(Received for publication, July 12, 1995)
From the
The possibility that histone H1 binds preferentially to DNA containing 5-methylcytosine in the dinucleotide CpG is appealing, as it could help to explain the repressive effects of methylation on gene activity. In this study, the affinity of purified H1 for methylated and non-methylated DNA sequences has been tested using both naked DNA and chromatin. Based on a variety of assays (bandshifts, filter-binding assays, Southwestern blots, and nuclease sensitivity assays), we conclude that H1 has no significant preference for binding to naked methylated DNA. Similarly, H1 showed the same affinities for methylated and non-methylated DNA when assembled into chromatin in a Xenopus oocyte extract. Thus potential cooperative interaction of H1 with polynucleosomal complexes is not enhanced by the presence of DNA methylation.
The major DNA modification in vertebrates is methylation at the
5 position of cytosine in the dinucleotide CpG. About 60-90% of
genomic CpGs contain 5-methyl cytosine (mC). Transfection
assays with methylated genes have shown that CpG methylation often
causes repression of
transcription(1, 2, 3, 4) . The
severity of repression depends on a number of parameters, particularly
the location of methylation relative to the promoter (1, 5; but see (6) ), the local density of methyl-CpG pairs, and the strength
of the promoter under test(7) . In general, high density
methylation strongly inhibits transcription, whereas low density
methylation can only inhibit weak promoters.
Several lines of
evidence suggest that proteins that bind preferentially to methylated
DNA are important components of the repression mechanism. Indeed,
binding of the methyl-CpG-binding protein (MeCP) ()MeCP1 (8) shows the same dependence on local density of methylation
as does transcriptional repression(4, 7) .
Methylation-associated transcriptional repression may also arise by
other routes. CpG methylation is known to prevent binding of some
transcription factors, and this is likely to contribute to repression
in some cases(9, 10) . Another potential cause of
methylation-mediated repression is direct alteration of chromatin
structure due to the presence of m
C. Involvement of
chromatin in mediating the effects of DNA methylation is suggested by
the experiments of Buschhausen et
al.(3, 11) . In addition, Keshet et al. found that methylated DNA is preferentially assembled into
nuclease-resistant chromatin after transfection(12) . While
these results could be explained by interaction between known
methyl-CpG-binding proteins and chromatin, it is also possible that an
ubiquitous component of chromatin, for example histones, interacts
differentially with methylated DNA leading to an altered higher order
structure that is incompatible with gene expression.
Attempts to detect an effect of DNA methylation on nucleosomal cores have not been successful(13, 14) . The linker histone H1, however, is an attractive candidate for mediator of the effects of methylation on chromatin. H1 plays a role in chromatin condensation(15, 16) . It is depleted in CpG islands, which are non-methylated and include the promoters of many genes, and in other potentially active genes, but is concentrated in inert chromatin(16, 17, 18) , and inhibits transcription in vitro(19, 20, 21) . If H1 were able to recognize methylated DNA, it could in theory contribute to the effects of methylation on chromatin structure and gene expression.
Studies using antibodies against mC
have suggested a link between DNA methylation and histone H1 by showing
that the modified base is preferentially localized in H1-containing
nucleosomes(22) . Whether H1 has a higher affinity for
methylated DNA is, however, an unresolved issue. Higurashi and Cole (23) did not detect any significant difference in the affinity
of H1 for methylated or non-methylated DNA. Nevertheless, they suggest
that the DNA complexed with histone H1 adopts a distinct conformation
when it is methylated, since the methylated MspI sites became
protected in the complexes. On the other hand, Levine et al.(24) reported that histone H1 bound preferentially to
methylated DNA and claimed that H1 suppresses transcription from
methylated templates in vitro. Jost and Hofsteenge (25) also implicated H1 as a methylated DNA-binding protein.
They obtained a protein fraction from chicken liver enriched in histone
H1 that showed binding to methylated CpG in a specific sequence
context. The activity, known as MDBP-2, was originally identified as a
40-kDa protein that was very tissue specific in distribution (26) but was subsequently attributed to a dimer of H1 (25) . Finally, Johnson et al.(27) have
concluded that H1 preferentially inhibits transcription from a
methylated template, and they propose that methylated sites on the DNA
serve as foci for long range chromatin condensation mediated by H1.
In this study, we have reinvestigated the interaction of purified chicken erythrocyte histone H1 (free of the erythrocyte variant H5) and rat kidney H1 with methylated and non-methylated DNA and chromatin using several assays. The results of comparative gel retardations, Southwestern blots, filter binding assays, and nuclease-protection assays indicate that histone H1 shows little or no preference for binding to naked methylated DNA. The apparent selective protection by H1 of methylated MspI sites can be explained by a characteristic of the endonuclease rather than the DNA itself. Most significantly, chromatin containing methylated DNA and chromatin containing non-methylated DNA have identical affinities for H1. We conclude that H1 is unlikely to be a primary mediator of the biological effects of DNA methylation.
Oligonucleotide DNA probes used in this study are shown in Table 1. The vitellogenin gene probes were synthesized according
to the sequence used by Jost and Hofsteenge (25) corresponding
to nucleotide positions -2 to +34 from the avian
vitellogenin gene and contained one CpG. Poly(GAC) and poly(GAM) are
ligated polymers of a synthetic 42-bp sequence containing 12 CGA
repeats, which in poly(GAM) have the cytosine substituted for
mC(28) . For the methylated vitellogenin and
poly(GAM) oligonucleotides, the m
C in the CpG sequences was
introduced during synthesis. The Sat probe corresponds to a repeat unit
of mouse satellite DNA cloned as a 234-bp insert of the pSat plasmids
described elsewhere(28) . The pAdomal plasmid (29) contains a fragment of adenovirus 2 major late promoter
cloned into pAT153. The plasmid contains 421 CpGs in 5785 bp total
length or one CpG every 14 bp on average. The Sat probe and the pAdomal
plasmid were methylated using SssI methyltransferase (CpG methylase) as
described in Boyes and Bird (4) . The 135-bp CG11 probe was
methylated in its 20 GCGC sites with HhaI methylase, or at 27 sites
using CpG methylase(8) . For the interaction of H1 with
chromatin, the plasmids pUC18 (2.7 kb) and pHsr11.9 (14.6 kb) were
used. The plasmid pHsr11.9 consists of an EcoRI fragment from
human rDNA cloned into pUC9 and was a gift from R. Anand.
Chick histone H1 was a generous gift from Jean Thomas (Cambridge). This histone H1 had been extracted from chicken erythrocyte nuclei by incubation with 0.65 M NaCl and purified by ion-exchange chromatography as described(30) . To check its purity, H1 was analyzed by discontinuous SDS-polyacrylamide gel electrophoresis. The H1 preparation was free from histone H5 and core histones. The rat histone H1 employed in the chromatin experiments was extracted from rat kidney nuclei that had been depleted of nonhistone proteins by low salt extraction (0.25 M NaCl), and subsequently extracted with 2 M NaCl, 5 M urea, 10 mM phosphate buffer, pH 7. This extract was fractionated on a Mono-S column and yielded pure H1, as evidenced by SDS-polyacrylamide gel electrophoresis.
For
the gel mobility assays using the vitellogenin promoter, increasing
amounts of chick H1 (5-50 ng) were mixed with 1-3 ng of
end-labeled probes, methylated or unmethylated, together with 100 ng of E. coli competitor DNA, and incubated for 30 min on ice. The
incubation medium consisted of 50 mM HEPES, pH 7.5,
50-100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol. The probe and conditions
corresponded to those used for MBDP-2 by Jost and
Hofsteenge(25) . The samples were loaded onto 5% polyacrylamide
gels prepared in 0.5 TBE and electrophoresed at 120 V and 4
°C in 0.5
TBE for 3-4 h. Finally, the gels were dried
and exposed. In other cases, a fixed amount of H1 was incubated with
the probes, together with various amounts of competitor DNA. In these
assays, 25 ng of chick H1 were incubated with the labeled vitellogenin
probes and 0-100 ng of competitor DNA, in 10 mM Tris-HCl
buffer, pH 7.5, containing 1 mM EDTA, 10 mM
-mercaptoethanol, 4% glycerol, and 0.1% Triton X-100 for 30 min on
ice. Samples were run in 5% polyacrylamide gels as described above.
For the Southwestern assays with the
vitellogenin probes, up to 1 µg of chick H1/lane was applied to
discontinuous polyacrylamide gels. As a control, a fusion protein
containing glutathione transferase plus the first 392 amino acids of
the methyl-CpG-binding protein MeCP2 was also loaded (see (31) ). After electrophoresis, gels were soaked 30 min in
transfer buffer (25 mM Tris, 190 mM glycine) to wash
off the SDS. The histone was transferred to nitrocellulose membranes
using a Bio-Rad transfer device. The proteins in the filters were
denatured in 6 M guanidine hydrochloride in binding buffer (20
mM HEPES, 40 mM KCl, 3 mM MgCl for 5 min, and renatured by rinsing the filters in five
successive 2-fold dilutions of guanidine-HCl in binding buffer, 5 min
each rinse. The filters were blocked for 30 min with 4% instant non-fat
dried milk in binding buffer and washed in the buffer. The filters were
incubated 10 min in binding buffer with 0.1% Triton X-100 and 2.5
µg/ml of competitor DNA. Then, about 500,000 counts/min of the
end-labeled vitellogenin probes were added and the filters incubated 1
h at room temperature with gentle shaking. After washing the filters
with 0.01% Triton X-100 in binding buffer, four times for 5 min, these
were dried and exposed. A Molecular Dynamics PhosphorImager was
employed to quantify the signals.
The DNA was assembled into
chromatin by the method described by Rodriguez-Campos et al.(32) which uses a high speed supernatant from Xenopus oocytes as source of the histones and factors required. Xenopus oocytes are deficient in histone H1; therefore,
minichromosomes packaged with this supernatant alone contain core
histones but no histone H1(33) . The chromatin assembly
reactions were carried out in 20 mM HEPES, pH 7.0, 5 mM KCl, 1 mM MgCl, 1 mM EGTA, 10
mM
-glycerophosphate, 10% glycerol, 0.5 mM DTT
(extraction buffer), supplemented with 3 mM ATP, 40 mM creatine phosphate, 2 µg/ml creatine phosphokinase, 60% oocyte
extract, the desired amount of labeled H1, and DNA. The DNA consisted
of equal amounts (0.75 µg/ml) of each of the two plasmids, pUC18
and pHsr11.9, only one of which was methylated in a given sample. In
any experiment, two different concentrations of labeled H1 (0.2-1
µg H1/µg DNA) were used for each pair of plasmids. A mixture
with all the components except the DNA was prepared and added to the
DNAs to start the reactions, which proceed overnight at 27 °C.
After chromatin assembly, part of each sample was loaded onto sucrose gradients, and part was digested with microccocal nuclease. Minichromosomes assembled on each plasmid were separated by loading 500 µl of chromatin samples onto 15-30% sucrose gradients made up in 20 mM HEPES, pH 7, 100 mM NaCl, 0.2 mM EGTA, followed by centrifugation in a SW40 rotor, at 40,000 revolutions/min and 4 °C, for 2 h 45 min. Fractions of about 730 µl were collected from the bottom of the tubes, and their radioactivity determined by Cherenkov counting. DNA and proteins were purified from these fractions. For analysis of the DNA, aliquots of gradient fractions were digested with 0.2 mg/ml proteinase K in 0.5% SDS for several hours and ethanol precipitated by standard methods. The DNA was resolved in 1% agarose gels and transferred to nylon membranes by alkaline transfer. For protein analysis the fractions corresponding to each minichromosome were pooled and the proteins precipitated with 20% trichloroacetic acid in the presence of 0.05% Triton X-100 and 10 µg/ml protamine sulfate as carrier. After incubation 20 min on ice, samples were spun in a microfuge. The pellets were washed with 20% trichloroacetic acid and HCl-acetone, dried, and dissolved in loading mixture for their resolution in Triton-acid-urea gels (TAU gels). This loading mixture included 1 M acetic acid, 8 M urea, 0.1% Triton X-100, glycerol, and methyl green. TAU gels contained 1 M acetic acid, 8 M urea, and 0.5% Triton X-100, with 10.5% acrylamide in the separating gels. The running buffer was 0.1 M glycine, 1 M acetic acid. After electrophoresis, the gels were Coomassie stained, dried, and exposed.
To check that
the DNA was organized into chromatin, after assembly part of each
sample was digested with 100 units/ml of Micrococcus nuclease
in the presence of 3 mM CaCl, at room temperature.
Aliquots were taken at 2, 10, and 30 min, and the digestion stopped
with 15 mM EGTA, 0.5% SDS on ice. These samples, plus an
undigested control and an equivalent aliquot taken before assembly,
were treated with 0.1 mg/ml RNase A, at 37 °C for at least 2 h, and
with 0.5 mg/ml proteinase K in 0.5% SDS for several hours. The DNA was
precipitated with ethanol and resolved in 1.5% agarose gels and
transferred to nylon membranes for Southern analysis. The membranes
were hybridized with two different probes. The EcoRI insert of
pHsr11.9 was a probe specific for the pHsr plasmid. With linear pUC18
as a probe, the signal was much higher for the pUC18 plasmid, but pHsr
was also detected due to homology of its vector portion with the probe.
Figure 1:
Gel retardation assays
of complexes between histone H1 and methylated or unmethylated DNA
sequences. A, assay with satellite DNA probes and different
amounts of histone. Various amounts of chick H1, as indicated in
nanograms above the lanes, were incubated with labeled satellite DNA
probes, either unmethylated (NM lanes), or extensively
methylated in its eight methylatable CpG groups (M lanes). The
samples were resolved in a 1.5% agarose gel with 0.5 TBE buffer
and the gel dried and exposed. The two first and last
lanes contained the free probes without histone. B, band
shift of H1 and CG11 probes in the presence of increasing amounts of
competitor DNA. In this case a fixed amount of H1 (50 ng) was incubated
with the 135-bp probe CG11, either unmethylated (NM lanes) or HhaI-methylated (M lanes) in the presence of various
amounts of E. coli DNA as indicated above the lanes. Samples
were electrophoresed in 5% polyacrylamide gels with 0.5
TBE
buffer. F refers to free probes, without
histone.
We next compared
the affinity of H1 for methylated and non-methylated DNA by competition
filter binding assays. A fixed amount of H1 (10 ng) was incubated with P-labeled non-methylated plasmid DNA (20 ng) in the
presence of increasing amounts of unlabeled competitor DNA. Competitor
was either non-methylated plasmid DNA or plasmid that had been
methylated at all CpGs. The mixtures were passed through nitrocellulose
filters to retain protein
DNA complexes, and the amount of labeled
DNA on the filters was measured in a scintillation counter. Each
competitor decreased the amount of labeled complex to a similar extent,
though the reduction was marginally greater with methylated DNA than
with non-methylated DNA (Fig. 2). The results of this and other
similar experiments indicated that methylated DNA was about 1.2-times
(20%) more effective than non-methylated DNA as a competitor.
Figure 2: Filter binding assays comparing methylated and non-methylated DNAs as competitors for complex formation with H1. Complexes between H1 (10 ng) and non-methylated labeled pAdomal (20 ng) were formed in the presence of various concentrations (0-60 ng) of unlabeled methylated or non-methylated competitor plasmid. The samples were filtered through nitrocellulose to retain only the DNA bound to H1. Points show percentage of labeled DNA complexed with the histone, taking the ``no competitor'' level as 100%. Filled circles, non-methylated competitor; open circles, methylated competitor. Short bars indicate two filters for the same concentration.
A
third assay for the affinity of histone H1 toward methylated and
unmethylated DNA was based on slot-Southwesterns. Unlike the previous
assays, where binding occurred in solution, Southwestern blots measured
the affinity of single immobilized molecules of H1 for DNA. Various
amounts of H1 protein were filtered onto a nitrocellulose membrane and
incubated with P-labeled methylated or unmethylated DNA.
The probes were poly(GAM) and poly(GAC), where M denotes m
C
(see Table 1). Poly(GAM) is a polymer of sequences containing 12
methyl-CpGs(28) . The results (Fig. 3) show that in the
absence of competitor dsDNA, H1 bound to the same extent with both
methylated (column 1) and unmethylated (column 4) probes, the signal
decreasing with the amount of protein in a non-linear fashion.
Non-linearity may indicate that the probe requires binding to several
H1 molecules to be retained in the filter. Competitor E. coli DNA also bound to H1 and was able to displace most of the probe
from the histone, but its effect was the same on poly(GAM) and
poly(GAC) (compare columns 2 and 3 with 5 and 6). This indicates that
the strength of the binding is equal irrespective of the methylation
status of the probe.
Figure 3: Slot-Southwestern blot of histone H1 probed with poly(GAM) and poly(GAC). Increasing amounts of chick H1 (125, 250, 500, and 1000 ng/slot, from top to bottom) were filtered through a nitrocellulose membrane. After renaturation and blocking, strips were incubated for 1 h with labeled poly(GAM), which is methylated (columns 1-3), or poly(GAC), which is unmethylated (columns 4-6). Non-methylated E. coli DNA was included as competitor for strips 2 and 3 and 5 and 6. Strips 1 and 4 did not include double-stranded competitor DNA. After washing, filters were dried and exposed to an x-ray film.
Figure 4: Gel mobility and Southwestern assays for effect of methylation on binding of H1 to the vitellogenin promoter. A, band shift assay with different proportions of competitor DNA. 25 ng of chick H1 was incubated with the vitellogenin promoter probe (see ``Experimental Procedures''), along with 0-100 ng of competitor DNA. Samples were resolved in 5% polyacrylamide gels. M, methylated probe; NM, unmethylated probe. Amounts of competitor E. coli DNA are shown above the lanes. Lanes 11 and 12 show the free probes without histone, ran in the same gel. B, Southwestern assay. Aliquots of chick H1 were run in polyacrylamide gels, transferred onto nitrocellulose membranes, and incubated with end-labeled vitellogenin promoter probe in the presence of 2.5 µg/ml of competitor DNA. Lanes 1-3, filter hybridized with methylated probe; lanes 4-6, unmethylated probe. Lanes 2 and 5, 300 ng of H1; lanes 3 and 6, 1 µg of H1. Control lanes 1 and 4 correspond to a fusion protein containing the first 392 amino acids of MeCP2 which binds to DNA methylated at CpG(31) .
The behavior of H1 with the vitellogenin probes was also studied in Southwestern assays (Fig. 4B). Different amounts of chick H1 were run in polyacrylamide gels and transferred onto nitrocellulose filters. As a control, recombinant MeCP2, a protein which shows a strong preference for binding to methylated DNA, was also included (31) . Equal counts/minute of end-labeled vitellogenin probes in either the methylated (left) or unmethylated (right) state were incubated with identical filters in the presence of competitor E. coli DNA. The two probes gave the same signal with H1 (compare lanes 3 and 6, for 1 µg of H1), indicating that the affinity of H1 for methylated and unmethylated DNA is the same. MeCP2, on the other hand, showed a much higher binding to the methylated oligonucleotide (see lanes 1 and 4).
Figure 5:
Digestion of histone H1DNA complexes
with restriction endonucleases. Methylated (left) or
unmethylated (right) pAdomal plasmids, linearized with SacI, were mixed with chick histone H1 at different H1:DNA
ratios (0-4 µg/µg DNA), as indicated on the top of the
lanes. H1
DNA complexes were formed for 150 min on ice and then
digested with MspI (100 units/µg DNA, upper
panel), HaeIII (100 units/µg, center panel),
or Taq
I (1000 nits/µg, lower
panel). The purified restriction fragments were resolved in 1.5%
agarose gels, transferred onto a nylon membrane, and hybridized using
labeled linear plasmid as probe. The films also show the linear
plasmids untreated with restriction enzymes (uncut, lanes 1 and 9) and a 123-bp DNA ladder as size markers (lanes
8).
To test whether
methylated DNA complexes were protected against all restriction
enzymes, we challenged them with HaeIII, which cuts the
sequence GGCC. The pattern of restriction with HaeIII was the
same for methylated and unmethylated plasmids (Fig. 5, central panel) indicating that methylated DNA complexes were
not generally protected against nucleases. The site for HaeIII
does not contain the methylated sequence CpG. We therefore tested the
sensitivity of the complexes to TaqI, which
cuts the sequence TCGA and is not blocked by CpG
methylation(36) . Surprisingly, the restriction patterns for
methylated and unmethylated pAdomal complexes were once more identical (Fig. 5, lower panel). At a ratio of 0.5 µg
H1/µg DNA cleavage was complete; at 0.75-1 µg H1/µg
DNA cleavage was partial; and at 2 µg H1/µg DNA cleavage of
both methylated and unmethylated DNAs was blocked. Thus CpG methylation
appears to be irrelevant to the Taq
I
sensitivity of H1
DNA complexes, implying that H1 does not
dramatically alter the conformation of methylated CpGs.
The above
results suggest that the protection of MspI sites is not due
to generalized inaccessibility of methylated CpGs in the complexes, but
to magnification of a pre-existing sensitivity of MspI to the
presence of mCpG within its recognition site. According to
this view, the preferential protection of methylated MspI
sites in the complexes is due to MspI itself and does not
reflect conformational inaccessibility of methylated DNA. In support of
this, we found that MspI digests naked DNA more slowly when it
is methylated (Fig. 6), in agreement with Butkus et al.(37) . For instance, an MspI concentration of 10
units/µg of DNA was enough to digest completely the unmethylated
plasmid, while the methylated DNA was only partially cleaved at this
enzyme concentration and required 100 units/µg to be completely
cut. Thus MspI, while able to cut methylated DNA, is inhibited
about 2-fold by CpG methylation, and this may contribute to the
inhibition of cleavage seen in complexes between methylated DNA and H1.
Figure 6:
MspI activity on methylated and
unmethylated naked DNA. Methylated and unmethylated pAdomal plasmids,
linearized with EcoRI, were cleaved with increasing amounts of MspI in the absence of histone, in 100 mM NaCl, 10
mM MgCl, 1 mM dithiothreitol, 10 mM Tris-HCl buffer, pH 7.5, at 37 °C for 75 min. The restriction
fragments were purified and analyzed on a 1.5% agarose gel, transferred
onto a membrane, and hybridized with labeled plasmid. Lanes
2-7 correspond to the methylated plasmid, and lanes
9-14 to unmethylated DNA. The concentration of MspI
increases from left to right as shown above the lanes (in units/µg
DNA). Lanes 1, 8, and 15 show DNA size
markers (123-bp ladder).
Higurashi and Cole (23) reported that H1 protects a subset of methylated MspI sites from cleavage. Our nuclease protection data did not show evidence for selective protection, as all sites were resistant to MspI at high H1 concentrations. To investigate this question more fully, we carried out H1 footprinting assays of methylated and unmethylated DNA. The pAdomal plasmids were labeled at one terminus, complexed with increasing amounts of H1, and treated with a constant amount of MspI. Increasing the amount of H1 conferred increasing resistance to MspI (Fig. 7), and this effect was stronger for methylated DNA as expected from the data in Fig. 6. However, when the bands resulting from partial digestion in the methylated and unmethylated lanes were compared, they were found to be the same (Fig. 7). Thus no site seemed to be specifically protected in the complexes with H1.
Figure 7: H1 footprinting of MspI sites. Methylated and unmethylated pAdomal plasmids were made linear with EcoRI and end-labeled. The plasmids were mixed with histone H1 in various ratios (as indicated in µg H1/µg DNA above lanes), and the complexes digested with MspI. The fragments were resolved in 1.5% agarose gels, and the gels were dried and exposed. Lanes 1-7, methylated plasmid; lanes 9-15, unmethylated DNA; lane 8, DNA size markers (123-bp ladder). Lanes 1 and 9 show the end-labeled probes, untreated with MspI. Each band corresponds to one MspI site.
To test if both plasmids were assembled into chromatin, the samples incubated with the extract were treated with nuclease from Micrococcus, and the resultant DNA fragments resolved in agarose gels and visualized by Southern analysis (Fig. 8A). Two different probes were used for this analysis: the insert of pHsr, as a probe specific for this plasmid (Fig. 8A), and linear pUC18, which gives a much stronger signal for pUC18 than for pHsr (data not shown). The results showed that both plasmids were efficiently assembled into chromatin whether methylated or not, as limited digestion with the nuclease gave an extensive ladder of fragments with a spacing of about 160 base pairs. The nuclease fragments gave the same pattern with both probes; no differences in degree of digestion or in spacing between the two plasmids were observed. This result demonstrated that both plasmids were properly organized into long stretches of regularly spaced nucleosomes. Thus, any cooperative binding of H1 to adjacent nucleosomes (perhaps enhanced by methylation) should occur in this system.
Figure 8: The incorporation of histone H1 into polynucleosomal chromatin is not affected by DNA methylation. Samples containing equal amounts of the plasmids pHsr11.9 (14.6 kb) and pUC18 (2.7 kb) were organized into chromatin using a Xenopus oocyte extract. One of the plasmids in each sample had been methylated in the CpGs with SssI methylase, the other was unmodified. The desired amount of radiolabeled rat histone H1 (0.2-1 µg H1/µg DNA) was mixed with the oocyte extract and supplemented with an ATP regenerating system, before its addition to the DNA. The assembly reaction proceed overnight at 27 °C. Part of each sample was then digested with Micrococcus nuclease, and part was loaded onto sucrose gradients. A, Micrococcus nuclease pattern of assembled chromatin. Chromatin samples were digested with 100 units/ml of Micrococcus nucleus for 0-30 min as indicated, and the reactions stopped with 15 mM EGTA, 0.5% SLS. The purified DNA was resolved in 1.5% agarose gels and transferred to membranes for Southern blot analysis. The representative patterns shown were obtained using the EcoRI insert of pHsr as probe. B, indicates samples before chromatin assembly, and M are DNA size markers. B, separation of the minichromosomes in sucrose gradients. The minichromosomes containing the pHsr11.9 or pUC18 plasmids were separated by centrifugation through sucrose gradients. DNA was purified from aliquots of the gradient fractions, resolved in 1% agarose gels and detected by Southern analysis. The film corresponds to one of the samples (closed circles in C) probed with pUC, which hybridizes with both plasmids. Using the EcoRI insert of pHsr as probe, only the longer plasmid gave signal (not shown). The amount of each plasmid was the same, although as the probe hybridizes only with the ``vector'' portion of pHsr (2.7 kb out of 14.6), the intensity of the signal is lower for pHsr. M stands for DNA size markers. C, distribution of H1 in the sucrose gradients. The content of labeled H1 in the gradient fractions was determined by Cherenkov counting. For each gradient, only one of the plasmids was methylated. Thus, either pHsr was methylated and pUC was non-methylated (open circles) or vice versa (closed circles). These profiles were obtained for 0.5 µg of added H1/µg DNA. D, as in C, but for a double amount of added H1 (1 µg H1/µg DNA).
Sucrose gradient centrifugation separated the fast sedimenting pHsr minichromosomes (14.6 kb) from the smaller pUC minichromosomes (2.7 kb), as shown by probing a blot of the DNA from the fractions resolved by agarose gel electrophoresis with plasmid-specific DNA sequences (Fig. 8B). Using pUC18 as probe, both plasmids were revealed since this probe also hybridizes with the vector part of pHsr although the signal from this plasmid is weaker. When the 11.9-kb insert of pHsr was utilized as probe, only the bigger plasmid was seen (data not shown). Most clearly for pUC, a faster migrating band of supercoiled (form I) DNA and a slower band of open circle (form II) are seen. We think that a certain amount of nicking occurs during the purification of the DNA from the fractions, particularly for the longer pHsr, giving rise to open circular forms.
The distribution of added histone H1 along the gradients was monitored by Cherenkov counting of the fractions. It is apparent from Fig. 8C that the profile of radioactivity did not depend on which plasmid was methylated. Two peaks of radioactivity were observed, corresponding to those fractions containing either pHsr or pUC minichromosomes (as seen by the DNA analysis of Fig. 8B). Analysis of the proteins in each peak by trichloroacetic acid precipitation and electrophoresis in TAU gels confirmed that all the radioactivity was due to histone H1 (data not shown). It is clear that the profiles for both combinations of plasmids can be superimposed. For instance, the amount of histone H1 incorporated into pHsr minichromosomes (left peak) was the same whether that plasmid is methylated (open circles) or non-methylated (closed circles). The same is true for the pUC plasmid (in this case the open circles indicating the non-methylated form). This demonstrated that the binding of the linker histone to chromatin is unaffected by methylation. Although the level of radioactivity was higher for the slower peak, this was seen for both samples and therefore cannot be attributed to methylation. This difference probably arose from some non-incorporated H1 trailing from the top of the gradient.
A possible trivial explanation for the absence of a methylation effect on H1 binding is that the concentrations of H1 were sufficiently high to saturate both minichromosomes. This seemed unlikely as, although the amount of added H1 was 0.5 µg/µg of DNA, which could give about 2.5 H1 molecules/nucleosome, most H1 did not get incorporated into chromatin and remained at the top of the gradients (Fig. 8C and data not shown). We suspect that H1 is sequestered by binding to a component of the extract, perhaps RNA ( (40) and data not shown). Thus the amount of incorporated H1 was a minority of that added, and the proportion of H1 in the minichromosomes was only of about 1 molecule each 8-10 nucleosomes. To rule out the possibility that both minichromosomes were saturated with H1, the amount of added H1 was doubled (Fig. 8D). This had the effect of doubling the amount of H1 incorporated into both minichromosomes, thereby confirming that H1 incorporation was not at saturating levels. Once again the affinity of H1 for methylated chromatin was the same as for unmethylated chromatin, as had been observed for naked DNA.
Another possible explanation for the results is that methylation is either lost or gained from the plasmids during incubation with the extract. Previous studies have shown that this does not happen in intact Xenopus oocytes, from which these extracts are derived(38, 39) . To check this, we isolated DNA from assembled minichromosomes and treated with the methylation-sensitive enzyme HpaII. The methylated plasmid was still resistant, and the unmethylated plasmid was still sensitive to this enzyme (data not shown). Thus no change in methylation had occurred during chromatin assembly.
Previous studies have
given conflicting impressions about the affinity of H1 for methylated
DNA. Southwestern assays of mouse and rat nuclear proteins failed to
detect differences in the affinities of methylated and non-methylated
probes for H1(28) . Higurashi and Cole (23) found
slightly enhanced binding of H1 to non-methylated DNA but considered
the magnitude of this preference too small to be significant. Our
filter binding assays showed preferential binding to methylated DNA,
but once again the effect was very small (1.2-fold). On the other hand,
Levine et al.(24) found that methylated DNA could
successfully disrupt a pre-existing H1DNA complex, whereas
non-methylated DNA could not. The failure of excess non-methylated DNA
to remove H1 is in fact more surprising than the success of methylated
DNA. This result would not be expected of soluble H1
DNA
complexes, but would be expected if the preformed complexes had
precipitated, or if H1 was in excess over DNA. Since complexes were
assayed only by filter binding, the presence of precipitates may have
gone undetected in the study. In view of the known tendency of
H1
DNA complexes to precipitate, and the disagreement with present
data and data of Higurashi and Cole(23) , it is important to
test this possibility.
The activity of MDBP-2, a protein that shows preferential binding to a methylated site in the promoter of the vitellogenin gene, has been attributed to the histone H1 fraction of chicken liver(25) . Our results show that purified histone H1 from chicken erythrocytes does not bind preferentially to the vitellogenin promoter when it is methylated. Thus, we conclude that erythrocytes lack the MDBP2-H1 form that was detected by others.