From the Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216
Received for publication, August 21, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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We investigated the relationship between linker
histone stoichiometry and the acetylation of core histones in
vivo. Exponentially growing cell lines induced to overproduce
either of two H1 variants, H10 or H1c, displayed
significantly reduced rates of incorporation of
[3H]acetate into all four core histones. Pulse-chase
experiments indicated that the rates of histone deacetylation were
similar in all cell lines. These effects were also observed in nuclei isolated from these cells upon labeling with
[3H]acetyl-CoA. Nuclear extracts prepared from
control and H1-overexpressing cell lines displayed similar levels of
histone acetylation activity on chromatin templates prepared from
control cells. In contrast, extracts prepared from control cells were
significantly less active on chromatin templates prepared from
H1-overexpressing cells than on templates prepared from control cells.
Reduced levels of acetylation in H1-overproducing cell lines do not
appear to depend on higher order chromatin structure, because it
persists even after digestion of the chromatin with micrococcal
nuclease. The results suggest that alterations in chromatin structure,
resulting from changes in linker histone stoichiometry may modulate the
levels or rates of core histone acetylation in
vivo.
Chromatin structure plays an important role in the control of gene
expression by limiting the accessibility of sequence-specific binding
proteins to DNA (1-3). The histone components of chromatin play an
integral role in this regulation. Recently, the reversible acetylation
of core histones has been recognized as a major mechanism by which
chromatin-mediated gene regulation is effected (4-6).
The relationship between histone hyperacetylation and transcriptionally
active chromatin was made many years ago (7, 8). Recently, it has been
demonstrated that many of the histone acetyltransferases (HATs)1 that acetylate core
histones are components of transcriptional activator or coactivator
complexes and are specifically targeted to genes to activate
transcription (9, 10). Transcriptionally silent chromatin is often
hypoacetylated and histone deacetylases (HDACs) have been shown to be
components of transcriptional corepressors and silencers that are also
targeted to the appropriate DNA sequences (11, 12). Several recent
reports provide direct evidence that the acetylation status of
nucleosomal core histones has a causal relationship to gene activity
(13-16).
The linker or H1 histones also modulate chromatin structure and gene
expression (17-24). H1 is often perceived to function as a general
repressor of transcription by stabilizing higher order structures (25,
26). Linker histones have also been demonstrated to directly occlude
factor binding (27), to limit nucleosome mobility (28, 29), and to
reduce the transient dynamic exposure of DNA on the nucleosome surface
(30, 31), all of which would be expected to have repressive effects on
transcription. Transcriptionally active chromatin has been reported to
display reduced H1 stoichiometry (32-34), and thus the removal or
reorganization of H1 may be a necessary aspect of gene activation (35,
36). However, recent in vivo studies indicate that the
linker histones have positive and negative influences on a subset of
genes and may function as specific gene regulators (17-19,
37-39).
The relationship between H1-mediated chromatin modulation and
reversible core histone acetylation has received little attention. Acetylation appears to alter the interaction of linker histones with
chromatin and may compromise the ability of H1 to promote the formation
of condensed structures (40, 41). H1 repression of transcription factor
binding to reconstituted nucleosomes was shown to be partly alleviated
by increased acetylation of the core histones prior to H1 addition
(27). In another study, linker histone-dependent
transcriptional repression was not affected by the acetylation state of
the core histones (42). However, these studies do not address the
converse relationship, that is, whether the presence of H1 in chromatin
influences the acetylation status of the core histones. Binding of H1
has been shown to result in subtle but significant rearrangements of
core histone interactions in the nucleosome (43, 44). Therefore, it is
reasonable to suppose that histone H1 may have an effect on the
properties of the amino termini tails of core histones, including their
acetylation status. A recent report demonstrated that the linker
histones H1 and H5 specifically inhibit the acetylation of
mononucleosomes and oligonucleosomes by the histone acetyltransferase
activity of p300/CBP-associated factor (PCAF) in vitro (45).
Here we have addressed the influence of H1 stoichiometry on core
histone acetylation in vivo.
We developed a system to overproduce H1 histone variants in homologous
mouse cells (17-20). Cell lines stably transfected with plasmids
containing the coding regions of either of two H1 variant genes,2 H10 or
H1c, under control of a mouse metallothionein gene promoter were
created. Treatment of these lines with the inducer ZnCl2 results in perturbations of the normal ratio of individual H1 variants.
This treatment also results in an increase in the total amount of
linker histone per nucleosome relative to that of control cells.
Variant-specific differences in cell-cycle progression and both
variant-specific and variant-independent differences in gene expression
associated with linker histone overexpression were detected in these
cell lines (17-19). In this study we utilized this system to
demonstrate that increased H1 stoichiometry inhibits core histone
acetylation in vivo.
Cell Culture--
The H10- and H1c-overexpressing
cell lines, MTH10 and MTH1c, and the control line MTA
(transfected with the expression vector lacking H1 sequences) were
described previously (17, 20). All experiments were initiated from
stocks of stable cell lines stored in liquid nitrogen and were
maintained as described previously (17). For the overexpression of H1
histone variants during exponential growth conditions, cells were
seeded at a low density; typically less than 10% of the total surface
area of the flask was covered. 12 h after the initial seeding,
cells were treated with 50 µM ZnCl2 for
12 h. This was followed by another 84 h of induction with 100 µM ZnCl2. The media was replaced every
24 h. Cells were harvested prior to confluence; i.e. no
more than 75% of the surface area of the flask was covered by cells at
the time of harvesting. Total chromatin-bound histones were extracted
with 0.2 M H2SO4 and separated by
high performance liquid chromatography (HPLC) as described previously
(17, 18) or separated on Triton-acid-urea (TAU) gels as described below.
TAU Polyacrylamide Gel Electrophoresis--
TAU polyacrylamide
gel electrophoresis was employed to separate post-translationally
modified forms of histone proteins, as described by Zweidler (47).
These gels contained 0.37% Triton X-100, 5% acetic acid, 8 M urea, 12% acrylamide, and 0.08% bisacrylamide. Gels
were fixed and stained for 1 h at room temperature in fixing and
staining solution (0.1% Coomassie Brilliant Blue R-250 in 50%
methanol and 7% acetic acid). They were destained for 48 h at
room temperature with multiple changes of the destaining solution (5%
methanol and 7% acetic acid) and processed for fluorography using
Entensify (PerkinElmer Life Sciences) fluor solution according to the
manufacturer's instructions. Dried gels were exposed to Kodak X-OMAT
AR autoradiography film with intensifying screen at Isolation and Treatment of Nuclei--
Nuclei were prepared as
described previously (48). Isolated nuclei were resuspended in an
appropriate volume of 1× wash buffer for nuclei (10 mM
Tris-HCl, pH 7.4, 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine) and processed
further as described in the figure legends and text.
For experiments involving acetylation of core histones following
microccocal nuclease treatment (Fig. 6), nuclei isolated as described
above were resuspended in 450 µl of wash buffer for nuclei
supplemented with 200 µM CaCl2 and 3 units/ml
microccocal nuclease (Sigma) and incubated at 37 °C for 15 min. The
digestion was stopped by pelleting the nuclei by centrifugation at
16,000 × g for 2 min at 4 °C. The supernatant was
discarded, and the nuclei were processed further as described in the
figure legends and text. For the analysis of nuclease-digested DNA,
parallel aliquots were stopped by adding 50 µl of 10× stop buffer
(20 mM EDTA, 10 mM EGTA, 5% SDS) and
proteinase K to a final concentration of 200 µg/ml. After overnight
digestion at 50 °C, the DNA was extracted with phenol:chloroform and
ethanol-precipitated. Samples were resuspended and electrophoresed on a
1.8% Metaphor-agarose gel.
In Vitro Assays for HAT Activity in Nuclear
Extracts--
Nuclear HAT activity in cultured mammalian cells is very
tightly bound within the nuclei and requires 2 M salt for
extraction (49). A strategy for preparing nuclear extracts containing
HAT activity was devised based on the observation that a mild digestion with nuclease renders the nuclear HAT activity susceptible to extraction with less than 0.4 M salt (50). All steps were
carried out on ice unless indicated otherwise. Nuclei were resuspended in DNase I digestion buffer (1× wash buffer for nuclei, 1 mM MgCl2, 0.5 mM CaCl2)
containing 200 units/ml of RNase-free DNase I (Sigma), incubated at
37 °C for 20 min, and then pelleted by centrifugation at 16,000 × g for 2 min at 4 °C. DNase I treatment renders some HATs soluble; therefore, the supernatant was concentrated ~10-fold in
a Microcon 10 concentrator by centrifugation at 16,000 × g for 60 min at 4 °C. The pelleted nuclei were
resuspended in 500 µl of high salt buffer (0.35 M KCl, 5 mM MgCl2, 10 mM Tris-HCl, pH 7.5)
supplemented with 1 mM DTT, 20 mM butyrate, 1%
thiodiglycol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF)
and incubated for 30 min on ice. The salt-extracted nuclei were
pelleted by centrifugation at 16,000 × g for 5 min at
4 °C. The supernatant was concentrated ~5-fold in a Microcon 10 concentrator by centrifugation at 16,000 × g for 45 min at 4 °C. The concentrated supernatants were pooled together and
used in HAT assays.
Soluble chromatin for substrate in HAT assays was prepared as follows.
Nuclei from exponentially growing cells were resuspended in 900 µl of
DNase I digestion buffer containing 200 units/ml of RNase-free DNase I
(Sigma). The nuclei were digested with DNase I at 37 °C for 10 min,
and the nuclear suspension was mixed by gentle tapping every 5 min.
Nuclei were pelleted by centrifugation at 16,000 × g
at 4 °C for 2 min, resuspended in nuclei lysis solution containing 2 mM EDTA and 1 mM EGTA, and incubated on ice for
15 min. Nuclei were pelleted by centrifugation at 16,000 × g at 4 °C for 10 min. The supernatant was collected and
concentrated 5-fold in a Microcon 30 concentrator and adjusted to
A260 = 10.
Assays for HAT activity in nuclear extracts were carried out in 1×
wash buffer for nuclei supplemented with 0.5 mM PMSF, 1 mM DTT, 1% thiodiglycol, 20 mM butyrate, 15 µCi/ml [3H]acetyl coenzyme A (1.88 Ci/mmol, 0.1 mCi/ml,
PerkinElmer Life Sciences), 150 µg of nuclear extract (based on the
assumption that an A280 of 1 absorbance
unit = 1 mg/ml protein), and 100 µl of soluble chromatin in a
total volume of 200 µl. Reactions were incubated for 2 h at
37 °C. Reactions were stopped by the addition of
H2SO4 and NH4OH to a final
concentration of 0.2 M and 0.75 M respectively,
and precipitated by the addition of 3 volumes of ice-cold 100%
ethanol. The pellet was washed twice with 70% ethanol, resuspended in
100 µl of water, and counted by liquid scintillation. For analysis of
the acetylated proteins, the assay was scaled up and the products
separated on TAU gels.
Overexpression of Histone H10 or H1c in Exponentially
Growing Cells Results in Decreased Incorporation of Acetate into Core
Histones--
Exponentially growing cultures of H1-overexpressing cell
lines (MTH10, MTH1c) and a control line (MTA) were treated
for 4 days with the inducer ZnCl2. In H1-overexpressing
lines this protocol results in major perturbations of the normal ratio
of individual H1 variants and in the total amount of linker histone per
nucleosome relative to that of control cells (Table
I). The two variants we investigated, H10 and H1c, normally comprise ~20% each of the total H1
population. In the appropriate overexpressing line, these variants
accumulate such that they account for ~70-80% of the total H1.
Furthermore, the expression of the endogenous variants is only
partially compensated such that the total amount of H1 per cell is
increased. Because we do not have extinction coefficients at 210 nm for
each of the variants, the absolute amount of H1 is an estimate.
However, in the overexpressing lines the levels of accumulation are 1.2 to 1.3 times that of control cells and are within the physiological range displayed by mouse tissues. We observed no effect on cell viability or cell cycle progression in these or previous studies (19).
Importantly, under these conditions we found no evidence of aberrant
chromatin structures, although subtle but significant changes in
nucleosome repeat length and nucleosome conformation were previously
documented (20).
Following this induction protocol cultures were labeled by the addition
of [3H]acetate to the culture medium. Parallel cultures
were treated during the labeling period with cycloheximide (CHX) to
prevent incorporation of label into proteins as acetate-derived amino acids and with trichostatin A (TSA) to inhibit HDAC activity. Total
chromatin-bound acid-soluble proteins were isolated and separated by
TAU gels (Fig. 1). The fluorograph shows
that, relative to control cells, the H1-overexpressing cell lines
displayed significantly lower levels of incorporation of label into the
core histones. This was observed both in the presence and absence of
TSA-mediated hyperacetylation, although the effect is better visualized
after TSA treatment due to incorporation of more label. Treatment with CHX alone does not affect acetylation of the core histones (19) (Fig.
1). This indicates that the incorporation of label most likely
represents the action of nuclear Type A HATs acting on histones in a
chromatin context. In a separate experiment, cultures of these cell
lines were treated as above but were not induced with ZnCl2
and therefore displayed similar H1 stoichiometries to one another. No
differences were observed among the cell lines in the incorporation of
label into core histones (data not shown). We detected no labeling of
any of the linker histone variants in this protocol.
Two distinct kinetic classes of histone acetylation have been described
previously (51). One class, representing ~10% of the histones,
displays rapid labeling and turnover. The second, which includes the
bulk of core histones, is labeled more slowly and is more stable. The
quantitative differences observed among the cell lines upon labeling
with [3H]acetate could reflect a specific effect on a
minor rapidly labeling subset of substrates. To investigate this
possibility, total chromatin-bound histones from the TSA-treated
samples displayed in Fig. 1 were fractionated by HPLC. Individual
fractions containing all the acetylated species of histone H4 were
collected and run on TAU gels. Equal amounts of material, based
on absorbance at 210 nm, were loaded from each cell line (Fig.
2). Coomassie Blue staining of the gel
showed significant differences in the relative amounts of individual
acetylated isoforms of these variants. This suggests that the
H1-overexpressing cells display reduced bulk levels of acetylated
histones under these conditions. However, these differences appear even
more dramatic in the resulting fluorographs. This may reflect, in part,
differences in the sensitivity of Coomassie Blue staining
versus fluorography. Alternatively, and perhaps more
interestingly, this may indicate that certain kinetic classes of
histone acetylation are differentially affected in H1-overexpressing cells.
As histone acetylation/deacetylation is a dynamic process, the observed
differences in label incorporation could conceivably be due to
alterations in HAT activity, HDAC activity, or both. Furthermore, at
this point we cannot differentiate between effects due to alterations
in the chromatin substrate or to changes in the amounts or activities
of the enzyme complexes. In the previously described experiments we
measured incorporation after a 6-h labeling period. Using a similar
protocol we measured the incorporation of label during multiple,
shorter labeling periods. Fig.
3A shows quantitation of label
incorporation into the tetra-acetylated form of H4. Incorporation was
fairly linear for the first 3 h, and the rate of incorporation was
approximately 3-fold greater in control cells relative to those
overexpressing H1. Analysis of Coomassie Blue-stained gels from the
same experiment, although not as sensitive, revealed a similar 3-fold
difference between the rate of appearance of tetra-acetylated H4 in
control cells relative to those overexpressing H1. Furthermore, the low
levels of tetra-acetylated H4 prior to TSA treatment allowed us to
determine that the specific activity of the label was roughly the same
in all cell lines and remained constant throughout the experiment. This
indicates that we are approaching initial rate conditions in this assay
and, as expected due to the inclusion of TSA, no significant turnover
occurred. Qualitatively similar results were obtained for the labeling
of each of the core histone variants in this assay.
We next measured the rates of deacetylation of labeled core histones in
these cells. Cultures were labeled for 6 h in the presence of CHX
and TSA, washed, and then incubated in fresh medium lacking the
inhibitors and label (Fig. 3B). Although we see some evidence of a biphasic loss of label, no differences between the cell
lines were observed. We conclude that the rates of HDAC activity on the
labeled histones are not significantly different among the cell lines
in this assay. These kinetic results suggest that the reduced levels of
labeled histones reflects decreased HAT activity on the chromatin of
H1-overexpressing cell lines.
Core Histone Acetylation in Isolated Nuclei--
Differences in
the incorporation of label into core histones among the cell lines
might be due in part to different rates of entry of acetate into the
cell or conversion of acetate to acetyl-CoA, the substrate for HATs.
Measurements of the rate of disappearance of the label from the culture
media and its appearance in the cytoplasmic and nuclear fractions
showed no significant differences among the three cell lines (data not
shown). This implies that the rate of isotope entry is likely to be the
same in the three cell lines. As an alternative assay, we measured acetylation in nuclei isolated from exponentially growing cultures that
were treated with ZnCl2 as described above. These nuclei were briefly labeled with [3H]acetyl-CoA followed by
separation of total chromatin-bound acid-soluble proteins on TAU gels
(Fig. 4). Due to the short treatment and labeling time, no differences among the cell lines were observed in the
Coomassie Blue-stained pattern of the core histones (lanes 1-3). However, there was a clearly less label incorporated into the core histones of nuclei isolated from MTH10 and MTH1c
cells relative to the control (compare lanes 8 and 9 to lane 7). We conclude that the reduction in
incorporation of tritium label into core histones either in whole cells
labeled with tritiated acetate, or in isolated nuclei labeled with
tritiated acetyl-CoA, reflects reduced core histone acetylation
upon histone H1 overexpression.
We also utilized isolated nuclei to investigate the effect of H1
depletion on core histone acetylation. Parallel samples from the
experiment described above were briefly extracted with 0.6 M KCl. This treatment releases H1 histones (and other
chromosomal proteins) but not core histones from the nucleus (Fig. 4,
compare lanes 4-6 to lanes 1-3). The extracted
nuclei were then labeled with [3H]acetyl-CoA. The
resulting fluorograph indicates that removal of H1 results in a slight
increase in labeling of control nuclei but, most importantly, a very
significant increase in labeling of the core histones of
MTH10 and MTH1c nuclei (compare lanes 11 and
12 to lanes 8 and 9). These results
are consistent with the notion that H1, in particular H1 at
stoichiometries greater than one per nucleosome, is antagonistic to
core histone acetylation.
It is obvious from the fluorograph that extraction with 0.6 M KCl alters the pattern of core histone acetylation in all
cell lines. Notably, there is a complete loss of detectable labeling of
H2B. The Coomassie Blue-stained gel indicates that these proteins are
present in the gel. We attribute the lack of labeling to a loss or
lability of the major H2B HAT activity during the salt extraction.
Results presented in a later section support this contention.
Regulation of Core Histone Acetylation by H1 Stoichiometry Is
Mediated through Chromatin Structure--
An important issue in the
interpretation of these results is whether the observed effects of H1
overexpression on acetylation are due to alterations in the chromatin
substrate or to changes in the levels or activities of nuclear HATs
and/or HDACs. Previously, we showed that overexpression of H1 variants
leads to changes in the expression of a number of genes (17-19). If
overexpression of H1 histones results in either a down-regulation of
HAT expression or an up-regulation of HDAC expression, this could
contribute to the inhibition of core histone acetylation. To measure
the nuclear HAT activities in these cells, crude nuclear extracts were
prepared from control and H1-overexpressing cells as described in
"Experimental Procedures." The substrate was a soluble mixture of
oligonucleosomes and mononucleosomes isolated from control (MTA) nuclei
following a mild DNase I digestion. HAT activity in the extracts was
measured as [3H]acetyl-CoA incorporation into total
acid-soluble proteins and was nearly identical in control and
H1-overexpressing cells (Fig. 5A), suggesting that H1
overexpression does not affect bulk nuclear HAT levels. We also assayed
the extract from control cells on chromatin substrates derived from the
H1-overexpresing cell lines. The control extract was found to be
approximately 3-fold less active on chromatin templates derived from
cell lines overexpressing H10 or H1c, relative to the
control templates (Fig. 5A). The values shown in Fig.
5A are the total amount of label incorporated into chromatin-bound acid-soluble material. Nuclear extract and chromatin substrate preparations were scaled up to allow analysis of the labeled
products on TAU gels (Fig. 5B). The fluorograph shows that
histones H2A, H3, and H4 are clearly labeled in the extracts but H2B is
not. The pattern is very similar to that obtained with salt-washed
intact nuclei (Fig. 4) and is consistent with loss or lability of HAT
activity toward H2B.
Total nuclear HDAC activity in the same extracts was assayed on free
hyperacetylated core histones, and no significant differences among the
extracts was detected (data not shown). Collectively, these data
suggest that the inhibition of core histone acetylation observed upon
H1 variant overexpression in vivo is due to differences in
the structure of the chromatin rather than a change in the level of
nuclear HAT or HDAC activities.
Overexpression of Histone H1 Variants Appears to Inhibit Core
Histone Acetylation at the Level of the Nucleosome--
Histone H1
could limit accessibility of the core histones to the HATs due to the
formation of higher order structures. H1 might also affect nucleosomal
structure through interactions with DNA or proteins at the nucleosomal
level. Isolated nuclei were prepared, and aliquots were digested with
micrococcal nuclease. Analysis of the DNA from these samples by agarose
gel electrophoresis revealed that most of the chromatin was digested to
mono- or dinucleosomes (Fig.
6A). Parallel aliquots of
nuclease-treated nuclei were washed and then labeled with
[3H]acetyl-CoA (Fig. 6B). The nuclei prepared
from cells overexpressing H10 or H1c incorporated
significantly less label than nuclei from control cells. It is
therefore likely that the histone H1-mediated inhibition of core
histone acetylation occurs primarily at the level of the nucleosome,
possibly as a consequence of H1 binding to the nucleosome. As in the
salt-washed nuclei and the extract experiments, labeling of H2B was not
observed.
The H1 histones are architectural components of chromatin capable of
modulating gene expression in general and specific ways (21-23). Here
we demonstrate that perturbation of H1 variant stoichiometry in
vivo affects the rate of core histone acetylation and that this is
likely mediated by alterations in nucleosomal structure. These results
confirm and extend in vitro observations (45) and suggest a
possible mechanism by which linker histones could directly influence transcription.
The simplest mechanistic explanation for H1-mediated inhibition of core
histone acetylation is that H1 is promoting the formation of higher
order structures in which the core histone tails are inaccessible to
the HAT enzymes. However, the results from the nuclease digestion
experiments suggest that the effect is mediated at least in part at the
nucleosomal level. A similar conclusion was reached from an in
vitro study of H1-mediated inhibition of PCAF HAT activity (45).
In seems unlikely that H1 physically occludes all the core histone
acetylation sites at the nucleosomal level. However, a number of
studies indicate that H1 binding to nucleosomes results in significant
rearrangements of core histone tails and in their interactions with DNA
(43, 44).
Although we cannot derive absolute values, the H1/nucleosome ratio
exceeds unity in the overexpressing cell lines implying that some
nucleosomes contain two H1 molecules. In vivo and in vitro evidence for a second H1-binding site has been presented (52-54), and some mammalian tissues naturally contain high H1
stoichiometries (1). The observation that removal of all H1 by high
salt extraction preferentially increased acetylation in the
overexpressing cell lines (Fig. 4) suggests that the H1 in excess of
one per nucleosome is critical for inhibition. Herrera et
al. (45) also noted that acetylation of H3 by a multiprotein
complex containing PCAF was only inhibited at relatively high H1
stoichiometries. The possibility that some chromatin subdomains have
higher H1 stoichiometries is of interest but has not been directly
demonstrated. Based on our results, these regions should be relatively
hypoacetylated. High H1 stoichiometry and core histone hypoacetylation
could conceivably work in concert to silence chromatin subdomains. The
observation that HDAC activity is not affected by H1 stoichiometry is
also interesting. Acetylated histones are generally believed to be associated with chromatin in a more active or open conformation. Thus,
even in the H1-overexpressing lines, the substrates for the HDACs might
be in accessible chromatin subdomains.
Biochemical studies have demonstrated that active or potentially active
genes are partially depleted in H1 (32-34) or that the manner of H1
binding is altered relative to bulk chromatin (55). Recruitment of HAT
activity is a likely early step in gene activation and may require
removal or remodeling of H1 (56). This might be accomplished by
phosphorylation of H1 (57) or by the involvement of molecules such as
high mobility group proteins (58, 59), nucleoplasmin (60), retinoic
acid receptors (36), or prothymosin
The differential temporal and spatial pattern of expression of
individual H1 variants or the number of H1 molecules bound to the
nucleosome could contribute to specific gene expression in
vivo. The results presented here suggest that one mechanism for
this regulation may be through modulation of core histone acetylation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
80 °C for 2 weeks to 6 months.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
H1 variant stoichiometries of control and H1 variant overexpressing
cell lines
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Fig. 1.
Effect of H1 variant overexpression on
incorporation of acetate into the core histones in exponentially
growing cells. Exponentially growing MTA, MTH10, and
MTH1c cells were treated with ZnCl2 as described under
"Experimental Procedures." Sodium [3H]acetate (4.1 Ci/mmol; 10 mCi/ml; PerkinElmer Life Sciences) was added to the culture
medium to a final concentration of 20 µCi/ml. Where indicated
cycloheximide (CHX) and trichostatin A (TSA) were
added to final concentrations of 50 and 100 ng/ml, respectively.
Cultures were labeled for 6 h at 37 °C. Total histones were
acid-extracted as described previously (17, 18) except that 10 mM sodium butyrate was added to all buffers to inhibit
endogenous HDAC activity. Aliquots from equal numbers of cells were
resolved on TAU gels and subjected to fluorography.
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Fig. 2.
H1 overexpression results in significantly
reduced levels of acetylated histone H4. Exponentially growing
MTA, MTH10, and MTH1c cells were treated with
ZnCl2 as described under "Experimental Procedures."
Cultures were labeled with sodium [3H]acetate in the
presence of CHX and TSA exactly as described in Fig. 1. Total histones
were acid-extracted, and all forms of H4 were separated by HPLC as
described previously (17, 18). From each cell line a single peak
containing all acetylated forms of H4 was collected, normalized on the
basis of absorbance at 210 nm, and resolved on TAU gels.
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Fig. 3.
Time course of acetylation and deacetylation
of core histones. Exponentially growing MTA, MTH10,
and MTH1c cells were treated with ZnCl2 as described under
"Experimental Procedures." A, cultures were labeled with
sodium [3H]acetate for 1, 2, or 3 h in the presence
of CHX and TSA exactly as described in Fig. 1. Coomassie Blue-stained
gels were subjected to densitometry to determine the total amount of H4
and the distribution of acetylated isoforms. The amount of label in
tetra-acetylated H4 was determined by densitometry of the resulting
fluorographs and is displayed in the graph using arbitrary units. Very
similar incorporation rates were observed for other variants.
B, cultures were labeled with sodium
[3H]acetate for 6 h in the presence of CHX and TSA
exactly as described in Fig. 1. The medium was then removed and
replaced with fresh medium lacking label, CHX, or TSA. Samples were
harvested prior to and following medium removal, resolved by TAU gels,
and subjected to fluorography. The graph represents the loss of label
from tetra-acetylated H4 and is expressed relative to the amount
present at the end of the labeling period.
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Fig. 4.
Effect of H1 variant overexpression on the
incorporation of acetyl-CoA into core histones of isolated nuclei.
Nuclei were isolated from exponentially growing MTA, MTH10,
and MTH1c cells after induction with ZnCl2 as described
under "Experimental Procedures." Lanes 1-3,
7-9: the nuclei were resuspended in 1× wash buffer
containing 10 mM butyrate, 1% thiodiglycol, 0.5 mM PMSF, 1 mM DTT, and 10 µCi/ml
[3H]acetyl-CoA (0.1 mCi/ml; 1.88 Ci/mmol; PerkinElmer
Life Sciences) and incubated at 37 °C for 30 min. The labeling was
stopped by pelleting the nuclei by centrifugation at 16,000 × g for 2 min at 4 °C and chilling on ice. Lanes
4-6, 10-12: nuclei from the same preparations as
above were incubated for 30 min on ice in nuclei wash buffer
supplemented with 0.6 M KCl, gently pelleted and then
labeled as described above. Total histones were acid extracted from the
pelleted nuclei and resolved on a TAU polyacrylamide gel. The gel was
stained with Coomassie Blue (lanes 1-6), and processed for
fluorography (lanes 7-12). The asterisks in
lanes 1-3 indicate the major H1 species expressed in each
cell line.
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[in a new window]
Fig. 5.
Regulation of core histone acetylation by H1
stoichiometry is mediated through chromatin structure.
A, nuclear extracts and soluble chromatin templates were
prepared from exponentially growing MTA, MTH10, and MTH1c
cells as described under "Experimental Procedures." Incorporation
of label from [3H]acetyl-CoA into total core histones was
measures as described under "Experimental Procedures." Values are
plotted relative to incorporation of label into control (MTA) chromatin
templates by control extracts. B, larger scale preparations
of control (MTA) chromatin template and nuclear extracts from each of
the cell lines was used in labeling assays exactly as described above
except that the resulting material was separated on a TAU gel and
subjected to fluorography.
View larger version (74K):
[in a new window]
Fig. 6.
Inhibition of core histone acetylation
persists in H1-overexpressing cells after digesting the chromatin with
micrococcal nuclease. Nuclei were isolated from MTA,
MTH10, and MTH1c cells after induction with
ZnCl2. Aliquots were digested with micrococcal nuclease as
described under "Experimental Procedures." A, following
nuclease treatment DNA was prepared and electrophoresed on a 1.8%
agarose gel, which was then stained with ethidium bromide. Lane
1 contains 1 µg of a 100-base pair DNA ladder (Life
Technologies, Inc.). Lane 2 contains DNA from MTA nuclei
partially digested with micrococcal nuclease to generate a nucleosomal
ladder. Lanes 3-5 contain DNA from nuclei of MTA,
MTH10, and MTH1c, respectively, that were treated with 3 units/ml micrococcal nuclease as described under "Experimental
Procedures." B, nuclei digested with 3 units/ml
micrococcal nuclease were resuspended in 1× wash buffer containing 10 mM butyrate, 1% thiodiglycol, 0.5 mM PMSF, 1 mM DTT, and 10 µCi/ml [3H]acetyl-CoA (0.1 mCi/ml; 1.88 Ci/mmol; PerkinElmer Life Sciences) and incubated at
37 °C for 30 min. The labeling was stopped by pelleting the nuclei
by centrifugation at 16,000 × g for 2 min at 4 °C
and chilled on ice. The supernatant was discarded, and total histones
were acid extracted from the pelleted nuclei in the presence of 10 mM butyrate. Total histones were resolved on a TAU gel,
stained with Coomassie Blue (left panel), and subjected to
fluorography (right panel).
(35). Interestingly,
prothymosin
was shown to bind to and mediate release of a portion
of H1 from chromatin (35). These results were interpreted as evidence
for two distinct interaction modes of H1 with chromatin and suggest a
role for prothymosin
in fine tuning H1 stoichiometry to allow gene
activation. The increased H1 stoichiometry in our overexpressing cell
lines could antagonize this system. The possibility that H1 depletion and core histone acetylation are linked in the process of gene activation is intriguing. Recently it was demonstrated that an HAT-containing transcriptional coactivator also possesses a
ubiquitin-conjugating activity toward H1 (61).
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FOOTNOTES |
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* This work was supported by Grant MCB-9305308 from the National Science Foundation, an institutional grant from the University of Mississippi Medical Center, and a donation from the F. D. Wade Research Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Chromosome Dynamics Laboratory, Imperial Cancer
Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms,
Potters Bar, Herts EN6 3LD, United Kingdom.
§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Mississippi Medical Center, 2500 North State St., Jackson, MS 39216-4505. Tel.: 601-984-1849; Fax: 601-984-1501; E-mail: dbrown@biochem.umsmed.edu.
Published, JBC Papers in Press, November 2, 2000, DOI 10.1074/jbc.M007590200
2 According to the nomenclature proposed by Parseghian et al. (46), H1c is a member of the H1S-1 class of somatic H1 variants.
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ABBREVIATIONS |
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The abbreviations used are: HAT, histone acetyltransferase; HDAC, histone deacetylase; PCAF, p300/CBP-associated factor; HPLC, high performance liquid chromatography; TAU, Triton acid-urea; PMSF, phenylmethylsulfonyl fluoride; CHX, cycloheximide; TSA, trichostatin A.
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