(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 -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.
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 (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.
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 (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.
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 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.
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.