Delayed methylation and the matrix bound DNA methylase

Vol. 126, No. 2, 1985

January 3 1, 1985

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 678-684

DELAYED MRTHYLATION AND THE MATRIX BOUND DNA MRTHYLASE

Terence Davis, David Kirk, Angela Rinaldi, Roy II. Burdon, and Roger L.P. Adams

Department of Biochemistry, University of Glasgow, Glasgow G12 SQQ U.K.

Received November 20, 1984

It is shown that the methylation of DNA that occurs in isolated nuclei is "delayed methylation". This methylation is not reduced in nuclei which have been pretreated with 0.2M NaCl to extract the soluble methylase suggesting that this methylation is the product of a firmly bound matrix associated DNA methylase. Evidence is provided that, like the methylase, the DNA substrate is associated with the nuclear matrix. 0 1985 Academic Press, Inc.

DNA methylation in eukaryotes involves the transfer of a methyl group

from S-adenosyl methionine to certain cytosine bases in DNA (1,2). This

reaction is catalysed by a specific DNA methylase, a number of examples of

which have been characterised (see 3). The bulk of DNA methylation occurs

rapidly after DNA synthesis (4-6) but a significant number of methyl groups

continue to be added to DNA for several hours after its synthesis (7-9).

This latter methylation is termed "delayed methylation". There is now

evidence for the presence of DNA methylase activity in two fractions in cell

homogenates; a low salt (<0.4M) extractable DNA methylase and a firmly

bound DNA methylase thought to be associated with the nuclear matrix

(10,111. it is not clear at present which of these activities is

responsible for either the delayed or the rapid methylation of DNA. This

manuscript presents evidence that the delayed methylation is a product of

the firmly bound form of the DNA methylase.

MATERIALS AND METHODS

Determination of delayed methylation: L929 cells in log phase were cultured in the presence of 5OhCi of

[6-3H]uridine (Amersham) for either 48 hours or 50 minutes and harvested while still in log phase. Another batch were labelled and harvested later when they had attained stationary phase. Nuclei were prepared by homogenisation of cells in 1% (v/v) Tween 80 (Sigma). DNA, purified as

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Vol. 126, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

described previously (121, was dissolved in 98% formic acid and hydrolysed at 170°C for l-l/2 hours. The excess formic acid was evaporated and the residue dissolved in 2OmM ammonium carbonate (pH1O.O) and the bases separated on an Aminex A6 HPLC column as described previously (12). The base8 cytosine and methylcytosine (mC) were collected and the proportion of cytosines methylated was determined. Nuclear Methylation

L929 cells were prelabelled with deoxy-[U-14C]cytidine (2nCi per ml> for three days prior to harvesting. In some cases 3mM hydroxyurea was present for the final hour. Nuclei were prepared and extracted separately with buffer M(tris-HCl pH7.8, 50mM; EDTA, 1mM; DTT, 1mM; glycerol, 10%) containing NaCl at concentrations varying from 0 to 0.35 Molar. The nuclei were then washed twice in buffer M and incubated with 5/.lCi of S-adenosyl-L-[methyl-3H]methionine (1.5Ci/mmole, Amersham) for 2 hour8 at 37oc. The DNA was purified and the 3H/14C ratio determined. Isolation of matrix DNA:

Following incubation nuclei were resuspended in buffer M + 2.OM NaCl and centrifuged at 165,000xg for 48 hours. The pelleted material was resuspended in buffer M + 5mM CaC12 and digested using micrococcal nuclease (Boehringer) at 1000 units per ml for 1 hour at 37OC. Sample8 were taken at each stage of the procedure and the 3H/14C ratio of the DNA was determined. Use of 5-azadeoxycytidine:

L929 cells were released from stationary nhase and cultured for 10 hours at 37OC. Then 5-azadeoxycytidine was-added to a final concentration of l.Oc(M and the cells grown for a further 10 hours at 37OC (11). By this means cells were produced containing hemimethylated DNA and only low levels of 'soluble' DNA methylase (11,13). The cells were harvested and nuclei prepared. These nuclei and control nuclei were methylated as before except that in some instances 40 units of a partially purified ascites DNA methylase (14) was added to the incubation. The DNA was purified and the amount of methyl groups incorporated was determined. DNA was assayed by the method of Burton (15).

RESULTS

Delayed methylation in vivo

Table 1 shows that DNA synthesised by mouse L929 cells during a 50

minute labelling period prior to harvesting is undermethylated by about 30%

Table 1 Extent of DNA methylation in mouse L929 cells

Incubation Percent mC expressed as Conditions bnc x 100)/(mC+c)

A. log phasecells 2.61, 2.85 50 minute label

B. log phase cells 3.88, 4.12 48 hour label

C. stationary phase cells 4.06, 3.70 (48 hour label)

A h B: Cells were labelled with [6-3H] uridine for the indicated times and harvested during log phase. C was labelled for 48 hours, the medium changed and the cells harvested later at stationary phase. DNA was purified from these cells and the methylcytosine (mC) content was determined. Duplicate results are presented.

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compared with DNA labelled for 48 hours. The conclusion is that a

considerable amount of methylation occurs more than 50 minutes after DNA

synthesis. This finding confirms previous results demonstrating the

occurrence of delayed methylation (7-9). For 1 to 2 minutes after

synthesis DNA is not methylated at all but this DNA represents less than 4%

of the DNA synthesised during a 50 minute labelling period and hence its

presence is insufficient to explain the lower level of methylation seen in

the present experiment. In contract to the findings of several authors

(16,171 we find no difference between the level of DNA methylation in log

phase cells and stationary phase cells when both are labelled for 48 hours

(table 1).

Delayed methylation in isolated nuclei

It has been shown previously that methylation in isolated nuclei occurs

predominantly on DNA more than 10 minutes old (50% is on DNA more than 4.25

hours old) i.e. it is delayed methylation (18). This was confirmed by

studying the effect of treatment of the cells with hydroxyurea for one hour

prior to nuclear isolation. Despite the fact that in this case DNA

synthesis has been almost completely inhibited, the methylation occurring on

DNA in nuclei from both hydroxyurea treated cells and control cells is the

same (figure 1). There is thus no distinction between total and delayed

methylation in this system (i.e. we are not looking at the rapid methylation

of nascent DNA).

Mouse cells contain two DNA methylase activities: one extractable at

low salt concentrations, and the other is thought to be a matrix bound form

of the readily extractable enzyme (10,ll). It is not known which activity

is responsible for DNA methylation in vivo, but the present communciation

reports experiments designed to discover which form of the enzyme is

responsible for the methylation which occurs in isolated nuclei. Nuclei

were prepared and extracted with increasing concentrations of NaCl in order

to remove the readily extractable DNA methylase. Figure 1 shows that

extracting nuclei with 0.35M NaCl prior to methylation results in, at most,

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0.4

0.3 0 \

G

!

#- .

= 2 0.2

7 NT

0.1

OI 0.1 0.2 0.3 0.4

LNaCll (Molar)

Figure 1 The effect of salt extraction on DNA methylation in isolated nuclei

Nuclei isolated from [U-L4C]deoxycytidine prelabelled L929 cells were extracted with buffer M containing NaCl at the indicated concentrations. These nuclei were then incubated with 13H] AdoMet (methods) and the 3H/14C ratio of the DNA determined. Open circles: cells treated with hydroxyurea for lh before nuclear isolation. Closed circles: control cells.

a 20% decrease in methylation compared with unextracted nuclei. As most of

the DNA methylase is extracted by 0.2M NaCl (11,14) the majority (>80%) of

the methylation in these nuclei is a product of the so-called matrix bound

DNA methylase.

Further evidence that the bound methylase is responsible for DNA

methylation comes from the use of 5-azadeoxycytidine (table 2). When cells

are treated with this drug, in vivo DNA methylation is inhibited by more

than 80% (19,201. Associated with this inhibition is a small increase in

the amount of bound DNA methylase and a dramatic decrease in the amount of

soluble DNA methylase (11,131.

Table 2 Effect of Pretreatment with Azadeoxycytidine

Sample p mole methyl groups incorporatedlpg DNA

endogenous methylase

with added methylase

control nuclei

nuclei from cells pretreated with 5-azadeoxycytidine

0.17, 0.18 0.21, 0.23

0.20, 0.25 1.00, 1.05

Methylation of DNA in nuclei from control L929 cells or from cells pretreated with 5-azadeoxycytidine for 10 hours. Duplicate results are presented (see methods for details).

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Table 3 Preferential methylation of matrix associated DNA

DNA methylation in isolated nuclei (3R/14C)

Control Extracted

Total DNA 0.11 0.10 Matrix associated DNA 0.22 (14%) 0.38 (7%)

Nuclei prepared from [U-14C]deoxycytidine prelabelled L929 cells were incubated with [3H1 AdoMet for 2h. Incorporation into total DNA and matrix associated DNA was measured (see methods section). Results are expressed as the tritium to 14C ratio. In the extracted nuclei the readily solubilised DNA methylase has been removed by extraction with 0.2M NaCl prior to in vitro methylation. The figures in parentheses represent the percentage of the total DNA ( 14C radioactivity) recovered in the matrix associated DNA fraction.

When nuclei from 5-azadeoxycytidine treated cells are incubated with

S-adenosylmethionine little difference in DNA methylation is observed

compared with nuclei from untreated control cells despite the marked

reduction in the amount of soluble enzyme present (table 2). Yet the DNA

in nuclei from treated cells is a very good acceptor of methyl groups when a

partially purified mouse DNA methylase is added to the incubation. The

implication here is that in addition to the normal delayed methylation there

are a considerable number of additional hemimethylated sites present which

have arisen as a result of the drug induced deficiency in the amount of

readily solubilised DNA methylase.

The site of nuclear methylation

The only base methylated in these experiments is methylcytosine

(results not shown). In isolated nuclei the DNA which is preferentially

methylated is that which is resistant to nuclease treatment following

dehistonisation with 2M NaCl i.e. matrix associated DNA (table 3). The

preference is shown to an apparently greater extent by nuclei from which the

readily solubilised DNA methylase has been removed by prior extraction with

0.2M NaCl, but this may be due to there being less DNA in the matrix

fraction in this case.

DISCUSSION

These results show that the DNA methylation which occurs in Isolated

mouse cell nuclei corresponds to the delayed methylation which occurs in -

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vivo on DNA up to several hours following replication. The enzyme

responsible for this delayed methylation is the tightly bound or matrix

associated DNA methylase which is the only DNA methylase remaining following

extraction of nuclei with 0.2-0.35M NaCl. It is a reasonable extrapolation

to suggest that the matrix bound enzyme is also responsible for the delayed

methylation observed in intact cells.

Moreover, the DNA methylated in isolated nuclei is also preferentially

associated with tight binding proteins in the so-called nuclear-matrix

fraction. The very presence of DNA methylase tightly bound to a region of

DNA may well render that DNA resistant to nuclease action while at the same

time allowing the sedimentation of the enzyme along with the DNA following

treatment with 2M NaCl. Such are the characteristics of the nuclear matrix

though it is by no means clear that the DNA:protein complex studied in the

present and related experiments is the same as that involved in replication

or transcription. Thus the term nuclear matrix is used purely to describe

the fraction resulting from a certain experimental procedure rather than to

imply a functional nuclear entity.

Some of the proteins associated in vivo with the DNA:methylase complex

may interfere with the action of the enzyme thereby allowing hemfmethylated

sites to persist for several hours after replication of the DNA. It is

these sites which are filled when isolated nuclei are incubated with

S-adenosylmethionine and this reaction does not require the 'soluble' DNA

methylase. In contrast, the hemimethylated sites arising following

treatment of cells with azadeoxycytidine are only filled when additional

soluble DNA methylase is added to the isolated nuclei. We would suggest

that normally the 'soluble' DNA methylase becomes firmly associated with the

DNA and is released again only following methylation of the DNA. Most

methylation takes place very shortly after replication but some is delayed

causing a proportion of the enzyme to be recovered in the matrix bound

fraction. The presence of azacytosine in the DNA leads to permanent

inactivation of enzyme when it interacts with the foreign base (21). There

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is, however, little effect on the nuclear DNA methylation perhaps because

the matrix bound enzyme equilibrates only slowly with the 'soluble' enzyme.

REFERENCES

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Adams, R.L.P. and Burdon, R.H. (1982) CRC Crit. Rev. Biochem. 13, 347-384. Doerfler, W. (1983) Ann. Rev. Biochem. 52, 93-124. Adams, R.L.P. and Burdon, R.H. (1983) in 'Enzymes of Nucleic Acid Synthesis and Modification' vol.1 p.119-144 (Jacob. S.T. ed) CRC Press Inc., Boca Ratan. Burdon, R.H. and Adams, R.L.P. (1969) Biochim. Biophys. Acta 174, 322-329. Kappler, J. (1970) J. Cell Physiol. 15, 21-32. Gruenbaum, Y., Szyf, H., Cedar, H. and Razin, A. (1983) Proc. Natl. Acad. Sci. USA E, 4919-4921. Adams, R.L.P. (1981) Biochim. Biophys. Acta 254, 205-212. Woodcock, D.M., Adams, J.K. and Cooper, I.A. (1982) Biochim. Biophys. Acta 696, 15-22. GeraccD., Eremenko, T., Cocciara, A., Scarano, E. & Volpe, P. (1974) Biochem. Biophys. Res. Comm. 57, 353-358. Qureshi, M.A., Adams, R.L.P. and Burdon, R.H. (1982) Biochem. SOC.

Trans. lo, 455-456. Burdon, R.H., Qureshi, M., Adams, R.L.P. and Brooks, W. (1985) (paper submitted). Adams, R.L.P., McKay, E.L., Craig, L.M. and Burdon, R.H. (1979) Biochim. Biophys. Acta 563, 72-81. Tanaka, M., Hibasami, H., Nagai, J. and Ikeda, T. (1980) Aus. J. Exp. Biol. Med. Sci. 2, 391-396. Turnbull, J.F. and Adams, R.L.P. (1976) Nucleic Acids Res. 3, 677-695. Burton, K. (19561 Biochem. J. 62, 315-323. Rubery, E.D. and Newton, A.A. (1973) Biochim. Biophys. Acta 324, 24-36. Kunnath, L. and Locker, J. (1982) Biochim. Biophys. Acta 699, 264-271. Adams, R.L.P. and Hogarth, C. (1973) Biochim. Biophys. Acta 331, 214-220. Jones, P.A. and Taylor, S.M. (1980) Cell 20, 85-90. Jones, P.A. and Taylor, S.M. (1981) Nucleic Acids Res. 2, 2933-2947. Santi, D.V., Garrett, C.E. and Barr, P.J. (1983) Cell 2, 9-30.

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Delayed methylation and the matrix bound DNA methylase