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(Received for publication, January 3,
1995; and in revised form, January 11, 1995) From the
We demonstrate that methylation of the 12 dinucleotide CpGs
within a GC-rich DNA fragment containing a Xenopus borealis 5
S rRNA gene does not influence histone H1 binding to naked or
nucleosomal 5 S DNA. Thus a simple mechanism in which histone H1
selectively associates with nucleosomes containing methylated CpG
cannot explain the repressive effects of methylation on gene activity. Methylation of eukaryotic DNA occurs at the dinucleotide
CpG(1) . DNA methylation is inversely related to
transcriptional activity(2, 3) . The reduction in
transcription could be attributed either to direct mechanisms, in which
a methylated CpG interferes with transcription factor recognition of a
binding site, or to indirect mechanisms, in which repressor proteins
selectively associate with sequences containing methylated CpGs.
Although some transcription factors are sensitive to methylation of
their recognition sites(4) , many are insensitive(5) .
There is considerable evidence for the operation of indirect
mechanisms. Human Bird and colleagues (10, 11) have characterized two proteins that
selectively recognize DNA sequences containing methylated CpG.
Methyl-CpG-binding protein 1 (MeCP-1) selectively represses
transcription from methylated DNA(12, 13) . How MeCP-1
would recognize extended DNA sequences containing methylated CpG within
chromatin remains unclear. Other proteins that have been reported to
selectively recognize methylated DNA and repress transcription are
histone H1 (14) and proteins similar to linker
histones(15) . Histone H1 selectively represses the
transcription of genes during Xenopus development(16, 17, 18) . In
vitro, the incorporation of histone H1 into chromatin can repress
the transcription of many genes including the Xenopus 5 S rRNA
gene(19, 20, 21) . We have examined the
consequences of methylating CpG dinucleotides within a DNA fragment
containing a Xenopus 5 S rRNA gene on the association of the
histone octamer and histone H1, both in isolation and together within
the nucleosome. We make use of 5 S DNA since it has many of the
characteristics of CpG-enriched sequences in the eukaryotic genome (CpG
islands; (1) ). These CpG islands are normally several hundred
base pairs in length and contain regulatory DNA sequences. The DNA
fragment containing the 5 S rRNA gene is 60% C + G, and contains
12 CpG dinucleotides. Furthermore, in earlier work we have developed a
simple in vitro assay for detecting the binding of linker
histones to model chromatin substrates including Xenopus 5 S
RNA genes. The binding of linker histones to reconstituted
mononucleosomes occurs preferentially relative to naked
DNA(22, 23) . This binding fulfills all classical
criteria for the correct incorporation of linker histones into the
nucleosome(22, 24) . We have therefore investigated
the consequences of DNA methylation on the incorporation of histone H1
into a nucleosome using this assay. We find that DNA methylation is
without effect on the association of histone H1 with nucleosomal DNA.
Figure 3:
Chromatosome stop with methylated or
unmethylated DNA reconstituted histone octamers and histone H1.
Reconstitutes were prepared and digested as described (22) (see
``Materials and Methods''). A, chromatosome stop
with methylated and unmethylated 5 S nucleosome cores and histone H1.
Reconstituted 5 S nucleosome cores on methylated DNA in the absence of
histone H1 were digested with 0.0375, 0.075, and 0.15 units of
micrococcal nuclease (5 min, 22 °C) as indicated by the triangles above the lanes 2-4. Lane1 contains size markers, the positions of 147, 160, and
180 bp length DNA fragments are indicated. CP indicates core
particle size DNA (
Figure 1:
Methylation of pXP10 by M. SssI methylase. pXP10 plasmid was methylated or mock
methylated (see ``Materials and Methods''). Complete
methylation was assayed through inhibition of restriction endonuclease HpaII. Lane1, MspI cleavage of
pBR322 for markers. Lane2, HpaII cleavage
of mock methylated pXP10. Lane3, HpaII
failure to cleave methylated pXP10. Lane4,
undigested methylated pXP10. DNA was stained with ethidium bromide
after resolution on a 1% agarose gel.
Figure 4:
Structure of DNA in the 5 S nucleosome is
not changed significantly by CpG methylation. A, DNase I
footprinting. DNA fragments, methylated or unmethylated, were
radiolabeled at the XbaI site before reconstitution with
histone octamers and H1 as described (see ``Materials and
Methods''). Lanes1 and 6 are markers
generated by G-specific Maxam-Gilbert cleavage(31) . Lanes2 and 7 are C-specific cleavage reactions. The dots between lanes6 and 7 indicate
methylcytosine not cleaved by this chemistry. Lanes3 and 8, naked DNA; lanes4 and 9, octamer-DNA complexes; lanes5 and 10, octamer and H1 bound to DNA after cleavage with DNase I. B, hydroxyl radical footprinting. Unmethylated (lanes1-3) and methylated DNA fragments radiolabeled at
the EcoRI site (lanes 4-6) were reconstituted
into histone DNA complexes (see ``Materials and Methods'').
Cleavage patterns for naked DNA (lanes 1 and 4),
octamer-DNA complexes (lanes2 and 5), and
H1-octamer DNA complexes (lanes3 and 6) are
shown. The horizontalarrow at +10 shows the
approximate dyad axis of the reconstituted
nucleosome(22) .
DNA fragments containing a Xenopus 5 S rRNA gene
were prepared either with all CpGs free of methylation by growth of
plasmid pXP10 in Escherichia coli, or with all CpGs methylated
following treatment of naked plasmid pXP10 DNA with M. SssI
methylase. We controlled the quality of our methylation reaction by
digesting unmethylated and methylated pXP10 with the
methylation-sensitive restriction endonuclease HpaII (Fig. 1). Whereas unmethylated pXP10 was completely cleaved with HpaII (lane2), methylated pXP10 was
resistant to cleavage (lane3). After radiolabeling
of the purified DNA fragments used for the histone binding experiments,
we also determined the efficiency of methylation by chemical
sequencing(31) . Methylation of cytosine appears as a gap in
the C-ladder (Fig. 4, compare lanes2 and 7, gaps are marked by dots), this is because
hydrazine does not react with 5-methylcytosine in the C-specific
chemical modification reaction (see also (32) ). Having
established that our DNA fragments were unmethylated or completely
methylated at the CpG dinucleotides, we next examined the consequence
of methylation for the binding of histone H1 and core histones. We
find that histone H1 binds equivalently to unmethylated or methylated 5
S DNA (Fig. 2A, compare lanes 1-4 with 5-8). A number of intermediate
nucleoprotein complexes (H1-DNA) are resolved in the nondenaturing gel
prior to the appearance of large aggregates at the origin of the gel.
These presumably are similar to the linker histone-DNA structures
assembled with linear DNA that have been previously
described(33) . There are no significant differences in the
abundance of these intermediate H1-DNA complexes between unmethylated
and methylated DNA. More aggregation appears to have occurred in the
unmethylated sample than in the methylated sample at the highest H1 to
DNA ratio (0.8:1.0 by mass) (Fig. 2A, compare lanes4-8). However, this feature was not reproducible.
We conclude that calf thymus H1 binds equivalently to this specific DNA
sequence whether or not it is methylated. This is in contrast to the
results of Levine et al.(14) , who concluded that calf
thymus H1 would selectively interact with methylated DNA at comparable
ratios of H1 to DNA (0.5-1.5:1 by mass). These authors made use
of a different DNA sequence containing the adenovirus major late
promoter and the mouse c-fos gene in a Bluescript vector. Thus
any selective association of histone H1 with methylated DNA must be
highly sequence-specific since the adenovirus major late promoter and
the 5 S DNA fragment contain a comparable density of CpG dinucleotides.
Figure 2:
Gel retardation assays for H1 binding to
naked and nucleosomal methylated and unmethylated DNA. A,
naked DNA. The methylated or unmethylated EcoRI-XbaI
fragment of pXP10 (
Linker histones prefer to associate with 5 S DNA wrapped around an
octamer of core histones rather than with naked DNA(22) . This
preferential binding might be due to changes in DNA conformation
following association with the histone octamer, or it might be due to
protein-protein interactions with the core
histones(34, 35) . We find that core histones
reconstitute onto methylated and unmethylated DNA with comparable
efficiency (Fig. 2B, compare lanes2 and 8). This is in agreement with earlier
data(36, 37, 38) . Incorporation of histone
H1 into nucleosomes containing methylated or unmethylated DNA occurs
equivalently (Fig. 2B, compare lanes 2-7 with 8-13). Minor variation between H1 binding to
methylated and unmethylated nucleosomal DNA at low ratios of linker to
core histone (Fig. 2B, lanes4, 5, 10, and 11) was not reproducible. Since
assembly into the nucleosome more closely resembles the natural
physiological association of histone H1 with chromatin, we suggest that
histone H1 will not discriminate between chromatin containing
methylated versus unmethylated DNA. An important control
for the correct incorporation of linker histones into the nucleosome is
the appearance of kinetic intermediate in micrococcal nuclease
digestion known as the chromatosome(39) . A chromotosome
contains about 166 bp of DNA, a histone octamer, and a single molecule
of linker histone. In contrast, a nucleosome core contains 146 bp of
DNA and a histone octamer. We find that incorporation of histone H1
into nucleosomes containing methylated or unmethylated DNA leads to the
appearance of chromatosome stops during micrococcal nuclease digestion
with equivalent efficiency (Fig. 3A, CH,
compare lanes 6-8 with 10-12). In our
experiments two bands appear as kinetic intermediates of micrococcal
nuclease digestion in the presence of histone H1, the lower band
labeled CH is In earlier work we have found
qualitative differences in the structure of methylated and unmethylated
DNA following reconstitution with tetramers or octamers of core
histones(38) . We next wished to examine whether the histone
octamer in the presence or absence of histone H1 would organize 5 S DNA
differently dependent on CpG methylation. Close comparison of the DNase
I cleavage patterns of naked methylated and unmethylated DNA (Fig. 4A, compare lanes3 and 8) reveals very minor differences around the sites of CpG
methylation (Fig. 4A, indicated by the dots between lanes6 and 7). The rotational
position of the DNA on the surface of the histones is unchanged by DNA
methylation, as determined by the sites of preferred DNase I cleavage (Fig. 4A, lanes4, 5, 9, and 10), or by hydroxyl radical cleavage (Fig. 4B, lanes2, 3, 5, and 6). We conclude that nucleosome positioning on Xenopus 5 S DNA is unaffected by DNA methylation ( Fig. 3and Fig. 4). We initiated this study because of
experiments suggesting that CpG methylation might influence the
association of histones, and especially histone H1 with DNA. There is
clear evidence that methylated CpG preferentially accumulates in H1
containing nucleosomes in vivo(40) . Although the
molecular mechanisms responsible for this selective accumulation are
unknown, chromatin assembly appears necessary for methylation-sensitive
repression of genes(7, 8) . Levine et al. (14) found that histone H1 bound preferentially to certain
methylated DNA sequences selectively repressing transcription from
methylated templates in vitro. Jost and Hofsteenge (15) also found that a protein similar to histone H1
(MDBP-2-H1) selectively bound methylated DNA. These results led to the
hypothesis that a simple mechanism in which histone H1 selectively
associates with nucleosomes containing methylated CpG explains the
repressive effects of methylation on gene
activity(14, 15) . In contrast, we find that histone
H1 does not discriminate between naked DNA that is methylated or
unmethylated (Fig. 2A). This result is in agreement
with earlier work(41) . Since the 5 S DNA used in these studies
has the same characteristics as a typical CpG island(1) , we
believe that this result is physiologically relevant. Thus any
preference of histone H1 for methylated compared to unmethylated
sequences must be highly sequence-selective. We have made use of the
assembly of defined nucleosomal structures on 5 S DNA to establish that
the histone octamer is positioned identically on methylated and
unmethylated 5 S DNA. More importantly we establish that histone H1
does not discriminate between nucleosomes containing methylated or
unmethylated DNA. Thus, histone H1 seems unlikely to have a role as a
general repressor selective for chromatin containing methylated DNA.
Perhaps other DNA-binding proteins that recognize methylated DNA such
as MeCP-1 or -2 have such a role in a chromatin environment.
Volume 270,
Number 9,
Issue of March 3, 1995 pp. 4197-4200
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-globin transcription is sensitive to DNA
methylation, but not through the modification of specific
sites(6) . Others have suggested that chromatin assembly might
be involved in repressing transcription of methylated
DNA(7, 8, 9) . These observations have led to
the search for proteins that selectively recognize DNA sequences
enriched in methylated CpG.
DNA Fragments
Radiolabeled DNA fragments
contained the Xenopus borealis 5 S rRNA gene (see Fig. 3B). A 271-bp (
)EcoRI-XbaI fragment derived from plasmid
pXP10 (25) was used for nucleosome reconstitution after
radiolabeling, either at the EcoRI site 78 bp upstream from
the initiation site for transcription of the 5 S gene (+1), or at
the XbaI site 193 bp downstream from the start of
transcription.
147 bp). Reconstituted 5 S nucleosomes
containing histone H1 and unmethylated (lanes 6-9) or
methylated DNA (lanes 10-12) were digested with 0.0375,
0.075, and 0.15 units of micrococcal nuclease. Lanes5 and 9 contain size markers. The positions of chromatosome
size (CH, 166 bp) and core particle (CP,
147 bp) DNA fragments are indicated. DNA was resolved on an 8%
nondenaturing polyacrylamide gel after end labeling with
[-
P]ATP and T4 polynucleotide
kinase(22) . B, mapping of core and chromatosome
boundaries. The EcoRI-XbaI fragment used in the
experiment is shown. The verticalarrows indicate the
sites of CpG methylation. The horizontalopenarrow indicates the 5 S RNA gene, +1 is the start
site of transcription, and +120 the site of transcription
termination.
Methylation of DNA Templates
Cytosines in the CpG
sequences were methylated with the CpG methylase M. SssI (New
England Biolabs). Incubation was as recommended by the manufacturer
except that the enzyme concentration was increased 5-fold. The extent
of methylation was monitored by resistance to cleavage with HpaII (New England Biolabs) (Fig. 1) and by Maxam and
Gilbert cleavage chemistry (Fig. 4). Methylation reactions were
carried on until complete resistance to cleavage was achieved, then
phenol-chloroform-extracted and ethanol-precipitated. Mock methylation
reactions were carried out in the absence of M. SssI.
Nucleosome Reconstitution and Footprinting
Histone
H1 was prepared from calf thymus as described previously(22) .
Nucleosome cores were reconstituted onto radiolabeled DNA fragments by
exchange with chicken erythrocyte core
particles(26, 27) , with the modification that only
four additions of diluent were used. The original 1 M NaCl
20-µl 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 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
mononucleosomes cores without detectable dinucleosome complexes with
this procedure as monitored by electrophoresis. Cleavage of DNA in the
reconstituted nucleosome with hydroxyl radical or DNase I was as
described(28) . All footprinting was accomplished by gel
isolation of the nucleoprotein products of digestion, followed by
deproteinization and denaturing gel electrophoresis.Linker Histone Binding Experiments
Approximately 5
ng of labeled 5 S DNA, either entirely naked in the presence of 100 ng
unlabeled calf-thymus DNA, or with 50% 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 10-20 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 15-30 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 0.1-0.5 unit of enzyme/sample at 22 °C.
Samples contained 100 ng of reconstituted chromatin, and the
incubation with H1 was as described (22) except that EDTA was
adjusted to 0.5 mM by omitting it from the final dilution
buffer (see above). Ca
was adjusted to 1 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(22) .
Restriction endonuclease cleavage to determine the exact position of
micrococcal nuclease cleavage sites was as
described(22, 29, 30) .
6 ng) was end-labeled using Klenow fragment of
DNA polymerase at the EcoRI site and mixed with 100 ng of
unlabeled sonicated calf thymus DNA (see ``Materials and
Methods''). The DNA mixture was incubated with 10 ng (lanes1 and 5), 20 ng (lanes2 and 6), 40 ng (lanes3 and 7), and 80
ng (lanes4 and 8) of histone H1.
Nucleoprotein complexes were resolved in a 0.7% agarose gel. An
autoradiograph is shown. B, nucleosome DNA. The same DNA
fragments as in A were reconstituted with histone octamers
such that 50% of the fragment remained naked. This mixture of
reconstitute and naked DNA (6 ng total) containing 50 ng of nonspecific
chromatin was mixed with 0, 1.25, 2.5, 5, 10, and 20 ng of histone H1
in lanes 2-7 for unmethylated DNA and lanes
8-13 for methylated DNA. Nucleoprotein complexes were
resolved in a 0.7% agarose gel (see ``Materials and
Methods''). An autoradiograph is shown. Lane1 contains naked DNA alone.
166 bp in length, and the other is 180
bp in length. Both are linker histone-dependent and presumably reflect
selective contacts by H1 with linker DNA. In the absence of histone H1,
digestion of methylated DNA associated with the histone octamer
generates only core particle size DNA (Fig. 3A, CP). In earlier work, we have determined that linker histones
are asymmetrically incorporated into the 5 S
nucleosome(22, 23, 24) . Mapping of the
boundaries of the nucleosome core and chromatosomes for methylated and
unmethylated DNA confirm these earlier results (Fig. 3B, data not shown). The positions of
methylated CpGs within the 5 S DNA are also indicated in Fig. 3B. We conclude that the presence of 11 methylated
CpGs within the boundaries of the chromatosome defined by micrococcal
nuclease digestion do not influence the efficiency with which histone
H1 is incorporated into the nucleosome (Fig. 2B). Nor
does DNA methylation influence the translational position of the
histones relative to DNA sequence.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
