(Received for publication, November 28, 1994; and in revised form, February 14, 1995)
From the
We have previously shown that in developing chicken embryos and
differentiating mouse myoblasts, the demethylation of 5-metCpGs occurs
through the replacement of 5-methylcytosine by cytosine (Jost, J.
P.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4685-4688;
Jost, J. P. & Jost, Y. C. (1994) J. Biol. Chem. 269,
10040-10043). We have now purified over 30,000-fold a
5-methylcytosine-DNA glycosylase from 12-day-old chicken embryos. The
enzyme copurifies with a mismatch-specific thymine-DNA glycosylase and
an apyrimidic-endonuclease. The reaction product of the highly purified
5-methylcytosine-DNA glycosylase is 5-methylcytosine. The copurified
apyrimidic-endonuclease activity cleaves 3` from the apyrimidic sugar.
A 52.5-kDa peptide, isolated as a single band from preparative
SDS-polyacrylamide gels, has both the 5-methylcytosine-DNA glycosylase
and the mismatch-specific thymine-DNA glycosylase activities.
5-Methylcytosine-DNA glycosylase has an apparent pI of 5.5-7.5
and maximal activity between pH 6.5 and 7.5. The K for hemimethylated oligonucleotide substrate is 8
10
M with a V
of 4
10
mol/h/µg protein.
5-Methylcytosine-DNA glycosylase binds equally well to methylated and
non-methylated DNA. The enzyme reacts six times faster with the
hemimethylated DNA than with the same bifilarly methylated DNA
sequence, and single-stranded methylated DNA is not a substrate. The
action of the enzyme is distributive.
Through DNA methylation, it is possible to change the
information content of DNA that can affect differentiation and
development(1, 2, 3) . The specific DNA
methylation pattern results from the combination of maintenance/de
novo methylation, demodification of the methylated CpGs, by
sequence-specific DNA binding proteins (transcription
factors)(1, 2, 3) , and possibly by
cis-acting DNA elements (4, 5) . Targeted disruption
of the DNA methyltransferase gene in mice has given strong evidence for
the pivotal role of DNA methylation during embryonic
development(6) . Active enzymatic demodification of methylated
CpGs may be operational in several systems and may also play a crucial
role during differentiation and development ( (7) and
references therein). Recently, we have shown that a nuclear cell-free
system from chicken embryos can promote the active demethylation of DNA (8) . One enzyme, coined 5-methyl-CpG endonuclease, has no
sequence specificity and cleaves only the methylated CpGs. A similar
enzymatic activity was also observed in differentiating mouse myoblasts (7) and in preimplantation and postimplantation mouse
embryos. More recently, Vairapandi and Duker (9) have identified in HeLa cell lysate a 5-methylcytosine-DNA
glycosylase, which may be also involved in the active demodification of
DNA.
While purifying the 5-methyl-CpG endonuclease, we discovered
that this enzymatic activity is a combination of 5-methylcytosine-DNA
glycosylase and AP()-endonuclease. In addition,
5-methylcytosine-DNA glycosylase copurifies with a mismatch-specific
thymine-DNA glycosylase.
Bands in the gel were visualized by the
CuCl staining procedure, cut out, and
destained(12) . Proteins were extracted and denatured by the
guanidium hydrochloride procedure as described by Hager and
Burges(13) . The relative amount of protein per band was
determined by comparison with a silver-stained dilution series of
specific protein standards.
Liquid-phase isoelectrofocusing was carried out in a mini-Rotofor cell (Bio-Rad) for 4 h at 4 °C at 8 watts(14) . After electrofocusing, 20 fractions were collected, and the pH was measured. Samples were dialyzed for 3 h against 500 ml of 20 mM Hepes, pH 7.5, 10 mM EDTA, 100 mM NaCl, 1 mM benzamidine, 1 mM DTT, and 10% glycerol (v/v). Activity of 5-methylcytosine-DNA glycosylase was determined as outlined below.
For UV-cross-linking experiments, oligonucleotide D had in the vicinity of the mCpG on the opposite strand two bromodeoxyuridine and one adenosine labeled at high specific activity. Upon formation of the complex, the reaction mixture was separated by gel shift in the dark. Band ``a'' was UV irradiated for 10 min with a transilluminator, and upon extraction and DNase I treatment, the residual protein-DNA complex was separated on a 10% SDS-polyacrylamide gel.
Figure 1: Analysis of the reaction product (20% urea polyacrylamide sequencing gel) of the 5-methylcytosine-DNA glycosylase (assay with oligonucleotide A) and the mismatch-specific thymine-DNA glycosylase (assay with oligonucleotide G) purified on Mono-S (upper panel) and from the 52.5-kDa peptide eluted from 10% SDS-polyacrylamide preparative gel (middle panel). The lowerpanel shows the cleavage product obtained with a fraction from Mono-S, incubated with the oligonucleotide F (Table 1), containing on the same strand a G/T mismatch and a methylated CpG (methylcytosine (mC) + G/T). Cleavage at the G/T mismatch and 5 met-C gives, respectively, 26- and 29-base-long-labeled oligonucleotides. G/T of the lowerpanel is the incubation of the same enzyme preparation with the oligonucleotide G (Table 1) labeled on the upperstrand (strand containing the mismatched G). For lanes1 and 5, the reaction product was heated for 30 min at 90 °C in Hepes-HCl, pH 6.6, whereas for lanes2 and 6, the reaction product was incubated for 30 min at 90 °C in 0.1 M NaOH. Lanes3 and 4 are the corresponding controls to 1 and 2 incubated without protein, and lanes7 and 8 are the controls of lanes5 a nd 6 incubated without protein.
Figure 5: Kinetics of the cleavage reaction with hemimethylated oligonucleotide A and the bifilarly methylated oligonucleotide B. Fullcircles show the kinetics of the reaction at a specific CpG site in the presence of 50 µg of bovine serum albumin and 2 µg of protein fraction eluted from the Mono-S column. Fullsquares show the same reaction mixture incubated in the absence of bovine serum albumin. The opensquares and opencircles are the product of reaction obtained with bifilarly methylated DNA in the presence of 2 µg of the same enzyme preparation and 50 µg of bovine serum albumin per 50 µl of reaction mixture. The opensquares represent the cleavage on the upperstrand, and the opencircles are the reaction on the lowerstrand. The reaction product was denatured and separated on a 20% urea-polyacrylamide DNA sequencing gel. Upon autoradiography, bands were cut out and measured for radioactivity. Values of the corresponding blanks were subtracted from the test values. Each point represents the average of two independent tests.
Figure 3: 5-Methylcytosine-DNA glycosylase binds to hemimethylated and non-methylated DNA. PanelA, gel mobility shift assay on 5% native polyacrylamide gel of post-affinity chromatography purified 5-methylcytosine-DNA glycosylase. Lane1 is the reaction with the hemimethylated oligonucleotide A, and lane2 is the reaction with the same non-methylated oligonucleotide. a and b are the protein-DNA complexes, and c is the free unbound DNA. PanelB, 5-methylcytosine-DNA glycosylase activity recovered from the complexes a and b and free DNA (c) from hemimethylated DNA (1 on figure) and non-methylated DNA (2 on figure). S are the size standards. The product of reaction was separated on a 20% urea-polyacrylamide denaturation gel. PanelC, UV cross-linking of protein-DNA complexes formed in band a from hemimethylated oligonucleotide D (1a) and from the same non-methylated oligonucleotide (2a). 1c is a control of hemimethylated DNA incubated without protein. The reaction product was analyzed on a 10% SDS-polyacrylamide gel. bp, base pairs.
Figure 2: Polypeptide profiles (on 10% SDS-polyacrylamide gel) at different stages of purification of 5-methylcytosine-DNA glycosylase. 3 µg of protein (for lanes1-4) were separated and silver stained. Lane1 is the crude nuclei extract, lane2 is the fraction post-heparin-Sepharose, lane3 is the post-DEAE-CM-Sepharose fraction, lane4 is the post-Mono-S chromatography fraction, and lane5 is the 5-methylcytosine-DNA glycosylase eluted from a preparative SDS-polyacrylamide gel. The molecular mass markers (Bio-Rad) are phosphorylase b, 97 kDa; bovine serum albumin, 69 kDa; ovalbumin, 46 kDa; and carbonic anhydrase, 30 kDa. On the rightside, we show the activity of 5-methylcytosine-DNA glycosylase (mC) and mismatch-specific thymine-DNA glycosylase (G/T) recovered from single peptide bands. Enzyme assays were carried out as outlined under ``Materials and Methods.'' The arrows represent the band(s) that were excised, extracted, and tested for enzymatic activities
Figure 4: 5-Methylcytosine-DNA glycosylase reacts preferentially with hemimethylated DNA (lane 1 with corresponding control in lane 2) and very poorly with single-stranded methylated DNA (lane 3) or with bifilarly methylated DNA (lane 4). Lane5 is the control of lane4, incubated without protein. For hemimethylated DNA (oligonucleotide A) and bifilarly methylated DNA (oligonucleotide B), both strands were labeled at the 5`-ends with the kinase reaction. The expected cleavage product for the upper and lower strands are 21 and 28 bases, respectively. For this experiment, 2 µg of a post DEAE-CM-Sepharose fraction were incubated in the presence of bovine serum albumin. The cleavage product was analyzed on a 20% urea-polyacrylamide DNA sequencing gel. In laneS, the size standards in base pairs are shown. bp, base pairs.
Figure 6:
Determination of the Kand V
for the
hemimethylated DNA substrate A incubated with purified
5-methylcytosine-DNA glycosylase (fraction post-CM-Sepharose). Each 50
µl of incubation mixture (in duplicates) received 2 µg of
protein and increasing concentrations of end-labeled oligonucleotide A.
After 1 h of incubation at 37 °C, samples were analyzed on a 20%
urea-polyacrylamide DNA sequencing gel. Upon autoradiography, bands
were cut out and counted for radioactivity. Corresponding controls,
with the DNA substrate only, were also incubated, and an area
corresponding to the protein-DNA complex of the test was cut out,
counted, and subtracted from the test values. Results are expressed as
a Lineweaver-Burk plot.
In vivo modification/demodification of DNA has been shown to start at discrete positions in the promoter region and to proceed bidirectionally(3) , suggesting, in the case of active demethylation, that the enzymes may be processive. To test this hypothesis, a 69-base pair-long oligonucleotide containing 5 mCpGs was synthesized (Table 1, oligonucleotide H), and the end-labeled DNA was incubated for various times with purified 5-methylcytosine-DNA glycosylase (post-CM-Sepharose chromatography). The results of Fig. 7show the reaction product obtained after 15, 30, 45, and 60 min of incubation. Even for the shortest incubation time, there is no sign of processivity of the enzyme for the five different mCpGs. However, some sites seem to be a better substrate for the enzyme than others (Fig. 7, compare mCpGs at positions 21 and 28). The differences observed were not due to the extent of DNA methylation (data not shown).
Figure 7: 5-Methylcytosine-DNA glycosylase is distributive. Oligonucleotide H (Table 1) was end-labeled and incubated with 2 µg of post-CM-Sepharose fraction. Incubation was carried out at 37 °C of 15, 30, and 45 min, respectively (lanes1-3). A control incubated without protein for 45 min is shown in lane4. S values are the size standards. Samples were processed and analyzed on a 20% urea-polyacrylamide DNA sequencing gel, as indicated under ``Materials and Methods.''
Figure 8:
Reaction product of 5-methylcytosine-DNA
glycosylase analyzed by TLC. 2 µg of enzyme preparation eluted from
an affinity matrix were incubated with H-labeled
(5-methylcytosine) hemimethylated DNA for 1 h at 37 °C, and the
reaction product was separated as indicated under ``Materials and
Methods.'' Each bar diagram represents the
average of three separate analyses. bp, base
pairs.
Figure 9: Nature of the base present 3` from the apyrimidic deoxyribose (at the CpG site). Hemimethylated DNA was incubated under the same conditions as in Fig. 8, and the resulting cleavage product was analyzed by TLC as described under ``Materials and Methods.'' Each bar represents the average of four different determinations.
If we compare the present results with our previously published data(8) , there is an apparent contradiction. While using crude nuclear extracts from chicken embryos, we could show a specific cleavage at the mCpGs, but we could not demonstrate the presence of a specific 5-methylcytosine-DNA glycosylase. Now, upon purification of the mCpG-cleaving activity, we can clearly see the presence of a specific 5-methylcytosine-DNA glycosylase. The reason for this discrepancy, between our previous and actual results, is that the incubation of the labeled substrate with crude nuclear extracts had to be carried out for several hours at high concentrations of proteins, where the released 5-methylcytosine was immediately converted into thymine by a 5-methylcytosine deaminase. As far as we can judge, there is very little to no 5-methylcytosine deaminase activity in our highly purified 5-methylcytosine-DNA glycosylase preparations. The presence of very active 5-methylcytosine deaminase in vertebrate cells has already been reported recently by Vairapandi and Duker (9) who found in HeLa cells a very rapid conversion of 5-methylcytosine into thymine. We also reported (8) that the reaction product after lengthy incubations with crude nuclei extracts was not sensitive to alkaline hydrolysis, presumably because the apyrimidic sugar had been removed during the incubation. In contrast, we now show that the reaction product from an incubation mixture containing the highly purified 5-methylcytosine-DNA glycosylase is 5-methylcytosine, and the DNA is sensitive to the alkaline hydrolysis, indicating that the apyrimidic deoxyribose is still present in the DNA.
However, there is still a major difference between our results and those reported by Vairapandi and Duker(9) . The 5-methylcytosine-glycosylase purified from chicken embryos cleaved preferentially hemimethylated DNA, whereas Vairapandi and Duker (9) found just the opposite. In their case, bifilarly methylated DNA is clearly a better substrate than hemimethylated DNA. The reasons for this difference are, at present, not known. Like the DNA-methyltransferase, the purified 5-methylcytosine-DNA glycosylase reacts much faster with hemimethylated DNA than with the non-methylated or fully methylated DNA, respectively, suggesting that the two enzymes may compete for the same substrate during replication. In this case, the mole ratio of the two enzymes combined with their respective binding affinities to DNA may decide which of the two enzymes will react with the newly replicated DNA, i.e. methylation or demethylation. It is interesting to note that prior to the genome-wide demethylation occurring in some differentiating systems, there is an abrupt drop in the methyltransferase activity(7, 17) . In this case, it is possible that the drop in DNA methyltransferase activity prevents the maintenance methylation of the newly replicated DNA, which then becomes the substrate for the 5-methylcytosine-DNA glycosylase. This way it may be possible to demethylate completely a specific site on DNA after just one round of replication. The site specificity of the reaction may be directed by specific transcription factors (18) and/or cis-acting elements(4, 5) .
Our most surprising result is the copurification of 5-methylcytosine-DNA glycosylase with the mismatch-specific thymine-DNA glycosylase. At present, it is not possible to decide whether a single peptide has the two enzymatic activities or whether two very close related peptides with different enzymatic activities copurify throughout the purification scheme. As we already mentioned under ``Results,'' we also observed on SDS-polyacrylamide gels that some preparations of the highly purified 5-methylcytosine-DNA glycosylase had minor bands migrating with an apparent molecular mass of 50 kDa. These minor bands had also the same enzyme activity ratio of 5-methylcytosine-DNA glycosylase and mismatch-specific thymine-DNA glycosylase and could represent either a degradation product of the 52.5-kDa peptide or covalent modified forms of the larger peptide. Clearly, we will have to wait until we have microsequenced these peptides and cloned the cDNAs to decide whether we are dealing with one peptide having the two activities or whether we have two separate enzymes.