From the Department of Biochemistry and Microbiology, University of
Victoria, Victoria, British Columbia V8W 3P6, Canada
The folding ability of chromatin fractions
containing approximately identical nucleosome numbers and the same
linker histone composition, but with different extents of core histone
acetylation, were analyzed by analytical ultracentrifugation. It was
found that the acetylated fractions consistently exhibited a relatively small but significantly lower extent of compaction than that of their
native nonacetylated counterparts. This was regardless of the extent of
the size distribution heterogeneity of the fractions analyzed.
Furthermore the acetylated chromatin fibers exhibited an enhanced
solubility in both NaCl and MgCl2, which is neither the result of a differential binding affinity of the linker histones to
chromatin nor of an alteration in the relative amounts of the histone
H1 variants.
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INTRODUCTION |
The possible implications of histone acetylation for eukaryotic
gene transcription were recognized almost from the discovery of this
post-translational histone modification more than 35 years ago (1, 2).
The finding 15 years later that n-butyrate could increase
the levels of histone acetylation in HeLa and Friend erythroleukemic
cells (3) represented an important landmark for the structural studies
designed to elucidate the structural implications of acetylation
because it allowed for the production of the large amounts of material
that are usually required for this kind of analyses. However, despite
extensive experimental effort in the following years, no significant
differences could be found either at the level of the nucleosome
chromatin subunit (4, 5) or at the level of chromatin fiber folding (6, 7).
The discovery that histone acetyltransferases (8) are an integral part
of the basal transcription complexes has rekindled interest in histone
acetylation as an important factor in the modulation of eukaryotic gene
expression (9). Histone acetylation has been linked to cancer (10-13),
and histone deacetylase inhibitors are being used now for the treatment
of certain cancer types (14).
Despite this, the structural implications of histone acetylation in
mediating eukaryotic transcription remain to be established. Thus,
although the functional implications seem clear, the mechanisms remain
to be unraveled. Although the current coding hypothesis (15) would
explain the localized "short range" effects, an example of a
transacting factor that requires histone acetylation for its
interaction with the chromatin template has not yet been identified. Furthermore the fact that acetylation can occur over long stretches (several kilobases) of DNA (16) ("long range" effect) argues against the coding hypothesis being the only structural role for histone acetylation.
In the present paper we have revisited some of the earlier structural
analyses using well defined chromatin fractions that differ only in
their extent of core histone acetylation. Although our results
generally agree with most of the earlier data, they also underscore the
structural differences and constraints that may be important for
understanding the mechanisms by which histone acetylation exerts its
functional effects.
 |
MATERIALS AND METHODS |
Chromatin Preparation--
Chromatin was obtained from HeLa
cells grown in the absence (native) or presence (acetylated) of 5 mM sodium butyrate using a protocol based on the method
originally described by Perry and Chalkley (17, 18). Briefly, the cells
from 4-liter cultures at ~5 × 105
cells/ml were harvested at 1400 × g for 10 min at
4 °C. The pellets were suspended in 120 ml of 0.137 M
NaCl, 10 mM phosphate buffer (pH 7.2, phosphate-buffered
saline with or without 10 mM sodium butyrate) plus protease
inhibitor mixture "Complete" from Roche Molecular Biochemicals (19)
and centrifuged at 3000 × g for 10 min at 4 °C. The
pellets were resuspended in 120 ml of 0.25 M sucrose, 60 mM KCl, 15 mM NaCl, 10 mM
MES,1 pH 6.5, 5 mM MgCl2, 1 mM CaCl2,
0.5% Triton X-100 (buffer A, with or without 10 mM sodium
butyrate) plus the protease inhibitor mixture and centrifuged as
before. This step was repeated twice without the protease inhibitor.
The pellets thus obtained were combined and resuspended in 50 mM NaCl, 10 mM Pipes (pH 6.8), 5 mM
MgCl2, 1 mM CaCl2 (buffer B, with
or without 10 mM sodium butyrate) to a final
A260 = 40. This absorbance was determined as described elsewhere (14). The nuclear suspension was then incubated
at 37 °C for 10 min and digested at this temperature for an extra 5 min with micrococcal nuclease (Worthington) at 5 units/ml. The
digestion reaction was stopped by the addition of 500 mM
EDTA to a final EDTA concentration of 10 mM (on ice) and
centrifuged at 10,000 × g for 10 min at 4 °C to
yield a supernatant ("SI") and a pellet. The pellet thus
obtained was suspended (by repeated pipetting to lyse the nuclei) in
0.25 mM EDTA (with or without 2 mM sodium
butyrate) using half the volume used for the nuclease digestion. The
nuclear lysis was allowed to continue for 1 h at 4 °C with
continuous stirring. Nuclear debris were removed by centrifugation at
10,000 × g for 10 min, and the chromatin thus obtained
in the supernatant ("SE") was run on a 5-20% sucrose gradient in 25 mM NaCl, 10 mM Tris-HCl (pH
7.5), 0.2 mM EDTA (with or without 3 mM sodium
butyrate) at 85,500 × g for 3 h at 4 °C. Fractions along the sucrose gradient were collected and dialyzed against the appropriate buffer.
Gel Electrophoresis--
SDS-polyacrylamide gel electrophoresis
(PAGE)was carried out according to Laemmli (20). Acid-urea PAGE was
carried out as described elsewhere (4). In this later case the
chromatin samples to be analyzed were dissolved in 4 M
urea, 5% acetic acid, and 0.5% (w/v) protamine sulfate (to displace
the histones from DNA) and incubated at 65 °C for 15 min before
loading them onto the gel (21).
Reversed-phase HPLC--
Reversed-phase HPLC analysis was
carried out using a 5-µm Vydac C18 column (25 × 0.46 cm) as described elsewhere (22) and an acetonitrile gradient in
the presence of 0.1% trifluoroacetic acid according to Ref.
23.
Analytical Ultracentrifuge Analysis--
Analytical
ultracentrifuge analyses were carried out on a Beckman analytical XL-A
ultracentrifuge using an An-55 aluminum rotor. Analyses of the
sedimentation velocity runs were carried out as described elsewhere
(24, 25).
Histone H1 Solubility--
Long native or acetylated chromatin
at an A260 nm = 8-9 in 10 mM
Tris-HCl, 2 mM sodium butyrate (pH 7.5) was brought to
different NaCl concentrations by addition of a 5 M NaCl
solution while vortexing (26), and in some instances (as indicated in
the figure legends) the final salt concentration was reached by mixing
chromatin in the 10 mM Tris-HCl buffer with an equal volume
of a 2× NaCl solution prepared in the same buffer. After a 30-min
incubation at room temperature, the samples were then centrifuged in a
Beckman Optima TLX ultracentrifuge with a TLA 100.3 at 200,000 × g for 2.5 h at 4 °C. Aliquots of the supernatants
were run on SDS-PAGE, and after staining with Coomassie Blue the gels
were scanned using an Alpha Innotech Chemi Imager 4000 (Alpha Innotech
Corp., San Leandro, CA).
Magnesium and Sodium Chloride Solubility--
Chromatin
solubility analysis in the presence of MgCl2 was carried
out in 1 mM Tris-HCl (pH 7.5) as described previously (22). The chromatin samples had an A260 nm = 0.8. For the NaCl studies the chromatin samples had an
A260 = 6-8 in 10 mM Tris-HCl, 2 mM sodium butyrate (pH 7.5). The different NaCl
concentrations were achieved by mixing equal volumes of the chromatin
samples in the 10 mM Tris-HCl buffer with an equal volume
of a 2× NaCl in the same buffer while vortexing.
 |
RESULTS AND DISCUSSION |
Fig. 1, A and
B, shows the ionic strength dependence of the sedimentation
coefficient of native and acetylated chromatin fractions with different
average nucleosome content (Nw) and size distribution (see Fig. 1C). To facilitate the comparison
between the native and acetylated counterparts as well as to account
for differences in Nw, the sedimentation
coefficients at any given ionic strength
(s20,w) for a given chromatin sample
were normalized by dividing them by the sedimentation coefficient of
the sample in plain buffer in the absence of salt
(s20,w (b)] (27). As can be
seen the dependence of the sedimentation coefficient on the NaCl
concentration adopts a different shape that depends on the size
distribution of the sample analyzed (24). However, independently of
this, the sedimentation coefficients of the acetylated chromatin fibers
consistently exhibited lower values than their corresponding native
counterparts. Under solution conditions approaching physiological
values a decrease of 11-15% was observed. Such decrease in the S
values reflects a decrease in the folding of the acetylated chromatin
fragments (28) when compared with that of the native fragments. The
decrease observed is similar to that which had been reported earlier
(27) and although relatively small is not negligible (29).

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Fig. 1.
Dependence of the sedimentation coefficient
(s20,w) of chromatin on the
NaCl concentration. A, two fractions with a narrow size
distribution (Nw = 31 nucleosomes (acetylated) and
Nw = 35 nucleosomes (native)) are compared.
B, two fractions with a broad size distribution
[Nw = 16 (acetylated) and Nw = 12 (native)] are compared. The average number of nucleosomes
(Nw) was determined as described in Ref. 43 and
using an average nucleosome repeat length of 192 base pairs for HeLa
cell chromatin (44). , acetylated fractions; , native chromatin
fractions. C, 1% agarose electrophoresis of DNA from
chromatin fractions with a narrow (lanes 1 and 3)
or broad size distribution (lanes 2 and 4).
Acetylated fractions in lanes 1 and 2 and
lanes 3 and 4 correspond to the native
counterparts. Lane M is a BstEII digest of DNA.
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Early studies on chromatin from butyrate-treated HeLa cells had shown
that histone acetylation increased the overall solubility of chromatin
(18). However, little attention was paid to the histone H1 composition
of the samples analyzed at that time. Therefore we decided to revisit
this using long chromatin fibers that had been fractionated previously
through sucrose gradients and that had exhibited the same linker
histone composition. It is important to note that the method we have
used to prepare chromatin fragments yields two chromatin fractions (SI
and SE) (see "Materials and Methods"). In the case of the cells
grown in the absence of butyrate (native), SI consists mainly of
mononucleosomes that are almost completely depleted of histone H1. In
contrast fraction SI from the butyrate-treated cells (acetylated)
consists of an oligonucleosome (mainly 1-6 nucleosomes) fraction that
(i) is histone H1-deficient (results not shown),
(ii) contains highly hyperacetylated core histones (4), and
(iii) is enriched in transcriptionally active sequences
(30). Only the SE fraction that had been size fractionated using
sucrose gradients was used in our experiments.
To assess whether the folding and solubility differences could be the
result of differences in the binding affinity of linker histones, an
analysis of the salt-dependent dissociation of these histones from native and acetylated chromatin was carried out (see Fig.
3). These results conclusively show that
the binding affinity of histone H1 to chromatin is independent of the
extent of the core histone acetylation. This is regardless of the more dynamic nature of the association between histone H1 and acetylated chromatin (33). As shown in Fig. 3B the relative
stoichiometry of histone H1 to core histones in the native and
acetylated chromatin fractions used for these analyses was the
same.
It was argued several years ago that the reason why only relatively
small changes were observed for the folding of the acetylated chromatin
fiber could be the result of an increased amount of histone
H10 (34, 35). The results shown in Fig.
4 show that under the experimental
conditions used by us the histone H1 variant composition of the native
and acetylated chromatin samples analyzed in this work was identical.
Hence none of the structural differences described above are the result
of an altered linker histone composition.
We thank John D. Lewis for skillful computer
assistance in the preparation of the figures.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M100501200
The abbreviations used are:
MES, 4-morpholineethanesulfonic acid;
Pipes, 1,4-piperazinediethanesulfonic
acid;
PAGE, polyacrylamide gel electrophoresis;
HPLC, high performance
liquid chromatography.
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