.O. Box 334, Canberra, Australian Capital Territory 2601,
Australia. 2Department of Biochemistry, The University of Texas Health
Science Center, San Antonio, Texas 78229, USA. 3Department of
Biochemistry and Molecular Biology, Colorado State University, Fort Collins,
Colorado 80523-1870, USA
Published online: 19 February 2002, DOI: 10.1038/nsb767.
Explaining the determinants involved in regulating the equilibrium between different chromatin structural states is fundamental to understanding differential gene expression.
Histone variant H2A.Z is essential to chromatin architecture
in higher eukaryotes but its role has not yet been explained.
We show here that H2A.Z facilitates the intramolecular folding of nucleosomal arrays while simultaneously inhibiting
the formation of highly condensed structures that result
from intermolecular association. This makes a case for
H2A.Z playing a fundamental role in creating unique chromatin domains poised for transcriptional activation. These
results provide new insights into understanding how chromatin fiber dynamics can be altered by core histone variants
to potentially regulate genomic function.
Within the eukaryotic nucleus, DNA and histones are assembled into chromatin to form a
highly dynamic nucleoprotein fiber. Chromatin
a
must be stable enough for efficient packaging of
DNA into the nucleus while at the same time
labile enough to allow access of DNA to DNAbinding proteins like transcription factors. The
nucleosome is the fundamental unit of chromatin, consisting of DNA wrapped around an
octamer of core histones. Cells exploit alterations in histone–DNA and histone–histone
Fig. 1 Characterization of H2A- (control) and H2A.Zcontaining nucleosomal arrays. a, Structure of the 12
repeats of 208-12 DNA template used in this investigation. b, Micrococcal nuclease digestion of assembled
arrays. 208-12 DNA (300 µg) was assembled into an
array using either control or H2A.Z-containing
octamers (300 µg). Arrays containing an equivalent of
3 µg of DNA were cleaved with micrococcal nuclease in
the presence of 1.0 mM CaCl2. c, EcoRI digestion of
nucleosomal arrays. The same reconstitutes were
digested with EcoRI. The digested array (0.5 µg and
1.5 µg) was then loaded and electrophoresed on a 1%
agarose gel. The percentage of octamer-free DNA was
quantitated using densitometry. d, Sedimentation
velocity analysis. The same reconstitutes, in low salt
buffer, were analyzed by sedimentation velocity in the
XL-A analytical ultracentrifuge, and the data were
analyzed to yield the integral distribution of sedimentation coefficients.
172
c
interactions within and between nucleosomes to regulate gene
expression, partly through modulation of the equilibrium
between open and highly condensed conformational states. One
extensively studied mechanism for altering the higher order conformation of chromatin is posttranslational modification of histones1. However, there are probably other equally important
ways to modify chromatin structure, such as altering the biochemical composition of nucleosomes by the replacement of
major histone types with specific histone variants.
The structural consequences of having the variant histone
H2A.Z incorporated into chromatin is not known, but its presence and high level of conservation from yeast to humans
(>90% sequence identity among different organisms) indicate
that H2A.Z has an important, specific role in chromosome function2. Consistent with this proposal, H2A.Z is essential for
Drosophila3, Tetrahymena4 and mouse survival5. Recently, we
defined the region of Drosophila H2A.Z required for its essential
function6. This region is part of the docking domain, which is
involved in maintaining the interactions between the histone
H3/H4 tetramer and the histone H2A/H2B dimer within the
nucleosome. The docking domain also contributes to surface
features of the nucleosome. Most recently, the crystal structure
of a nucleosome containing H2A.Z was solved7. Amino acid
changes in the docking domain of H2A.Z result in an altered
nucleosomal surface that could potentially modulate interactions between nucleosomes. Here, we addressed the issue of
whether H2A.Z can modulate nucleosome–nucleosome interactions involved in higher-order chromatin condensation.
H2A.Z facilitates intranucleosomal interactions
To determine the role of H2A.Z in this nucleosome–nucleosome
interactions, we employed a highly defined in vitro chromatin
model system. This model system uses the 208-12 DNA template,
which consists of 12 repeats of a 208-base pair (bp) nucleosome
positioning sequence from the sea urchin 5S RNA gene (Fig. 1a).
One major advantage of using this system to investigate the influ-
b
d
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letters
a
b
c
d
Fig. 2 H2A.Z facilitates the intrinsic 29S–55S folding pathway.
a, Sedimentation coefficient distribution plot of control and H2A.Zcontaining arrays in 0.25 mM MgCl2. b, Sedimentation coefficient distribution plot of control and H2A.Z-containing arrays in 1.5 mM MgCl2.
c, Effect of MgCl2 on the average S of H2A (control) and H2A.Z nucleosomal arrays. The average Save (Smidpoint) is defined as the boundary fraction = 0.5 of the sedimentation coefficient distribution plot. d, Effect of
ZnCl2 on the average S of control H2A and H2A.Z nucleosomal arrays.
The same reconstitutes, in different ZnCl2 concentrations, were analyzed
by sedimentation velocity. Save was determined for each sedimentation
coefficient distribution plot. Plasmid linear DNA 2.74 kb in length (pGEM
digested with EcoRI) was assembled into a nucleosomal template using
control or H2A.Z-containing octamers. e, Sedimentation coefficient distribution plots of control and H2A.Z-containing nucleosomal templates
in low salt buffer. f, Sedimentation coefficient distribution plots of control and H2A.Z nucleosomal templates in 1.0 mM MgCl2.
repeats released by EcoRI digestion gives a precise determination
of the extent of nucleosome assembly9–11. This analysis revealed
that the nucleosomal arrays (Fig. 1b) have 93.0 and 93.5% of 208
DNA repeats occupied with H2A and H2A.Z histone octamers,
respectively (Fig. 1c; Table 1). H2A-containing mononucleosomes migrate at two distinct positions (Fig. 1c). The faster
migrating band may be due to some partial dissociation during
e
f
electrophoresis under the gel-shift conditions used here. H2AZ
mononucleosomes run at single position, perhaps indicating
that they are more stable (see discussion).
Next, we performed sedimentation velocity studies of H2Aand H2A.Z-containing nucleosome arrays under low salt conditions using the analytical ultracentrifuge. This analysis provides
an extremely sensitive measure of the sedimentation coefficients
and degree of saturation of all species in a sample. Previously,
208-12 DNA was shown to be assembled with 12 histone
octamers adopting an extended 29S conformation in low salt
ence of H2A.Z on chromatin dynamics is that regularly spaced buffer8–10. Because the arrays do not fold under these conditions,
nucleosomal arrays can be assembled using only purified DNA any differences in the sedimentation coefficients of arrays
and histones, which yield a highly homogenous population of assembled with H2A or H2A.Z would indicate a difference in
nucleosomal arrays that can be characterized rigorously by ana- nucleosome density8–11. The nucleosomal arrays assembled with
lytical methods8–11. In addition, this model system has been used either H2A or H2A.Z display identical sedimentation distributo extensively analyze the macromolecular determinants involved tion profiles (Fig. 1d). Because the degree of DNA wrapping is
in chromatin fiber condensation8–11. Here, we compare the solu- identical for both types of nucleosomes, as shown by the crystal
tion dynamics of chromatin model systems assembled from structure of an H2A.Z-containing nucleosome7, both templates
either control Xenopus histone octamers or Xenopus histone have been assembled to the same nucleosome density (Fig. 1d).
Because the average sedimentation coefficient for both temoctamers containing H2A.Z7 (Fig. 1a).
A major concern in this analysis was to ensure that the 208-12 plates is 28.5S, the majority of both the control and H2A.Z
DNA templates contained an identical degree of saturating H2A arrays are fully saturated (≥50% loaded with 12 nucleosomes per
or H2A.Z histone octamers. To accomplish this, three approach- DNA template) (Table 1).
es were used. First, we cleaved the H2A or H2A.Z nucleosomal
Finally, to confirm these results, quantitative agarose gel
arrays with EcoRI and subjected the digestion products to non- electrophoresis was carried out to determine the effective radius
denaturing gel-shift analysis. A pair of EcoRI digestion sites flank (Re) and gel-free mobility (µo) of these arrays. Both of these
each 208-bp repeat (Fig. 1a). Consequently, digestion of a parameters are sensitive measures of the level of nucleosome satnucleosomal array with EcoRI releases 208-bp fragments of DNA uration8,9. Both the Re and µo values are indistinguishable for
that are either histone-free or contain a histone octamer (and control and H2A.Z-containing arrays and are similar to reported
partially digested di-nucleosomes). Following gel-shift analysis, values obtained for nucleosomal arrays containing 12 histone
careful quantitation of the amount of histone-free 208 DNA octamers per 208-12 DNA (Table 1)8,9,11.
Having established that control and
H2A.Z
nucleosomal arrays are assembled
Table 1 Properties of purified 208-12 control and H2A.Z-containing nucleosomal
with histone octamers to the same extent,
arrays in low salt buffers
we used sedimentation velocity analysis to
Save (S)1
Re (nm)2
–µo (× 10–4 cm2 V–1 s–1)
% free DNA3
address whether H2A.Z influences
H2A
28.5
28.33 ± 0.4
1.84 ± 0.33
7.0 ± 0.3
intramolecular folding of nucleosomal
H2A.Z
28.5
28.55 ± 0.9
1.81 ± 0.05
6.5 ± 0.3
arrays. The addition of increasing concen1Values represent the S
trations of Mg2+ ions to an unfolded array in
20,W obtained at the boundary fraction midpoint in TE buffer (Fig. 1d).
2Quantitative agarose gels were carried out as described9. Values represent the mean ± standard
low salt was previously shown to cause a
deviation of three experiments at Pe ≥ 200 nm.
series of conformational changes that result
3The mean ± standard deviation of the percentage of unoccupied 5S rDNA sites (Fig. 1c).
in the formation of a highly folded state9,10.
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Specifically, with increasing salt, the extended 29S array first
compacts into a moderately folded 40S intermediate and finally
to a highly folded 55S conformation12. Linker histones stabilize
the array in the 55S conformation, which is as condensed as the
canonical 30 nm fiber9–11. Under physiological conditions, a fully
saturated nucleosomal array lacking linker histones is in equilibrium between the 29S, 40S and 55S conformational states9,10,12.
The sedimentation coefficient distribution profiles of control
and H2A.Z arrays in the presence of 0.25 mM (Fig. 2a) and
1.5 mM MgCl2 (Fig. 2b) indicate that H2A.Z facilitates the
intrinsic 29S–55S folding pathway. In both MgCl2 concentrations (Fig. 2c), the H2A.Z-containing arrays adopted a significantly more folded conformation than the control H2A arrays,
as indicated by the right-shifted sedimentation coefficients at all
points in the distribution plot. H2A.Z facilitates the formation
of both the 40S intermediate and the highly folded 55S structure. In 1.5 mM MgCl2, the sedimentation coefficient distribution of the H2A.Z arrays, but not the control arrays, reached 55S
(Fig. 2b). To determine whether the effect of H2A.Z on higher
order folding is ion-specific, the sedimentation velocity experiments were repeated using ZnCl2. At all salt concentrations
tested, H2A.Z increased the average degree of folding (Fig. 2d),
consistent with the results obtained in MgCl2. Given that the
role of cations in the folding process is neutralization of DNA
charge, ZnCl2 was more efficient than MgCl2 in promoting the
folding of both types of arrays (Fig. 2c,d), because Zn2+ binds
more tightly to DNA than Mg2+. Taken together, these data
demonstrate unequivocally that the conformational equilibrium of H2A.Z arrays is shifted toward the folded states relative
to control H2A arrays.
Next we determined whether H2A.Z facilitates folding of a
nucleosomal array when a different DNA sequence is used. To
address this, we assembled linear plasmid DNA of 2.74 kb in
length (pGEM digested with EcoRI) with control or H2A.Zcontaining histone octamers to the same nucleosome density
(≥50% loaded with 13 nucleosomes per DNA template, equivalent to one nucleosome per 210 bp of DNA) (Fig. 2e). In the
presence of 1.0 mM MgCl2, plasmid DNA containing H2A.Z
adopted a more folded conformation (Fig. 2f). Therefore, we
conclude that H2A.Z can promote intramolecular folding
independent of DNA sequence and the positioning of nucleosomes in a regular array.
H2A.Z inhibits internucleosomal interactions
We noted another feature during the sedimentation velocity
experiments. At 0.6 mM ZnCl2 and 2.0 mM MgCl2, control
arrays pelleted within seconds in the analytical ultracentrifuge,
whereas H2A.Z arrays sedimented much more slowly. Above the
range where folding occurs, divalent cation concentrations
induce a cooperative, reversible oligomerization reaction in
which 208-12 nucleosomal arrays self-associate to form large,
soluble nucleoprotein complexes. These complexes share many
features of interphase chromosomal fibers12. The N-terminal
domains of the core histones are required for oligomerization10,
and acetylation impedes the oligomerization process in vitro13,14.
To investigate whether H2A.Z also impedes oligomerization, we
used a microfuge-based assay15 to determine the extent of
oligomerization as a function of Zn2+ concentration for both
control and H2A.Z-containing nucleosomal arrays. Using the
same control and H2A.Z nucleosomal arrays as the sedimentation velocity experiments (Fig. 2a–d), H2A.Z arrays were shown
to oligomerize less effectively than control arrays, both in the
absence (Fig. 3a) and presence of 50 mM NaCl (Fig. 3b), as
revealed by the requirement for greater amounts of divalent
cations to achieve equivalent levels of oligomerization. We conclude that H2A.Z, like histone acetylation, impedes the formation of highly condensed chromatin that results from
intermolecular associations in vitro. This finding may explain
why H2A.Z-containing arrays are more sensitive than control
arrays to micrococcal nuclease digestion (carried out in the presence of CaCl2, Fig. 1b).
H2A.Z can alter nucleosome positioning
To investigate the possibility that H2A.Z may alter nucleosome
positioning, we determined whether H2A or H2A.Z histone
octamers occupy different positions on the 208-12 DNA template. Studies have shown that ∼50% of chicken erythrocyte histone octamers occupy a single translational position, with the
remaining octamers distributed in alternate translational positions16. To measure nucleosomal positioning we exploited a
unique AluI restriction site located 48 bp from the 5′ boundary
of the 5S positioning sequence (Fig. 1a). Control and H2A.Z
arrays were digested with micrococcal nuclease to produce a
homogeneous population of nucleosome core particles. The
core particle DNA was purified and digested with AluI, and the
DNA fragments were run on a polyacrylamide gel (Fig. 4).
Consistent with previous studies16, control histone octamers are
located primarily at two major locations, with position 1 being
the favored position (44% of the total positions). Although
H2A.Z-containing octamers occupy the same positions, a
a
b
Fig. 3 H2A.Z inhibits the formation of highly condensed chromatin fibers.
The percentage of control and H2A.Z-containing nucleosomal arrays that
remained in the supernatant after a centrifugation step (using the same
reconstitutes as in Fig. 2) are shown. Each data point represents the average and standard deviation of two independent experiments. a, Control
and H2A.Z-containing nucleosomal templates in low salt buffer.
b, Control and H2A.Z-containing nucleosomal templates in 50 mM NaCl.
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Fig. 4 H2A.Z alters nucleosome positioning. Control and H2A.Z nucleosomal arrays were digested with micrococcal nuclease (in 1.0 mM CaCl2)
into trimmed mononucleosomes. The DNA was dissociated from histones
and purified on a 6% polyacrylamide gel. A single DNA fragment, with
an apparent length of 155 bp, was excised, purified and digested with
AluI (because the 5S DNA is structured, core particle DNA runs at
∼150–155 bp). Following digestion, the products were run on a 12%
polyacrylamide gel, and the digestion products were quantitated using
densitometry. Shown is a representative densitometer trace. P1 and P2
refer to the AluI digestion products, which define the two major nucleosomal positions as described16. U is undigested nucleosomal DNA. Shown
below the traces are the position of the two major nucleosomal positions and the percentage of nucleosomes located at these positions.
significantly greater fraction of the H2A.Z histone octamers are
found in the major positional frame (position 1 = 70%). Thus,
H2A.Z can influence nucleosome positioning in vitro, which
may also be important to the biological functions of this variant
histone in vivo.
Conclusions
Modulating the equilibrium from a compacted to a decondensed
chromatin state is a key step in gene expression17. Removal of
linker histones destabilizes folded chromatin fibers9–11. However,
the intrinsic folding of nucleosomal arrays is sufficient to repress
transcription13. Therefore, identifying the molecular determinants that regulate nucleosomal array condensation is of fundamental importance to understanding gene regulation. Here we
show that the histone variant H2A.Z is critical to the chromatin
condensation process. We show that H2A.Z promotes intramolecular folding of nucleosomal arrays (Fig. 2). In contrast, H2A.Z
also significantly impedes in vitro oligomerization (Fig. 3). No
other known effector of chromatin condensation shows these
characteristics.
Changes in intramolecular folding involve local alterations
that influence fiber conformation, whereas oligomerization is
correlated with global chromosomal fiber condensation. This
later stage of compaction involves interactions between chromatin fibers, yielding a highly condensed state. Our data suggest
that H2A.Z may create a transcriptionally poised higher order
nature structural biology • volume 9 number 3 • march 2002
chromatin domain. The effect of H2A.Z to promote global
decondensation mimics the effect of hyperacetylation in vitro13.
As such, regions of decondensed genomic chromatin enriched in
H2A.Z will be, in principle, marked as a functionally primed
chromatin domains18. Assuming that the histone acetyltransferase Gcn5 participates in global acetylation of the yeast
genome19, this could explain why disruption of the histone
acetyltransferase GCN5 gene in yeast made cells highly dependent upon H2A.Z for viability20. At the same time, the globally
decondensed fiber containing H2A.Z will be more stably folded.
This stable folding may explain the need for more localized
structural changes caused by SWI/SNF and histone-modifying
enzymes. Alternatively, higher order folding induced by H2A.Z
may be required for establishing a specific ‘active’ chromatin
architecture at the promoter regions of certain genes20. This ability of H2A.Z to inhibit the formation of highly condensed chromatin while facilitating intramolecular folding can explain the
role of H2A.Z in both gene activation21 and the formation of
specialized structures at the HMR locus22.
H2A.Z probably affects higher order folding by altering core
histone tail domain interactions with nearby nucleosomes. The
tail domains are required for both higher order folding and
oligomerization8,10,11. H2A.Z-containing nucleosomes have a
more extensive acidic patch on the nucleosome surface compared to nonvariant nucleosomes7. This may promote tail
domain-dependent nucleosome–nucleosome interactions,
thereby facilitating the close approach of neighboring nucleosomes and concomitant intramolecular folding. The basis for
the inhibitory effect of H2A.Z on oligomerization remains to be
explained, but may be related to competition between folding
and oligomerization for the requisite tail domains12.
During the preparation of this manuscript, a conflicting
study was published, which reported that H2A.Z-containing
nucleosomes were less stable compared to nucleosomes containing major H2A, and, because of this, H2A.Z arrays were
less folded23. This study differs from ours in several ways.
First, we used a histone expression and purification system
shown to produce nucleosomes that yield high-resolution
crystallographic structures. Second, their study used a monovalent cation instead of divalent cations to study array folding,
and monovalent cations have been clearly established to be
unable to induce the formation of a stable 55S structure9.
Third, their study did not demonstrate that the arrays were
fully in complex with nucleosomes. In addition, H2A.Z was
shown to stabilize the association of H2A.Z/H2B dimers in
nucleosomes isolated from chicken erythrocyte chromatin24.
Consistent with this enhanced nucleosome stability, we have
also found that H2A.Z strengthens the interaction between
the H2A.Z/H2B dimer and the H3/H4 tetramer using our
in vitro assembled nucleosomal arrays in salt dissociation
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experiments (data not shown). Potentially, enhanced nucleosome stability can explain why H2A.Z-containing octamers
are found in one major position on the 5S RNA gene (Fig. 4);
however, we cannot rule out that intranucleosomal interactions during the folding process may facilitate positioning of
H2A.Z nucleosomes.
In summary, we report the first biochemical analysis of a
homogenous preparation of H2A.Z-containing nucleosomal
arrays. H2A.Z uniquely effects chromatin condensation;
higher order folding is accentuated, whereas oligomerization
is inhibited. These data suggest that a major function of
H2A.Z is to regulate the conformational equilibria of the chromatin fiber to promote specialized functional chromosomal
domains.
Methods
Nucleosome array reconstitution and sedimentation velocity
analysis. H2A.Z and control histone octamers were assembled
from recombinant histones25. These different octamers were used
to assemble the 208-12 DNA template into nucleosomal arrays26.
Sedimentation velocity studies using a Beckman XL-A and analysis of boundaries were carried out as described15. These data yielded
the integration distribution of sedimentation coefficients plotted as
the boundary fraction versus S20,W (sedimentation coefficient corrected for water at 20 °C). Average sedimentation coefficients (Save)
were determined from the rate of sedimentation at the boundary
midpoint (boundary fraction = 0.5).
Determination of nucleosome array saturation. Nucleosomal
arrays (∼2 µg) were digested with EcoRI and subjected to native
agarose gel electrophoresis as described15. Densitometry was used to
quantify the extent of nucleosome assembly, accounting for the
population of partially digested di-nucleosomes. To correct for histone quenching of fluorescence, the ethidium signal from nucleosomal bands was multiplied 2.5-fold15. Quantitative gel electrophoresis
was performed as described9.
Microcentrifuge assay for oligomerization self-association.
The ability of H2A.Z and control arrays to oligomerize at different
ZnCl2 concentrations was determined using a differential centrifugation assay15.
176
Nucleosome positioning. Nucleosomal arrays were extensively
digested with micrococcal nuclease. The monomer was DNA purified and digested with EcoRI and AluI. The DNA digestion products
were analyzed by polyacrylamide gel electrophoresis26.
Acknowledgments
This work was supported by a Human Frontier Science Program grant to K.L. and
D.J.T., and an NIH grant to J.C.H.
Competing interests statement
The authors declare that they have no competing financial interests.
Correspondence should be addressed to D.J.T. email:
[email protected]
Received 12 November, 2001; accepted 24 January, 2002.
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