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(Received for publication, December 5,
1995; and in revised form, January 19, 1996) From the
We have reconstituted nucleosomes containing the Xenopus
borealis 5 S rRNA gene, a single histone octamer, and 1 or 2
molecules of histone H1. We determine that the 1st molecule of histone
H1 to associate with the 5 S nucleosome binds with high affinity (K
In vivo during the maturation of the chromatin of
chicken erythrocytes, multiple molecules of linker histones have been
shown to become associated with a single nucleosomal repeat length of
DNA containing a single histone
octamer(1, 2, 3, 4) . In contrast,
the chromatin of mammalian tissue culture cells contains on average a
single molecule of histone H1 per histone
octamer(5, 6) . In vitro deconstruction and
reconstruction experiments indicate that two H1-binding sites exist
within each nucleosome and that the presence of 2 molecules of H1 per
nucleosome generates a more compact structure than native
chromatin(7) . The biological and structural significance of
chromatin potentially consisting of a mixture of nucleosomes containing
0, 1, or 2 molecules of histone H1 has not been resolved. More
recently several investigators have made use of Xenopus or Drosophila chromatin assembly extracts to investigate the
influence of chromatin structure on transcriptional regulation. These
extracts are deficient in the normal somatic form of histone
H1(8, 9, 10, 11, 12) and
at least for Xenopus contain maternal H1
variants(13, 14) . Deficiency in the normal somatic
form of histone H1 should have allowed these extracts to prove useful
in determining the influence of this variant of histone H1 on
transcription. However, the results of such experiments are
controversial. Histone H1 has been variously reported to act as a
general inhibitor of transcription within chromatin (10, 12, 15) or not to influence the
transcription process (11, 16) or to selectively
inhibit the transcription of particular genes(9, 17) . In vivo the selective inhibition of particular genes by the
normal somatic variant of histone H1 is observed(18) . One
possible explanation for the discrepancy in results is that chromatin
templates with different structural properties are being assembled as a
consequence of including histone H1 in varying stoichiometries. The
standard assay for the assembly of histone H1 into chromatin in the Xenopus and Drosophila assembly systems requires
measurement of the change in nucleosome spacing(19) . The
nucleosomal spacing increases from 180 bp ( A concern with these
studies is whether an unusual or atypical chromatin structure is being
assembled as a consequence of including much higher stoichiometries of
histone H1 than are normally present in chromatin. The spacing of
nucleosomes in a normal somatic cell is typically 180-200
bp(20) . Thus multiple molecules of histone H1 must be
physically associated per nucleosome to generate the increase in
nucleosome spacing to 220 bp. How might these additional molecules bind
to nucleosomal DNA? We have investigated this issue using a positioned
nucleosome containing a fragment of the Xenopus 5 S rRNA
gene(21, 22, 23, 24, 25, 26) .
We establish conditions under which a 2nd molecule of histone H1 can be
stably incorporated into a 5 S nucleosome protecting additional linker
DNA from micrococcal nuclease digestion. We find that the association
of this 2nd molecule of histone H1 with nucleosomal DNA occurs with
comparable affinity to the association of H1 with naked DNA. Thus, only
a single high affinity histone H1 binding site exists on the 5 S
nucleosome.
Figure 1:
Histone H1 association with nucleosomal
DNA. Gel retardation assays for H1 binding to a mixture of naked and
octamer-associated DNA are shown. The 237-bp HpaII-DdeI fragment of pJHX1 was end-labeled using
Klenow fragment of DNA polymerase at the HpaII site and
reconstituted with purified core histones (see ``Materials and
Methods'') such that approximately 50% of the DNA mass was
associated with histone octamers. This mixture of reconstituted and
naked DNA (50 ng total mass) was mixed with 0, 10, 20, 40, and 80 ng of
histone H1 (lanes 2-6). Nucleoprotein complexes were
resolved on a 0.7% agarose gel (see ``Materials and
Methods''). An autoradiograph is shown. Lane 1 contains
naked DNA alone (F). The positions of free DNA, octamer-bound (Oct) DNA, and nucleosomes containing 1 molecule of H1 (H1-Oct) or multiple molecules of H1 (2H1-Oct) and
aggregates of H1 with DNA (Aggregates) are
indicated.
We wished to further
substantiate the stoichiometry of histone H1 association with
nucleosomal DNA(21) . This was accomplished by recovery of
nucleoprotein complexes from the non-denaturing gels followed by
radioiodination of the associated proteins. Control iodinations of
known masses of histone H1 and core histones allowed the stoichiometry
of histone H1 to the histone octamer to be determined as 1 under the
reconstitution conditions shown in Fig. 1, lane 3 (data
not shown). The formation of aggregates of histone H1 with naked DNA
that migrate as a diffuse smear (see Fig. 2C) prevents
the exact stoichiometry of linker histones within the second
octamer-containing complex to be determined; however, our iodination
experiments indicate a stoichiometry of 2 or 3 molecules of histone H1
per octamer (not shown). These methodologies also allowed the
demonstration that no histone H1 was associated with the DNA migrating
at the position indicated as Free DNA in Fig. 1. We
conclude that it is possible to form three types of nucleoprotein
complexes in our H1 reconstitution experiments: complexes of the
histone octamer and a single molecule of histone H1, complexes of the
histone octamer and at least 2 molecules of histone H1, and complexes
of histone H1 with naked 5 S DNA. We next examine both the nature and
strength of association of histone H1 with DNA in these different
nucleoprotein complexes.
Figure 2:
Gel retardation assays for the measurement
of the binding affinity of histone H1 to DNA when associated with a
histone octamer (A) or when associated with a histone octamer
and a single molecule of histone H1 (B) or when naked (C) (see ``Materials and Methods''). A, gel
retardation assays for H1 binding to a mixture of naked and
octamer-associated DNA (see ``Materials and Methods'' and
note that carrier chromatin is present). Final histone H1
concentrations were 0, 0.19, 0.39, 1.0, 2.0, 3.0, 6.0, 13.0, 26.0, and
52.0 nM in lanes 1-10, respectively. B, gel
retardation assays for H1 binding to DNA already associated with a
histone octamer and a single molecule of H1 (see ``Materials and
Methods'' and note that carrier chromatin is present). Lane 1 shows naked DNA; lane 2 shows DNA associated with a
histone octamer; lanes 3-9 contain DNA associated with a
histone octamer and final histone H1 concentrations of 10, 11, 13, 14,
17, 20, and 25 nM, respectively. C, gel retardation
assays for H1 binding to naked DNA (see ``Materials and
Methods'' and note that carrier DNA is present). Lane 1 shows naked DNA; lanes 2-9 contain histone H1 at
concentrations of 11, 13, 14, 17, 20, 25, 33, and 50 nM,
respectively.
Figure 3:
Binding titration for histone H1 binding
to naked DNA (
Figure 4:
Dissociation of histone H1 from
nucleoprotein complexes. Left panel, dissociation of histone
H1 from nucleosomal DNA. A mixture of naked and octamer-bound 5 S DNA
(10 ng) (shown in lane 1) was incubated with 100 ng of histone
H1 for 15 min before the addition of increasing amounts of linearized
competitor DNA. Lanes 2-8 contain 0, 8, 16, 32, 63, 125, and
250 ng of competitor DNA, respectively. Right panel, as in left panel except 10 ng of naked 5 S DNA was
used.
A feature of histone H1 association with 5 S
nucleosomal DNA is that the additional linker DNA protected from
micrococcal nuclease digestion is asymmetrically distributed with
respect to the nucleosome core(21) . We next mapped the
micrococcal nuclease digestion boundaries of the 175- and 200-bp DNA
fragment using denaturing gel electrophoresis. We found the boundaries
of histone-DNA interactions in the 175-bp DNA fragment to be tightly
distributed as previously determined ((21) , not shown).
However, the boundaries of the 200-bp fragment were much more diffuse
and weaker than those obtained with the 175-bp fragment (not shown).
The variation in the position of the boundaries of the 200-bp fragment
indicates that either significant heterogeneity in the position of
histone H1-DNA contacts exists or that these additional histone-DNA
contacts are easily displaced during digestion. Finally we examined
the influence of incorporating 2 molecules of histone H1 into the
nucleosome for the wrapping of DNA around the core histones. Previous
work with 5 S monosomes and dinucleosomes had not detected any change
in DNA structure on the surface of the histone octamer following
inclusion of histone H1(21, 37, 38) .
Hydroxyl cleavage of the 5 S nucleosome does not reveal any change in
the helical periodicity of DNA on the surface of the histone octamer or
in the extent of histone DNA interactions following inclusion of a
single molecule of histone H1 (Fig. 5, compare lanes 3 and 4, and 8 and 9). Neither does
addition of a 2nd molecule of histone H1 alter the wrapping of DNA
within the nucleosome (Fig. 5, compare lanes 3 and 5, and lanes 8 and 10). We conclude that the
incorporation of a 2nd molecule of histone H1 into the nucleosome does
not significantly alter pre-existing contacts made by the core histones
and histone H1 with DNA.
Figure 5:
Structure of DNA in the 5 S nucleosome is
not changed significantly by conclusion of multiple histone H1
molecules. Hydroxyl radical footprinting is shown. Reconstitution
reactions as shown in Fig. 1, lanes 2, 3, and 5, were cleaved with hydroxyl radical (see ``Materials
and Methods'') before resolution on a non-denaturing gel, excision
of the appropriate complex, deproteinization, denaturation of DNA, and
resolution on a denaturing gel (see ``Materials and
Methods''). Lanes 1-5 and lanes 6-10 show the same samples subjected to electrophoresis for different
times. Lanes 1 and 6 show a Maxam-Gilbert G reaction
for markers (G); lanes 2 and 7 show hydroxyl
radical cleavage of naked DNA (N); lanes 3 and 8 show cleavage of octamer-bound DNA (C); lanes 4 and 9 of H1-octamer show cleavage of bound DNA (A); and lanes 5 and 10 show cleavage of
2H1-octamer-bound DNA (B). The ellipsoids show the position of
core (shaded) and chromatosome (open) boundaries for lanes 6-10. The asterisks mark the dyad
position for the nucleosome core.
These experiments were designed to examine the nature of a
defined sequence nucleosome containing 1 or more molecules of histone
H1. The major conclusion is that although multiple molecules of histone
H1 can bind to DNA wrapped around a single histone octamer, only a
single preferential high affinity binding site exists for histone H1 (Fig. 1Fig. 2Fig. 3Fig. 4). Structural
changes to this defined sequence nucleosome following inclusion of more
than one histone H1 molecule are minor (Fig. 5). Although these
results are obtained with a particular nucleosome they have general
implications for studies interrelating the influence of histone H1 on
chromatin structure and transcription.
The binding of a 2nd molecule of histone H1 to
nucleosomes may occur in vivo(4) and has been
documented in vitro (Refs. 7, 15, 16, and 19, and this work).
The association of additional molecules of basic protein with
nucleosomal DNA is not surprising, since additional histone octamers
can also bind to the nucleosome(46) . The association of
additional molecules of histone H1 during chromatin assembly does,
however, lead to a decreased density of nucleosomes on the DNA molecule
and an increased length of DNA bound by H1 away from core histone-DNA
contacts(15, 16, 19) . The additional
molecules of histone H1 bound to nucleosomal DNA under these conditions
are likely to be bound more weakly than the 1st molecule to bind to the
nucleosome (Fig. 1Fig. 2Fig. 3Fig. 4). Thus
it is possible that the repressive character of chromatin will decrease as more histone H1 is reconstituted into chromatin.
This result is in fact observed by Sandaltzopoulos et
al.(16) .
Volume 271,
Number 12,
Issue of March 22, 1996 pp. 7090-7094
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
2 nM), and the 2nd
molecule of H1 binds with a reduced affinity (K
10 nM). This latter binding is comparable with the
association of histone H1 with naked DNA. Neither molecule of histone
H1 alters the helical periodicity of DNA in the nucleosome as revealed
by hydroxyl radical cleavage. We conclude that although multiple
molecules of histone H1 can associate with nucleosomal DNA, there is
only a single high affinity binding site for histone H1 within the 5 S
nucleosome.
)without addition
of exogenous H1, to a repeat of 205 bp when 2 molecules of histone H1
are present per nucleosome, to a repeat of 220 bp when an excess of 5
molecules of histone H1 per nucleosome is present(19) . Without
histone H1, transcription in the Xenopus extract is repressed
by histone octamers alone only at very high densities (one per 160 bp),
but with histone H1 transcription was repressed at a ratio of 1.5
molecules of H1 per nucleosome, at a nucleosomal repeat length of 205
bp(10, 19) . Comparable results in which exogenous
histone H1 increases the spacing of nucleosomes were reported in Drosophila extracts; 1 molecule of histone H1 per nucleosome
increased nucleosome spacing from 190 to 210 bp, and 3 molecules per
nucleosome increased spacing to 220 bp(15, 16) .
However, transcription results were very different; Kamakaka and
colleagues (15) find that 3 molecules of H1 per nucleosome are
necessary to repress transcription, whereas Sandaltzopoulos and
colleagues (16) find that histone H1 makes a negligible
contribution to transcriptional repression.
DNA Fragments
Radiolabeled DNA fragments
contained the Xenopus borealis 5 S rRNA gene. A 237-bp HpaII-DdeI fragment derived from plasmid pJHX1 (27) was used for nucleosome reconstitution after
radiolabeling, either at the HpaII site 102 bp upstream from
the initiation site for transcription of the 5 S gene (+1) or at
the DdeI site 137 bp downstream from the start of
transcription.Nucleosome Reconstitution and Footprinting
Histone
H1 was prepared from calf thymus as described previously(21) .
Nucleosome cores were reconstituted onto radiolabeled DNA fragments
either by exchange with chicken erythrocyte core particles (28, 29) or by dialysis using purified histone
octamers(30) . The original 20 µl of exchange reaction
containing 3.0 µg of donor chromatin, 0.5 µg of naked
nonspecific DNA, and 10-100 ng of labeled 5 S fragment was
incubated for 1 h (all incubations at room temperature). This was then
diluted from 1 M NaC1 with two 5-µl additions of TE (to
0.8 and 0.66 NaCl, respectively, where TE is 10 mM Tris, pH
8.0, 1 mM EDTA), each for 1 h. The salt concentration was then
diluted to 0.2 M with 170 µl of TE for 15 min and later
finally diluted to 100 mM NaCl with 200 µl of TE. About
50-60% of the labeled 5 S fragment was assembled into
mononucleosome cores without detectable dinucleosome complexes with
this procedure as monitored by electrophoresis. Cleavage of DNA in the
reconstituted nucleosome with hydroxyl radical was as
described(31) . All footprinting was accomplished by gel
isolation of the nucleoprotein products of digestion, followed by
deproteinization and denaturing gel electrophoresis.Linker Histone Binding Experiments
Unless
indicated otherwise in the figure legends, approximately 5 ng of
labeled 5 S DNA, either naked in the presence of 50 ng of unlabeled
calf thymus DNA or reconstituted with a single histone octamer in the
presence of 50 ng of unlabeled ``nonspecific'' chromatin,
were incubated with various amounts of linker histone H1 (see figure
legends) in 10 µl of binding buffer (10 mM Tris-HCl, pH
8.0, 50 mM NaCl, 0.1 mM EDTA, 5% (v/v) glycerol).
Samples were incubated at room temperature for 15 min and loaded
directly onto running 0.7% agarose gels in 0.5 
TBE (1
TBE is 90 mM Tris base, 90 mM boric acid, 2.5 mM EDTA). After electrophoresis, the gels were dried and
autoradiographed.Micrococcal Nuclease Digestion Analysis
Digestions
were for 5 min with 0.01-0.5 unit of enzyme/sample at 22 °C.
Samples contained 100 ng of reconstituted chromatin, and the
incubation with H1 was as described(21) . Ca
was adjusted to 0.5 mM concomitantly with addition of
micrococcal nuclease. Digestion was terminated by the addition of EDTA
(5 mM), SDS (0.25% w/v), and proteinase K (1 µg/ml). The
DNA was recovered and 5`-end labeled with polynucleotide kinase, and
fragments were separated by electrophoresis in nondenaturing 8%
polyacrylamide gels (21) . Restriction endonuclease cleavage to
determine the exact position of micrococcal nuclease cleavage sites was
as described(21, 32, 33) .
Radiolabeling of Linker and Core Histones
For
these experiments purified histones were used to reconstitute
nucleosomes (30) . Nucleoprotein-containing bands were excised
from the gel, and proteins were electroeluted in 2 TE, 0.1%
SDS, 0.5 mM diisopropyl fluorophosphate for 25 min at about 5
V/cm and dialyzed thoroughly to 0.01% SDS, freeze-dried, and dissolved
in 5 µl of 5 mM HEPES-Na, pH 7.4, 1 mM EDTA. The
eluted proteins (or the reference protein mixtures) were radioiodinated
by adding an aliquot of the protein solution to a test tube with
10-40 µCi of Bolton-Hunter reagent (Amersham Corp., freshly
dried by a stream of dry nitrogen). The reaction was allowed to proceed
for 20 min at room temperature, stopped by 10 µl of 1 M Tris/glycine, pH 8.0, incubated at 95 °C for 1 min, and
adjusted to 60 µl of 0.2% SDS. The labeled proteins were
precipitated by 27 µl of 5 M NaCl on ice, redissolved in
30 µl of TE containing 1% 
-mercaptoethanol on 0.1
µg/µl protein carrier (see below), adjusted to 0.3 M NaCl and precipitated by 6 volumes of acetone, dissolved in the
appropriate sample buffer, and resolved by gel electrophoresis. We used
a mixture of peptides derived from histone H5 by prolonged hydrolysis
in 2.5% acetic acid at 105 °C as a carrier protein. The advantage
of this carrier is that although the component polypeptides closely
resemble the histones by molecule size and composition, none of them
match the histones by electrophoretic mobility.
Histone H1 Forms Two Distinct Complexes with 5 S DNA
Associated with a Histone Octamer
Earlier work has examined the
structure of a nucleosome in which a single molecule of histone H1
associates with the X. borealis 5 S RNA gene wrapped around an
octamer of core histones(21, 24) . Association of
histone H1 is dependent on the length of linker DNA available for
binding; removal of linker DNA eliminated the selective association of
histone H1 with nucleosomal DNA compared with naked DNA(21) .
The association of histone H1 with DNA in the 5 S nucleosome is
asymmetric(21, 24) . Since one model of asymmetric
histone H1 association with nucleosomal DNA would potentially leave a
second histone H1 binding site exposed(26) , we examined
whether we could bind a 2nd molecule of histone H1 to the 5 S
nucleosome containing a long linker DNA segment (Fig. 1). At a
stoichiometry of 1 molecule of histone H1 per 5 S nucleosome, a clear
preferential binding of histone H1 to nucleosomal compared with naked
DNA is apparent (Fig. 1, compare lanes 2 and 3). At higher ratios of histone H1 molecules per 5 S
nucleosome, two phenomena occur: a second complex containing
nucleosomal DNA is formed, and naked DNA begins to be bound by histone
H1 (Fig. 1, compare lanes 4 and 5). The second
complex containing nucleosomal DNA contains at least 2 molecules of
histone H1 per histone octamer (see below). At higher excesses of
histone H1, both naked and nucleosomal DNA begin to form aggregates (Fig. 1, lane 6)(34) .
A Single Molecule of Histone H1 Associates with the 5 S
DNA Wrapped Around the Histone Octamer with High Affinity, and
Additional Molecules of Histone H1 Bind Nucleosomal DNA with the Same
Lower Affinity as That with Which H1 Binds Naked DNA
We made use
of the gel retardation assay to determine the affinity with which
histone H1 interacts with naked and nucleosomal DNA ( Fig. 2and
3). The 1st molecule of histone H1 binds to nucleosomal DNA with a K
of 2 nM (Fig. 2A and Fig. 3). Assuming that only a 2nd molecule of linker
histone H1 binds to the nucleosome leads to an estimation of a K
of 10 nM for this association (Fig. 2B and Fig. 3). The binding of histone H1
to naked DNA was also with a K
of 10 nM (Fig. 2C and Fig. 3). The initial
association of histone H1 with the naked 5 S DNA fragment leads to the
appearance of specifically retarded nucleoprotein species (Fig. 2A, lane 9; Fig. 2C, lanes 2-5). This may reflect the stable association of
linker histone at a single site before additional molecules of linker
histones associate. Note that the binding curve for H1 association with
naked DNA (Fig. 3) is consistent with cooperative
binding(34) . The binding constants obtained in these assays
are consistent with the preferential association of the 1st molecule of
histone H1 with DNA wrapped around a histone octamer and the
disappearance of naked DNA only at high H1 excess (Fig. 2A). The ability of histone H1 to bind naked DNA
and form aggregates is unaffected by the presence of octamer-bound DNA (Fig. 2, compare B with C). Dissociation
experiments yield the same relative affinities (Fig. 4), and the
2nd molecule of histone H1 is displaced from nucleosomal DNA at the
same level of competitor DNA required to release naked DNA from
association with histone H1 (Fig. 4, compare lanes 4 and 12). These results lead to the conclusion that a
single high affinity site exists on each nucleosome for the binding of
histone H1. Importantly the additional molecules of histone H1 that
bind to the nucleosome do so with comparable affinity to the binding of
H1 to naked DNA.
), octamer-bound DNA (), and
H1-octamer-bound DNA (
). Autoradiographs of gel mobility shifts
were scanned with a laser densitometer, and the fraction of histone
H1-bound DNA or nucleoprotein complex was plotted against the H1
concentration used.
Structural Consequences of Binding 2 Molecules of Histone
H1 per Nucleosome
The original assay for the stable inclusion of
histone H1 into a nucleosome is the generation of a ``chromatosome
stop'' (35) . In this assay, histone H1 association leads
to the appearance of a new kinetic intermediate during the digestion of
chromatin by micrococcal nuclease. Typically for nucleosomes containing
a mixture of a large number of DNA sequences, 1 molecule of histone H1
will protect a total of 20 bp of linker DNA immediately contiguous to
the 146 bp in the nucleosome core(35, 36) . In earlier
work, we have documented that the histone octamer adopts a single
translational position on the 5 S RNA gene and that the association of
histone H1 extends this protection into the linker
DNA(21, 24, 37, 38) . We repeated
this analysis for nucleosomal structures containing 1 and 2 molecules
of histone H1. A 5 S nucleosome core without histone H1 protects
approximately 149 bp of DNA from micrococcal nuclease whereas a 5 S
nucleosome containing a single molecule of histone H1 protects
approximately 175 bp of DNA (21) and a 5 S nucleosome
containing 2 molecules of histone H1 protects both 175 and 200 bp of
DNA from micrococcal nuclease (not shown). The variation in the size of
fragments protected between 5 S nucleosomes (149 bp) and 5 S
chromatosomes (175 bp) from those of mixed sequence nucleosomes (146
bp) and chromatosomes (166 bp) is due to sequence-specific variation in
micrococcal nuclease cleavage of a defined sequence DNA
fragment(24) . The 200-bp DNA fragment derived from the 5
S nucleosome is much less abundant than the 175-bp DNA fragment (not
shown). This may reflect the necessity of 2 molecules of histone H1
binding to nucleosomal DNA to protect 200 bp and the reduced binding
affinity of the 2nd molecule of histone H1 to the nucleosome (Fig. 2Fig. 3Fig. 4). Weaker association of
histone H1 with DNA is less likely to impede the digestion of DNA by
micrococcal nuclease.
A Single High Affinity Binding Site for Histone H1 in the
Nucleosome
We have determined that a single molecule of histone
H1 prefers to bind to nucleosomal DNA with a 5-fold higher affinity
than the affinity for nucleosomes already containing H1 or for naked
DNA (Fig. 1Fig. 2Fig. 3Fig. 4). This
increased affinity of histone H1 for nucleosomal compared with naked
DNA may depend on contacts the linker histone makes with the core
histones (39, 40) or a preference for DNA that is
curved through interaction with the core histones (24) or that
contains two DNA helices that are juxtaposed in
space(41, 42, 43) . The preferential binding
of a single molecule of histone H1 in a nucleosomal context indicates
that studies examining the binding of histone H1 to naked DNA as a
model for chromatin structure (34, 44) might not
recapitulate the same quality of histone H1 association with DNA as
seen within the nucleosome(21) . This might account in part for
the failure of histone H1 complexes with naked DNA to repress
transcription, under conditions where histone H1 incorporated into a
nucleosome will repress transcription(12, 45) . A
weaker association of histone H1 with DNA will make the repressive
``chromatin'' complex more accessible to the transcriptional
machinery.The Structure of Nucleosome Containing 2 Molecules of
Histone H1
We make use of two established assays to demonstrate
the association of multiple molecules of histone H1 with nucleosomal
DNA. The first is the gel retardation assay, which clearly resolves a
second nucleoprotein complex that migrates more slowly than nucleosomal
DNA containing a single molecule of H1 (Fig. 1, Fig. 2,
and Fig. 4). The second assay is the protection of linker DNA
from digestion with micrococcal nuclease. Each molecule of histone H1
protects an additional 25 bp of linker DNA from digestion by
micrococcal nuclease. The exact basis for this protection is not known;
it could derive from direct contacts made between histone H1 and DNA,
or it could derive from allosteric changes in contacts made between the
core histones and DNA(25) . Mapping the boundaries of histone
DNA contacts in the nucleosome containing at least 2 molecules of
histone H1 reveals that strong histone-DNA contacts are not apparent
(data not shown). Moreover, inclusion of 1 or more molecules of histone
H1 has no apparent influence on the wrapping of DNA on the surface of
the histone octamer (Fig. 5)(21) . Thus major changes in
chromatin structure attributed to the incorporation of multiple
molecules of linker histone per nucleosome repeat (7) are
unlikely to derive from structural changes at the level of the single
nucleosome.
Conclusion
Chromatin templates containing either
histone H1 bound to naked DNA, a single molecule of histone H1 bound
per nucleosome, or 2 molecules of histone H1 bound per nucleosome will
differ significantly in the quality and stability of histone H1
association. It is probable that these differences will contribute to
variation in the transcriptional properties of these chromatin
templates(15, 16, 19) .
)
We thank Drs. Peter Becker, David Clark, and Stefan
Dimitrov for useful discussions. We are grateful to Thuy Vo and Genine
Williams for manuscript preparation.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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