Received for publication, December 12, 2000, and in revised form, January 31, 2001
The MgCl2-induced folding of
defined 12-mer nucleosomal arrays, in which ubiquitinated histone H2A
(uH2A) replaced H2A, was analyzed by quantitative agarose gel
electrophoresis and analytical centrifugation. Both types of analysis
showed that uH2A arrays attained a degree of compaction similar to that
of control arrays in 2 mM MgCl2. These results
indicate that attachment of ubiquitin to H2A has little effect on the
ability of nucleosomal arrays to form higher order folded structures in
the ionic conditions tested. In contrast, uH2A arrays were found to
oligomerize at lower MgCl2 concentrations than control
nucleosomal arrays, suggesting that histone ubiquitination may play a
role in nucleosomal fiber association.
 |
INTRODUCTION |
Although for many years histones were thought to be merely
structural components of nucleosomes, the primary level of DNA organization required to compact the genome in the nucleus, they are
now recognized as important players in the mechanisms underlying gene
expression. One of the keys to chromatin's dynamic nature is
post-translational modification of the flexible histone tails. These
modifications include acetylation, phosphorylation, methylation, and
ubiquitination (1-3). Ubiquitin is a small, mainly globular and highly
conserved protein consisting of 76 amino acids found, as its name
implies, in most living organisms. Ubiquitin has been found to be
conjugated in vivo to histones H2A, H2B, H3, and H1 (4-7).
Ubiquitin is reversibly attached to bovine H2A by means of an
isopeptide bond between its terminal glycine and the
amino group of
H2A lysine 119 (8), which lies in the trypsin-accessible region of the
carboxyl-terminal tail (9). Histones are among the most abundant
ubiquitin-protein conjugates in higher eukaryotes, where 5-15% of the
total H2A is ubiquitinated (10). The function of histone ubiquitination
remains unclear. Although ubiquitin has been shown to play an important
role in the degradation of many short-lived proteins (for reviews see
Refs. 11, 12), two independent studies have shown that ubiquitination
does not tag histones for degradation (13, 14). Nucleosomes can be reconstituted with two molecules of uH2A or
uH2B1 without obvious
perturbation of the nucleosomal structure (15, 16). Although some
studies have reported an enrichment of uH2A in transcriptionally poised
or active chromatin (17, 18) others do not find this correlation
(19-21). Moreover, inhibition of transcription does not alter the
levels of uH2A in a variety of cell lines (22-24), whereas inhibitors
of hnRNA synthesis were found to cause a decrease in uH2B levels (23,
24). Cell cycle studies have shown that, in cells undergoing mitosis,
uH2A levels decrease progressively to non-detectable levels at
metaphase but increase again in late anaphase (14, 25, 26). Based on
these and other observations, several authors have proposed that H2A
ubiquitination could perturb chromatin structure (e.g. Refs.
6, 17, 18), but until now this model has not been tested. In this
report we analyze the folding of defined nucleosomal arrays containing
uH2A in response to MgCl2 using quantitative agarose gel
electrophoresis and analytical centrifugation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Fresh calf thymus and whole chicken blood
were obtained from the local abattoir.
Ubiquitinated histone H2A was purified from calf thymus as described
previously (16, 27). All chemicals were of reagent grade.
Preparation of Template DNA--
The DNA template (208-12)
consisting of 12 tandem repeats of a 208-bp sequence derived from
Lytechinus variegatus 5 S rDNA was amplified and purified
from plasmid p5S 208-12 (a kind gift from Dr. R. T. Simpson (28)).
The plasmid was purified using Nucleobond (Machery-Nagel) columns
followed by HhaI digestion. Template DNA thus excised was
purified from the remainder of the plasmid by centrifugation through a
linear 5-12% (w/v) sucrose gradient in TE buffer (10 mM
Tris-HCl, 1 mM EDTA, pH 8.0) for 16 h at 4 °C at
30,000 rpm in a Beckman SW 40 Ti rotor. Template DNA was concentrated
from selected fractions by ethanol precipitation.
Octamer Reconstitution--
Reconstitution of control or uH2A
octamers containing either calf H2A or uH2A instead of H2A in the
chicken erythrocyte octamer was carried out as described previously
(16, 29).
Nucleosomal Array Reconstitution--
Nucleosomal
arrays were reconstituted from control or uH2A octamers and template
DNA by salt gradient dialysis (30) as described previously (31). A
ratio of 1.3 mol of histone octamer to 1 mol of 208-bp DNA was
used to generate saturated nucleosomal arrays. Reconstitutes were
stored at 4 °C no longer than 1 week.
Quantitative Agarose Gel Electrophoresis--
A nine-lane
multigel system as described by Hansen and co-workers (32-34) was used
to determine the electrophoretic mobilities (µ) of reconstituted
nucleosomal arrays in 0.2-3.0% (w/v) agarose. Running gels were
prepared in E buffer (40 mM Tris-HCl, 0.25 mM EDTA, pH 7.8) containing a final concentration of 0 or 2 mM
MgCl2. Samples containing 0.6 µg of bacteriophage T3
standard and 0.5 µg of nucleosomal array were dialyzed for 4 h
against running buffer prior to electrophoresis at 2.65 V.cm
1 at 20 ± 2 °C for 8 h with buffer
recirculation. Control and uH2A arrays were analyzed in parallel on
each multigel. Samples were visualized by UV illumination after
ethidium bromide staining. The gel free migration was calculated by
extrapolation of the line fitted by linear regression to a plot of
migration distance versus percentage agarose concentration
(
1% (w/v) agarose) to 0% agarose. The gel-free migration was
converted to the gel-free mobility (µ0'), which
was then corrected for electro-osmosis and normalized as described
previously (34) to obtain µ0. The average gel pore radius
(Pe) and effective radius
(Re) of nucleosomal arrays was determined as
described previously (32-34).
Analytical Ultracentrifugation--
Sedimentation velocity
analyses (35) were carried out on a Beckman XL-A analytical centrifuge
using an An-55 Al (aluminum) rotor and double sector cells with
aluminum-filled Epon centerpieces as described elsewhere (36, 37). Runs
were routinely carried out at 20 °C in 10 mM Tris-HCl
(pH 7.5) 0.1 mM EDTA or in E buffer. The UV scans (260 nm)
were analyzed using XL-A Ultra Scan version 4.1 sedimentation data
analysis software (Borries Demeler, Missoula, MT). The average
sedimentation coefficient (s20,w) values were determined by second moment analysis (38).
Differential Centrifugation Assay for Nucleosomal
Array Oligomerization--
The solubility of nucleosomal arrays at an
A260 of 1.6 in increasing MgCl2
concentrations was determined as described previously (39).
 |
RESULTS |
Reconstitution of Control and uH2A Nucleosomal
Arrays--
Ubiquitinated H2A (uH2A) and H2A were purified from an
acid extract of calf thymus and reconstituted with equimolar quantities of chicken H2B, H3, and H4 to form uH2A and control octamers, respectively (Fig. 1A). The
integrity of the octamers was confirmed by dimethylsuberimidate
cross-linking (29) (not shown). Control and uH2A nucleosomal arrays
were prepared by salt gradient dialysis reconstitution (30) of control
or uH2A octamers onto a 208-12 DNA template (28). The DNA template is a
tandem array of 12 copies of a 208-bp DNA fragment from the L. variegatus 5 S rRNA gene (28). Nucleosomal arrays must be
saturated with nucleosomes to fold maximally in response to
MgCl2, because previous studies have shown that the extent
of folding decreases significantly as the number of nucleosomes per
array decreases (32). Arrays are considered to be saturated when 12 nucleosomes are reconstituted, because each 208-bp repeat contains a
nucleosome-positioning sequence. The degree of array saturation was
determined by analysis of the products of AvaI digestion of
nucleosomal arrays (Fig. 1B). The AvaI site has
been shown to flank the principal nucleosome-positioning site on this
template (40, 41). Thus digestion of saturated arrays should give rise
to mononucleosomes whereas 208-bp free DNA monomers are also produced
upon digestion of subsaturated arrays. No free DNA was detected
following AvaI digestion of either nucleosomal array.
Densitometric quantitation of Fig. 1B showed that
mononucleosomes constituted 45 and 42% of total reconstituted species
in control and uH2A arrays, respectively, indicating saturation of both
arrays (31). uH2A nucleosomes exhibited reduced mobilities compared
with that of control nucleosomes in agreement with previous observations (15, 18). The oligomers of slower migration seen in Fig.
1B are the result of nucleosomes not being uniquely
positioned on 208-12 DNA and some minor positions blocking the
AvaI restriction site (41). Micrococcal nuclease digestion
of saturated control and uH2A arrays gave rise to a well-defined
nucleosomal ladder at intermediate stages of digestion (not shown).
There was no significant difference in the rate of digestion of the two
arrays, and extensive digestion produced mononucleosomes in both
cases.

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Fig. 1.
Reconstitution of control and uH2A
nucleosomal arrays. A, SDS-polyacrylamide gel
electrophoresis analysis of reconstituted control and uH2A hybrid
octamers (lanes 2 and 4). A total acid extract of
calf thymus nuclei is included as a standard in lanes 1 and
3. Bands were visualized by staining with Coomassie Blue
G-250. B, comparison of saturated control and uH2A
nucleosomal arrays. Nucleoprotein gel of native 167-bp core particles
(lane 1), AvaI digestion products of: naked
208-12 template DNA (lane 2), control nucleosomal arrays
(lane 3), and uH2A nucleosomal arrays (lane 4).
Bands were visualized under UV illumination after ethidium bromide
staining.
|
|
Quantitative Agarose Gel
Electrophoresis--
MgCl2-induced folding of nucleosomal
arrays was analyzed by quantitative agarose gel electrophoresis (32,
33). Nucleosomal arrays were electrophoresed in multigels of
concentrations ranging from 0.2 to 3% (w/v) agarose in E buffer or E
buffer containing 2 mM free Mg2+. Data from
multigels were used to generate Ferguson plots, which were convex in
shape (32, 34) for both control and uH2A arrays (Fig.
2, A and B). The
data from the linear portion of the Ferguson plots were used to
calculate µ0, the gel free mobility (Table I), which is a measure of the average
electrical surface charge density of a macromolecule (42). The extent
of array saturation has been shown to influence µ0
values. The µ0 value obtained for control arrays in low
salt buffer (Table I) lies within the range of
1.82 ± 0.04 to
1.92 ± 0.02 × 10
4
cm2.V
1.s
1 previously reported
for saturated arrays reconstituted on the same DNA template (31, 34,
43, 44) thereby providing further confirmation that array saturation
was achieved. In the presence of 2 mM Mg2+, the
gel free mobility of control arrays decreased by 45% as the arrays
adopted a more compact structure (32). The gel free mobility of uH2A
arrays was 10% lower than that of control arrays both in the absence
and in the presence of 2 mM free Mg2+. In E
buffer, this reduction in µ0 corresponds to either an
increase of 25 to 30 positive charges per octamer (32) or to the
shielding of an equivalent number of charges per octamer or a
combination of both effects. The later explanation is more likely,
because at pH 7.0, ubiquitin has 11 acidic and 11 basic residues, of
which only three of the seven lysine residues are not involved in
intramolecular contacts and are fully exposed on the surface of the
molecule (45). The 44% reduction of µ0 values observed
for uH2A arrays in E buffer + 2 mM Mg2+ was
comparable to that of control arrays.

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Fig. 2.
Ferguson plots of bacteriophage T3 ( ),
saturated control ( ) and uH2A ( ) nucleosomal arrays in E buffer
(A) and E buffer containing 2 mM free
MgCl2 (B). The gel free migrations
were converted to gel free mobilities to determine µ0 as
described under "Experimental Procedures."
|
|
Quantitative agarose gel electrophoresis data can also be used to
determine an average Re, which can be correlated
to the surface area of a rod-like nucleosomal array at low agarose
concentrations (<0.6% w/v) (32, 34) as well as to the frictional
coefficient derived from the average sedimentation coefficient (46).
The effective radii (Re) of control and uH2A
arrays remained essentially constant at all agarose concentrations in E
buffer without and with 2 mM MgCl2 (Table
II), whereas naked template DNA (data not shown) was found to reptate at smaller pore sizes as reported previously (32). The Re values obtained for
control arrays at pore sizes
200 nm (Table II) correlate well with
previous estimates of 26-28 nm in E buffer and 20.5-22 nm in the
presence of 2 mM MgCl2 (32, 34, 43, 44) for
equivalent arrays under the same electrophoretic conditions. No
significant difference in Re values of control
and uH2A arrays was observed (Table II), suggesting that uH2A has
little effect on the compaction of nucleosomal arrays under these
experimental conditions.
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Table II
Re values of nucleosomal arrays in low and high salt buffer
Values represent the mean ± S.E. of eight determinations.
|
|
Oligomerization--
Nucleosomal arrays have been shown to
oligomerize rapidly in response to increasing concentrations of
divalent salts (39, 47). This association is reversible upon removal of
the salt by extensive dialysis (39) and is distinct from the folding process (46). Some evidence suggests that the results obtained from
in vitro oligomerization of relatively short chromatin
fragments may be significant regarding the in vivo
interaction of chromatin fibers during chromosomal condensation (39).
Histone tails have been shown to play an important role in this process
as their absence (39, 48, 49) or acetylation (43) hinder
Mg2+-induced oligomerization of nucleosomal arrays. Fig.
3 shows that uH2A arrays oligomerized at
lower Mg2+concentrations than control arrays. Control
arrays were 50% oligomerized at ~4 mM MgCl2
in close agreement with previous results (48), whereas uH2A arrays were
almost fully oligomerized at this concentration. Because the gel free
mobility of uH2A arrays was 10% lower than that of control arrays
(Table I), ubiquitin may thus shield some of the DNA charge, thereby
facilitating the aggregation process. Ubiquitin itself may also provide
additional surfaces for inter-array contacts.

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Fig. 3.
Mg2+-dependent
oligomerization of saturated control ( ) and uH2A ( ) nucleosomal
arrays. Arrays were incubated in increasing concentration of
MgCl2 for 10 min at room temperature before centrifugation.
Unaggregated arrays remain soluble in the supernatant. Each point
represents the average of two to three determinations.
|
|
Analytical Ultracentrifugation--
Sedimentation velocity
experiments were next used to monitor the effect of H2A ubiquitination
on nucleosomal array folding in response to increasing
MgCl2 concentrations (Fig.
4). The 208-12 oligonucleosome complexes
used in these experiments consisted of 11-11.5 nucleosomes per DNA
template determined as described elsewhere (35). The histone loading
was kept slightly under-saturated because of the oligomerization
problems discussed in the previous section. The sedimentation
coefficients of uH2A arrays obtained in this way were on average 11%
lower than that of control arrays (Fig. 4B) regardless of
whether the experiments were carried out in 10 mM Tris-HCl
(pH 7.5 buffer) (Fig. 4, A and B) or in E buffer (40 mM Tris-HCl) (results not shown). This could be due to
the lesser histone loading of the DNA template (50) and/or of a slight
increase in the frictional parameters of uH2A arrays due to the
presence of two ubiquitin molecules per nucleosome. Nevertheless, increases in s20,w values of uH2A arrays
in response to MgCl2 paralleled those of control arrays,
and in 2 mM MgCl2 uH2A arrays attained a degree
of compaction similar to that of control arrays (Fig. 4C)
(31) where
s+MgCl2/s
MgCl2 = 1.35. This 35-40% increase in the sedimentation coefficient has been
correlated with the formation of an intermediately folded species such
as an open helix (31, 37, 47). The results presented in Figs. 2 and 4
show that the presence of ubiquitin attached to H2A does not hinder
this degree of nucleosomal array compaction.

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Fig. 4.
Magnesium dependence of the sedimentation
coefficient (s20,w) of 208-12 nucleosome arrays. A, integral distribution of the
sedimentation coefficient of uH2A containing nucleosome arrays in the
absence ( ) or in the presence of 2 mM MgCl2
( ). The integral distributions were obtained after
analysis of the sedimentation boundaries using the method of van Holde
and Weischet (58). B, uH2A-containing nucleosome arrays
( , ) are compared with nucleosome arrays reconstituted with
native histones ( ) (32). The data shown by ( , )
were obtained from two completely independent reconstitution
experiments. C, same results as in panel B
plotted as in (32). In this representation, the sedimentation
coefficient of the arrays at a given magnesium concentration
(S+MgCl2) are plotted relatively to the
sedimentation coefficient of the arrays in the starting buffer in the
absence of magnesium (S MgCl2).
Nucleosome arrays reconstituted with native histones ( )
average of the two sets of experimental data for nucleosome arrays
reconstituted with uH2A ( ). All experiments were carried out at
20 °C and at 18,000 r.p.m. ( ) or 20,000 r.p.m. ( ). The
composition of the buffer was 10 mM Tris-HCl (pH 7.5) 0.1 mM EDTA.
|
|
 |
DISCUSSION |
Histone modifications such as acetylation and phosphorylation
mediate changes in chromatin largely through alteration of the charge
of amino acid residues in the amino-terminal histone tails. Ubiquitination is, by comparison, a bulky modification that has led
researchers to postulate its function to lie in hindering chromatin
folding (e.g. Refs. 6, 17, 18). This postulate has been
difficult to confirm in vivo, because the enzymes involved in conjugating ubiquitin to histones are also required for the ubiquitination of many other proteins that may directly or indirectly affect chromatin folding. We have therefore used an in vitro
model system to assay the impact of histone H2A ubiquitination on the Mg2+-induced folding and oligomerization of nucleosomal
arrays. Moreover, the extent of H2A ubiquitination used in this study
was far greater than that in vivo where it is more common
for only one H2A molecule to be ubiquitinated per nucleosome (51). In
the absence of linker histones, nucleosomal arrays equilibrate between
moderately folded and extensively folded structures in buffers
containing 2 mM MgCl2 (31, 37, 47). The data
obtained from quantitative agarose gel electrophoresis (Tables I and
II) and analytical ultracentrifugation (Fig. 4) show that uH2A and
control arrays attained a similar extent of compaction in 2 mM MgCl2 relative to low salt conditions. This
indicates that uH2A does not affect this degree of nucleosomal array
folding. Thus, although the tail domains of histones are crucial for
the salt-induced folding of nucleosomal arrays (37, 48, 49, 52), the
carboxyl-terminal tails of H2A can be ubiquitinated without much impact
on the folding process. Furthermore, the results shown in Fig.
4A in 2 mM MgCl2 are almost
identical to those previously reported for unmodified arrays (see Fig.
3A of Ref. 47). Therefore, it is possible to conclude that
histone H2A ubiquitination neither affects the 28-40 S folding
transition, which is characteristic of the histone H1-depleted
chromatin in either the presence of monovalent (31, 37) or low
concentrations of divalent ions (47), nor the maximum folding (40-55 S
transition), which occurs at higher levels of histone saturation in the
presence of MgCl2 (43, 44, 47, 49).
Although support for uH2A playing a role in hindering the final stages
of chromatin compaction has been provided by reports of the loss of the
uH2A ubiquitin moiety at metaphase (25, 26), not all compact chromatin
structures are devoid of ubiquitin. In mice spermatocytes, uH2A has
been associated with the inactive sex body that contains the
heterochromatic X and Y chromosomes (53), and in Drosophila
ubiquitin has been shown to be mainly associated with the band domains
of polytene chromosomes (54). Further investigations are required to
determine if uH2A affects the higher degree of folding attained by
nucleosomal arrays containing linker histones in response to elevated
salt concentrations (44). It also remains to be investigated if
ubiquitination of H2A could affect the binding of other proteins
involved in the formation of mitotic chromosomes. Finally, it has been
suggested that histone ubiquitination could label specific chromatin
regions (26, 55) and as such could be part of the "histone code"
(56). This ubiquitin tag could direct as yet unidentified or known
cellular machinery such as chromatin remodeling complexes (57) to
uH2A-enriched chromatin regions such as the 5'-end of the mouse
dihydrofolate reductase gene (17) or the copia and hsp 70 genes in Drosophila (18).
We are very grateful to Dr. R. T. Simpson for the p5S 208-12 plasmid construct and Dr. J. C. Hansen
for providing details of the quantitative agarose gel electrophoresis apparatus.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M011153200
The abbreviations used are:
uH2A and uH2B, ubiquitinated histones H2A and H2B;
bp, base pair(s);
µ0, gel-free electrophoretic mobility;
Re, effective
macromolecular radius;
Pe, effective gel pore
size.
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