Volume 309, auml~r 1, 9%102 FEB$114'/3 O 199~ Fedenttionof European Bioehemi~l SocietiesO014STO3/9~dSS,00
Aught 1992
Vector methylation inhibits transcription from the SV40 early promoter M. Bryans*, S. Ka,~, C. Scivwright and R.L.P. Adams Drpartmrt~t of Biochemistry, Ut~iversityof Gta#gon', ~Ttaxgow,GI 2 ~QQ, OK Received I July 1092 Math~latior~ of a plasmid ¢ontainittll th= SV40 promoter linked to the ¢hlommpheni¢ol a=tyl trant,l'¢ra= (CATJ I~¢n¢, with ¢ith,r marine DNA m~thyl=r~ or methyla~ ,.~sl r~ults in inhibition of the cxpr~ion of the reporter =¢nc after transl.:lion into cultured ¢=11s,Mathylation of th= plasmid with the methyla~s H/me and l t ~ l t has no =fleet on the expre,'~ion of this ilene. Prot,in-DNA int,r, tctions in the SV40 promoter arc not affected by the pr©ten~ of ntethylcytofit~ suili~stin8 that illaetivalion r=sults from the formation of an inactive ¢hromatin structure that is d¢~nd©nt on the high eG content of the plasmid, DNA m¢thylation; SV40 proinoten CpG island; gpl: Cltromatin structure
I. I N T R O D U C T I O N Inhibition of transcription has been documented for
a large number of genes containing methylcytosine in the promoter region. However, to date there is insu..m.cient evidence for a single medianism of inactivation. Methylation can interfere dir¢ctly with the transcriptional machinery by pr,venting the binding of transcription factors. For example, the major late transcription factor [1], the adenovirus F~ factor [Z3]. the cAMP r~ponsiv¢ element binding protein [4] and AP2 [5]. However 'methylation sensitive" transcription factors are still in a minority of those studied. Methylations, nsitive genes such as the adenovirus E2a gone [6,7] and the herpes simplex virus tk gene [8] bind transcription factors regardless of the mrthTlatioa state of their promoters. In such gen,s the effect of methylation may rosalt from the formation of inactive chromatin struttares that are inhibitory to efficient transcription as observed for the herpes simplex virus thymidine kintts¢ gen, [9,10]. A protein factor has recently been purified that binds to clust,rs of methylcytosines in promoter regions resulting in inhibition of transcription [11,12]. On transfection of cultured cells, it has been shown in some cases that mrthylated DNA assumes an inactive, DNase I insensitive, chromatin structure whereas. unmethylated D1NA forms DNase I sensitive chromatin characteristic ofexpressing genes [13]. In other cases the pre-~enee of tranacription factors appears to be able to
*Prexent address: 124i]Jam=s Cancer Hospital, Columbus, OH 43210. USA, Co~rezpandence address: R,L,P, Adam=, Department of Bio~hmad=it3,. University of Gla,gow, Glasgow, (312 8QQ, UK. Fax: (.at) (41)
~30 462O.
Pubflzhcd hy Elacvicr $dcne¢ Pubh'dlcr,~ B, V,
override inhibitory effects of methylation and the binding sites arc actively dcmethylated [14,15], In this report we ~tudy the effect of methylation of the SV40 promoter and its flankinl~ s~uences on gone expre.~sion, This i.~ t~ promoter that i,~ nnt nnrmally methylatrd in vivo and that is regarded as a model for a hou,,~kecpin$ promoter. We confirm that mcthylation of CpO dinucleotides within the SV40 promoter sequence does not affect transcription factor binding yet quite low levels of methylation, generated largely in the CpG-rich ¢nvironn~ent of th~ prokaryotic vector DNA, significantly reduce transcription when the DNA is introduced into cultured cells. We propo.~ that inactivation most likely occurs via the formation of an inactive chromatin structure that does not involve promoter methylation. 2. MATERIALS AND METHODS 2,1, [.~'ahaim~of DNA methyi#s¢ DNA mcthyla~ ~',,a'~pr¢pared from mou~ Kr¢bs I1 arcit~ tumour calls as previously described [I 6]. Prokaryotic DNA mcthylas~ w~r¢ purrk~sed from New England Biolabs. 2.2, Comtrurticm mat c¢ltani~utiml of pVHCt pVHCl was constructed by i n ~ i n $ th= promotgr/enhan~r region of SV40 into th¢ promot¢rl¢~s CAT $¢n¢ containing platmid p200, p200 was ¢o,mructcd by replacing th= promoter ~qurn~ of the phtsmid pTK3CAT with a polylink=r scqu=n~ from pIC20H [17]. A map of tl~o plasmid and the location of the CG dinucleotid~ and CCGG and CGCG shrs is shown in Fill, 1-"-. 2.3. /tfethrlatt, ii qf phtxmM DNA (a) with murinr mrthyt, x¢: plasmid DNA (5 PB) was mgthylated in a 70 al reaction containing 30 a M S.adenosyl m¢lhionin¢ (in some cases this was tritiated at 0.~ CL/mmol). 0.1 mg/ml bovine r~rum albumin. 50 mM Tris-HCI, pH 7,8, 1 mM EDTA. 10% glycerol and I iO rd marine DNA mcihfia~¢ ton~ u oz ~n~-ff~ wit; i ~ ~ u-ira pmol of methyl group into denatured DNA from M, haeus in I h), After the appropriam incubation time at 3"/'C the DNA was r¢iso
97
'Volume 309, numl~r i i l l
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Fig. 1. (a) Lineari~¢d map of pVHCI show,nil xbe loft,on of the SV40 promoler and terminaxor regions and the CAT ~nc. A b o ~ and beJow the map. shun vcrti~'al lin~ show the distribution of CpG dinucl¢otid~ and Hpal[ and It/m| sites, r~pcetiv¢ly. (b) An expanded view of the promoter region d~owing, the legation of the three oliltonucleotid~. luted, The extent of mctlwlation was determined either by calculatinll
the namer of radioactive methyl groups incorpor:tted or b). mcasurinl~ r~istanc~ to f/pall and Cf.| ¢ndonacleases or by Maxum Gilbert ~qucncing. {b) with harlcrlal mcth,vktsrs: plusmid DNA ~ t s mcth).laxed a, per manufacturers instructions (sometimes usinll[~HJAdoMet) and completion of methylation assa),ed as above, Methylases (New Englaltd Uiolabs) .~sI and tllmt meth)'latcd to complexion but M//Fall was incompletely effective, 2.4. Trn#~f¢ctlml , f tlSSUe¢uhur~d cells m~d mmlysis uf CA T net,riO" Mcthylatcd or unmethylatcd plasmid DNA (5 fig) was introduced lion method [Itt]. Cells were grown in ~ cm dish~ in Eagle's medium (Glasgow modifi~ttion), supplemented with 10~ newborn call,cram. All transfcctions were perl'ormcd in duplicate. $outharn blot analysis of trunsl'ected cells indicated the presence of comparable amounts of plmsmid DNA whether or not it was methytated prior to transfcction. Cell extracts were prepared and a ~ y c d for CAT activity by following the transfer of butywl groups to v'C-labelled chlorampbeni¢ol a~ordinlt to the method of ~eed and Sheen [19]. Protein assays were per. formed on the r~'~mcextract using the method of Bradford ['2-0]. 2.S. Gr! ~l~bilit.v sh~[t .ssa.vs Oligonucleotides were prepared on an Applied Biosystems apl~tratug, The s ~ u c n ~ and Iocaxion of oligo 22, m22 and m40 i~ shown in Fig. Ib. Oligo 62 h,,': the unrdated sequea~ CAA GCT TGG CGT AAT CA, When required, oligonucleotides were end.labelled with [¥J"P]ATP using polynuclcotide kinase (Bochrinsar) under conditions recommended by the manuracturcr. HeLa ¢¢11nuclear extracts were prepared according to the method of Dignam et at. [21 ]. Protein-DNA complexes were formed in a reaction containing 0.S-l.0 ng of end. labelled oligonucleotid¢, lO/ag of HeLa cell nuclear extract, 2/Ji~ pnly dl-dC. 20 m M H E P E S pH 7.9,20% vlv glycerol.0.I M KCI, 0,2 m M EDTA. S mM DTT in a total volume of 10/al. Complexes were ~parutcd on a 7~ non-denaturinlt polyncrylamidc gel and detected by autoradlollraph)'.
3. R E S U L T S 3.1. Met/w/ation of p VH CI with re,rise DNA methy ',asc
reduces expresa'iot~fram the SV40 early promoter The plasmid pVHCI, containing the SV40 promoter linked to the chloramphenicol acctyl transferase (CAT) gone, was methylated in vitro using murine DNA moth. yla~. All ho',,'3h . . . . . "---" ' methylation were obtained (measured by tritiated methyl group transfer), more typical values after a 5-6 h incubation ranged from 20-30%. This is partly the 98
result of formation of unproductive, covalent complexes between tha mouse enzyme and substrata DNA [22]. Moreover. Hpull digestion of the population of methylated plasmid molecules (Fig. 2:0 showed some molecul~ that were larl~ely resistant to digestion, whilst others were completely dighted by/-/pail. Maxam and Gilbert sequencing on the plasmid which had been methylatcd in vitro showed little evidence for methylslion of promoter or adjacent resionn (Fig. 2b). It is not cl~ar from thc~ r~.~ult5 "'" - -.d~.LUl~. ' . . . . . . .u,,-~..tt., w , i c"". . . r .~ I , - . 3 ii~¢.|~lylated and unmcthylated molecules in present or whether plnsmid molecul~ with a range of methylation have bccn produced, although the latter is probably the CaSe.
When this DNA was introduced into mouse L929 cells transcription was prollressively inhibited (Fig. 3). $0% inhibition was achieved with plasmid that clearly showed less than ~",% methylation (Fig. 2). p~/HCI contains a total of 216 CpG pairs (,~laown in Fig. Is), g of which arc contained within the SV40 encoded promotor DNA. If mcthylation were random, then at this level of saturation fewer than 0.1% of plasmid molecules would have all the GC boxes in the promoter methylated and g0% of molecules would still have four unmethylated OC boxes. It seems unlikely that in the present situation inhibition of transcription can be m,'Aiated via promoter mcthyhttion. 3.2. Methylation of p VHCI with bacterial DNA ntethy-
lases The methylase SssI has the same specificity as the marine enzyme° methylatinB all cytosines in CG dinu. clone,des, but it acts in a de novo manner which results in a much faster transfer of methyl groups to unmethylatcd DNA. Complete methylation ofpVHC1 by M.Sssl (as assessed by resistance to HpalI and by Maxam and Gilbert sequencing; Fig. 2) led to almo.~t complete inhibition of transcription from the SV40 early promoter wl=n t ~ ~ z s m ~ w=s i n t r e d t = ~ in'~ mo-.~-. ~ L929 cet~ (Fig. 3). With the fully methylated plasmid, the percent inhibition of transcription was dependent on the time after transfection (Fig. 4): less than 50% inhibition being
Volume 309, numl~r I
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D
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4---11 Fill. 2..~_~_~__tm~ntof the ~tcnt or m~thylation, tramples m~thylatcd for $ h a, ¢Jczcribcd in the h:liend to Fill, 3 w~r¢ ~ith~r diip~te.d with ~Vpall (u) or subj~ctQd to partial r,equen¢.inll by flu; Maxam Gilbert method (b), In (a) the lan~ reprezent marker (I); undil~tea plat,mid (2); plasmid methylntr.d with M,S.rs] mM dil~tcd with llpul| (3); plasmid m¢thldatcd with routine methylene and dilleltcd with//'pall (4); mock methylat~ plasmid dill~itc.d with #pall ($). In (b) the lancet r~prcrcnt the G.rca¢lion; th~ A~G re.action; the C+T r~tetion 'and the C r~¢tion on the/-/it~dll l/Kbu~I liniment from th~ unm~thylated plat. mid and th~ C r~¢tion emrricd out on th~ fraBmgnt from tl~ plebe'aid methylat~ wi|h M.~td (S) OF the murir~ m~thyla~ (M). The frallm~nt was fndlaballfd at the Hindl[l end u~inB Klfnow polym¢~ts¢ and [s-nP]dCTP. The ~trro~ indicate the position of the ¢'/totin¢$ in CG dinu¢l~otid¢,t in the ~ix GC box~ of the SV40 promoter,
•, , . - - . l
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Inhibition af transcription is not mediated through GC box methylatian
The SV40 promoter contains 6 GC box motifs which influm~_¢¢t-r~u'~.r__"_xp_ti¢_n_to varying deg,r~es and are capable of binding the tran.~eription factor SpI [23]. We confirm here that $pl binding is unaffectcd by mcthylation.
,
.
.
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G found at 24 h whilst over 90% inhibition was observed at 48 h. This implies that formation of the inactive .~tructur¢ does not occur immediately following transfeetion. In contrast methylation with blpalI and Hhal mothyloses had no effect on transcription (results not shown), There are only 33 sites for these enzymes distributed evenly but spnrsely over the prokaryotic section of the plasmid (Fil~. la). None of these sites lie in the $V40 early promoter re,ion.
.
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3.3.1. Transfection exper#tzents. Oligonu¢leotides 22 or 40 residues Ions, containing one or three $pl binding sites resp~tively, were synthesized (Fi& I b), Cotransfcction of mouse L929 ~lls with pVHCI and the double stranded oligonuclcotides resulted in inhibition of gone expression, an effect which was independent of the methylation state of the synthetic oligonucleotides (Fig. 5). Both thQ rnethylated and unmethylatcd 22mers resulted in a 5-fold reduction in 8one expression while the methylated 40m=r inhibited ~xpression by 90%. A non-specific oligon_u_cl¢_otide_ (oligo62) or small restriction fragments of pAT153 had no effect. This indicates that oligonucleotides contain. inB $pl binding sites are equally effective at .~uester99
'Volume 309. number l
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Time of methylatlon {h) Fig. 3. Time coarse of mcthylation of pVHCI and the ell'tot on tran=¢ription, gpg ~mplcti ofpVHCI were rnethylatcd as dmmribcd in the ,~ectien 2 ulin$110 units of the marine methyltmt or I .S units of M.S.ul (note that th~ unit definitions are different), After the indicated time, the DNA was r=iwlated and uutd to t rand'~:t mouu: I..929cells, Other ~mples wcr¢ used to ass~ss the cxtont of mcthylation. CAT activity
was measured 411h after transl'¢ction or the ¢¢11s.
ing transcription factor whether or not they arc methylatcd. 3.3.2. Gel retardation assa2s Fig, 6 shows that protein factors present in Hela cell nuclear extract bind equally well to mcthylat¢d or unmcthylated oli$o22 in $,1 retardation assays. While we offer no formal proof that the protein involved is Spl. this would be the expectation from previous workers" findings [23] and from the competition experiments reported below. Both the m=thylatcd and non.mcthylattd non-labelled oligonucleotides compete equally well for Spl binding to labelled probe (Table I and [23.24]). Addition of an excess of the promc,eriess plasmid, p200. had no effect on binding while oligonucl¢otides including unrelated promoters had only a small competitive effect, These results confirm that the ubiquitous transcription factor Spl can bind to its target sequence independently of the state of methylation of that sequence.
fectH with the platmid, methylatH (or mock methylated) usitlg M,$=I and CAT activity was determined at 6. 24 and 411 h.
sin#c, key methylcytosines in the promoter of a gcn¢ may prevent the binding of an ¢sscntial transt;ription factor and hence reduce transcription. However, it has bccn shown previously [24.2S] and we have confirmed here that Spl can bind to its target sequence independently of its methylation status. This is despite the fact that each Spl binding site contains a CG dinucleotide. A.~Spl is involved in the activation of many hous, keepin$ genes it would be detrimental if these genes were to be inactivated by random methylstics, however infrequently this were to occur. On such grounds one would not expect promoters of housekeeping genes to be controlled via cytosine mcthylation. It is also pertinent that the SV40 enhancer is free of CO dinucleotid,s and so is not susceptible to inactivation by methylation. A previous study on the effect of methylatics on the SV40 early promoter in its natural environmeat, showed thai mcthylation of the 27 pairs of CpG dinucl=otidcs in the SV40 genome had no effecton early gent expression when the viral D N A was introduced into cells [26]. However, the $V40 gcnome is very deficient in CpGs and their distribution is very uneven; 19 Table l Competition in bandshift experiments, 2 nil cnd-lal~llcd 22rncr (double-strand=d) was incub;ttH with 10/.tg nud,ar oxtract (as tar Fig. fi}, in the pre~n~ of increasing concentrations of ¢omlmting oligonucieotidc., n:t indicntH. The retarded band watt cxcisH and counted Competitor DNA
4, DISCUSSION
p200 22mcr
D N A methylation controls the level of expression of differentgenes by differentmechanisms. In some cases,
22m~r.mcthyl,'ttcd 40m~r-m~thylatH
I00
Amount (n$) required to give 50% inhibition of binding
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of the 2"1 CpGs occurring within a region of 350 bp spanning the promoter region and the origin of replication [27I. These CpGs may be functionally important (e.g. 6 arc present in Spl binding yit~) but arc not normally methylated in viva. Nevertheless. we show her, that methylation does
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August Ig92
cause inhibition of transcription when the promoter is in a plasmid and this ctT~t is seen at quite low levels o f methylation. Greater inhibition is sccn the greater the extent of methylation and inhibition may be dependent on the presence of a critical number of methylcytosines in the D N A , 70~ inhibition is seen when pla~'nid molccul~s contain an average of about 50 methylcytosines, We propose that this inhibition is independent o f promater methylation but is mediated by the formation of inactive chromatin characteristic of that found in nonexpressing cells [13], However, formation of this inactive structure does not occur immediately and. even with the fully m~thylated plasmid. 50% of the control enzymic activity is found 24 h after tran~fcction (Fig. 4). Meehan et al. [l l] have shown that a methylcymsinebinding, protein binds to DNA only when 15 or more methylcytosines are present within a short region and the eft'cot we see may be mediated by the bindin$ of this, or a similar, protein, In the fully methylated plasmid, taking the distribution of CG dinucleotidcs into account, methyl groups will be present over most of the molecule (excluding the terminator) every 13 bp; i.e. the vector resembles a CpO island. However, as there are only 9 CGs in the promoter region, such inhibition cannot be mediated through the binding of thi~ protein to the promoter region of the playmid and must rely on binding to vector sequen~s. These results indicate a constraint that is imposed by the vector on the expression of recombinant genes in higher cukaryoten, Clearly G+C rich vectors may not be the most satisfactory for use in c¢11 transformation studies as there is evidenc¢ that CpG island DNA is progressively methylated in transformed cells [28.29].
[ 22 J[~m22 I 0 S10 ~S20 0 S ~0~S20
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Fij,6, Gel retardationanalysis:2 n$ ofend.lab~|led22mer or m22mer (double-stranded) were incubated with 0 to 20aB nuclear extrdctas descrilmd
in the section 2. The productswere~eparaled on a 7% polyacrylamidegel whichwas dried and autoradiographcd. lOl
Volume )09. number I
FEDS LETTERS
Funh©rmor¢, tallowing pn= it=nit'or..n iniroduccd gone c~[n inc¢ln~t¢ at r~ndom into the host chromosome and may become located in ~l G+C.rich ~nvironrr~ni, The Wcsenl iludy shows ih~i rneihyl~don o£ fhmkinB sequel.s cnn h~v~ zz dr-m~tic ¢1"r¢c~on ~ub:mqu~nl ~¢n¢ ¢xpr~ion. Arknmvlrd~¢mrmx: We would like I~ (hank the M¢ir~l K~-q~ar~h Council and lhi Welicorr~. Trust I'¢r funds thai r.upimrlid Ihls ~ork. We wo~uldIbm like to tll~nk Profit.sot Hourly and the Univcrs~l)~O£ Glasllow For the provision or t',cililics.
REFERENCES [I} '~'alhF, and Molloy. P.L. (19~ GCn~ Dcv. 2, 1136-I143, [-~JK©v~ski. 1,,Riichil,R. ~nd N~vins. J,R.{19117iPr~. Natl.Acad. ~i. USA iS4. 21ti0-2184. [:~] Hcnnann. R.. H~acvclcr. A. and [~mrllcr. %V.(19t~9).1. Moi. Lliol. .~10. 411-4lS. {4] lluchi.Ari|a.$,M.M, and SehulTner, W, (19B9} Genes D=~,, ~l. 61~.-619. {5] Comb. h4. ~nd Goodman. H.M. (1990) Nucleic Acids ges. ll~. 3915-3982, [6} Lanier. K.D., Vardiman, L., Rent, U. and Do©rflcr. W. (1984) Proc. Nail. Acid. Set. USA El. 2950-29S4. [?] Hoivctcr. A. ~nd Docrfl~r. W. (19117)DNA 6. 449-460. (~J /3¢n.Haitcr. L. Dcard. P. and Jiricny..1. (19~) I~uclci¢ Acids R~. ]?. 101?9-10190. [9} Grmessmann. A. aild Gr~c'ssmann, M, (19¢B)Gin¢ 74. 135-137, [10} l:~ob~skar. D.D.. Li~bllr. M.. Gnscum~.nn. M, ~nd Grncssrr~nn. A. (1990) Prec. Natl. Acid, $ci. USA ~?, i691-1~%, [[ [] Mcchan. R,R,. Lewis, J,D,. McKay. $., Klcinir. E.L. and Bird, A,t*. (1~69) C¢1[ 58. 499-50?. [12] ]~y~s. J. and Bird. A. (iggl) Cell 64. 1123-1134.
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(13] K~hth 1,, L~man-Hurwilx. J, and Cedar, H. {1916) Crdl, 44. [ 141 Yisracli. J,, Adiisilin, R,S,. Mdloni, D,. Nud©l, U,, YalTl, D, and Ccd~r, H, (19~) Cell 46. 409~15, [15| Pirouih, ~,,, Kcsh¢l, l.. Ylsraclir J. and Cedar, H. (1990) Call 6), [16| Adams, R.I_P,, Gardirmr. K., Rinaldi. A,, Bryam, M.. hlcGarv~, M. and Butdon. R,H, (191~) I~iochim, Biopll~, Aciil ~ , 9-16, liT] Marzh, J,L,, Erlll, M, and W~tk~6 E.I, (1984) Gcnl 3~. 41H-4ZiS, [ I ill Gorrnan. C, M.. MolTal, L,F. and Howard. B, H. (19112) Mol. Coil. Bi~l. 2. 1044-105l. [19] S~cd. B. and Sheen. J..Y. (19M) Gone 67, 271-277, [-sO} Bradford, M.M. (1976) Anat. 5io~hcm. 72, 241$-2~, [21] Di~am. $,D,, Lctmvi,~, R,M. ~,nd Rocdcr, R,G, {19153)Nucleic Acidl Rts. i 1. 14T$-141~9, (22] Adams. R,L,P., Lind~y. H.. R~I~> A.. Scivwrilhz, C,. Kau, S., Cumminp. M. and HouLslon.C',E, [n DNA mcthylation: biol~lilll iiinil~an~e (].P. JOSl and H,P, ~aluz. Eds.) Birkhauser, Basel, in press. [23i Kudonala, J.T,. Jones. K.A. and Tjian. R. (19~) Trends Bio, chore, Sci. Ih 20-23, [24} l'[arrinllon, M,A,, Jon¢~. P,A., Imall~wa. M, ~nd Karin, M, (19~ti) Pr~. Nod. Acad. Sci, USA 85, 20~6-2070. [25] l-[ollir, M,, Wcslin, G., Jiricny. J, and SchulTnir, W, (Igt[~) Gcncs I~v, 2, 1127-1135. [-~6| Gmc~'nann. M,, Gr~ssmann. A,, W-slncr, H.. Wern©r. E, and Simon. D. {19113)Pro~, Nazi, ~,~d, Sci, USA ~ , 6470-~74, ['27] Uuchman. A,R,, Burnltt. L. and Bit|. P, in: DNA turnout viruses (J. Tooze. Ed,). Cold Sprinl Harl~ur Pr~u. 19Z~O,N~w York, pp, 7g~8~,
[21~] J~ncs. P.A.. Wolkowiix. M..I.. ~idiout. W.M.. Gonxalct. F.A.. Marzia~. CM., Co¢1x¢¢.G.A. ~nd Tap~:o(¢. S.J. (1990) Proc. Nail. Acad. SoL USA. !~?. 8117-612L [29] Antcqucra. F.. Boy,s..[. ~nd Bird. A. (1990) Cell. 62. 503-$14.
Aught 1992
Vector methylation inhibits transcription from the SV40 early promoter M. Bryans*, S. Ka,~, C. Scivwright and R.L.P. Adams Drpartmrt~t of Biochemistry, Ut~iversityof Gta#gon', ~Ttaxgow,GI 2 ~QQ, OK Received I July 1092 Math~latior~ of a plasmid ¢ontainittll th= SV40 promoter linked to the ¢hlommpheni¢ol a=tyl trant,l'¢ra= (CATJ I~¢n¢, with ¢ith,r marine DNA m~thyl=r~ or methyla~ ,.~sl r~ults in inhibition of the cxpr~ion of the reporter =¢nc after transl.:lion into cultured ¢=11s,Mathylation of th= plasmid with the methyla~s H/me and l t ~ l t has no =fleet on the expre,'~ion of this ilene. Prot,in-DNA int,r, tctions in the SV40 promoter arc not affected by the pr©ten~ of ntethylcytofit~ suili~stin8 that illaetivalion r=sults from the formation of an inactive ¢hromatin structure that is d¢~nd©nt on the high eG content of the plasmid, DNA m¢thylation; SV40 proinoten CpG island; gpl: Cltromatin structure
I. I N T R O D U C T I O N Inhibition of transcription has been documented for
a large number of genes containing methylcytosine in the promoter region. However, to date there is insu..m.cient evidence for a single medianism of inactivation. Methylation can interfere dir¢ctly with the transcriptional machinery by pr,venting the binding of transcription factors. For example, the major late transcription factor [1], the adenovirus F~ factor [Z3]. the cAMP r~ponsiv¢ element binding protein [4] and AP2 [5]. However 'methylation sensitive" transcription factors are still in a minority of those studied. Methylations, nsitive genes such as the adenovirus E2a gone [6,7] and the herpes simplex virus tk gene [8] bind transcription factors regardless of the mrthTlatioa state of their promoters. In such gen,s the effect of methylation may rosalt from the formation of inactive chromatin struttares that are inhibitory to efficient transcription as observed for the herpes simplex virus thymidine kintts¢ gen, [9,10]. A protein factor has recently been purified that binds to clust,rs of methylcytosines in promoter regions resulting in inhibition of transcription [11,12]. On transfection of cultured cells, it has been shown in some cases that mrthylated DNA assumes an inactive, DNase I insensitive, chromatin structure whereas. unmethylated D1NA forms DNase I sensitive chromatin characteristic ofexpressing genes [13]. In other cases the pre-~enee of tranacription factors appears to be able to
*Prexent address: 124i]Jam=s Cancer Hospital, Columbus, OH 43210. USA, Co~rezpandence address: R,L,P, Adam=, Department of Bio~hmad=it3,. University of Gla,gow, Glasgow, (312 8QQ, UK. Fax: (.at) (41)
~30 462O.
Pubflzhcd hy Elacvicr $dcne¢ Pubh'dlcr,~ B, V,
override inhibitory effects of methylation and the binding sites arc actively dcmethylated [14,15], In this report we ~tudy the effect of methylation of the SV40 promoter and its flankinl~ s~uences on gone expre.~sion, This i.~ t~ promoter that i,~ nnt nnrmally methylatrd in vivo and that is regarded as a model for a hou,,~kecpin$ promoter. We confirm that mcthylation of CpO dinucleotides within the SV40 promoter sequence does not affect transcription factor binding yet quite low levels of methylation, generated largely in the CpG-rich ¢nvironn~ent of th~ prokaryotic vector DNA, significantly reduce transcription when the DNA is introduced into cultured cells. We propo.~ that inactivation most likely occurs via the formation of an inactive chromatin structure that does not involve promoter methylation. 2. MATERIALS AND METHODS 2,1, [.~'ahaim~of DNA methyi#s¢ DNA mcthyla~ ~',,a'~pr¢pared from mou~ Kr¢bs I1 arcit~ tumour calls as previously described [I 6]. Prokaryotic DNA mcthylas~ w~r¢ purrk~sed from New England Biolabs. 2.2, Comtrurticm mat c¢ltani~utiml of pVHCt pVHCl was constructed by i n ~ i n $ th= promotgr/enhan~r region of SV40 into th¢ promot¢rl¢~s CAT $¢n¢ containing platmid p200, p200 was ¢o,mructcd by replacing th= promoter ~qurn~ of the phtsmid pTK3CAT with a polylink=r scqu=n~ from pIC20H [17]. A map of tl~o plasmid and the location of the CG dinucleotid~ and CCGG and CGCG shrs is shown in Fill, 1-"-. 2.3. /tfethrlatt, ii qf phtxmM DNA (a) with murinr mrthyt, x¢: plasmid DNA (5 PB) was mgthylated in a 70 al reaction containing 30 a M S.adenosyl m¢lhionin¢ (in some cases this was tritiated at 0.~ CL/mmol). 0.1 mg/ml bovine r~rum albumin. 50 mM Tris-HCI, pH 7,8, 1 mM EDTA. 10% glycerol and I iO rd marine DNA mcihfia~¢ ton~ u oz ~n~-ff~ wit; i ~ ~ u-ira pmol of methyl group into denatured DNA from M, haeus in I h), After the appropriam incubation time at 3"/'C the DNA was r¢iso
97
'Volume 309, numl~r i i l l
1
I l l l l l l l
FEBS LETTERS I
I I I
lgllli
i~¢I
• II
t
III
l
I|rlll|r| |11|
|II
Aught 1992 OIIgO,lUCll~gtldn ~ , m32 ona m4g
lll::::~lllllll
I II]ll
147s4 I
I
Fig. 1. (a) Lineari~¢d map of pVHCI show,nil xbe loft,on of the SV40 promoler and terminaxor regions and the CAT ~nc. A b o ~ and beJow the map. shun vcrti~'al lin~ show the distribution of CpG dinucl¢otid~ and Hpal[ and It/m| sites, r~pcetiv¢ly. (b) An expanded view of the promoter region d~owing, the legation of the three oliltonucleotid~. luted, The extent of mctlwlation was determined either by calculatinll
the namer of radioactive methyl groups incorpor:tted or b). mcasurinl~ r~istanc~ to f/pall and Cf.| ¢ndonacleases or by Maxum Gilbert ~qucncing. {b) with harlcrlal mcth,vktsrs: plusmid DNA ~ t s mcth).laxed a, per manufacturers instructions (sometimes usinll[~HJAdoMet) and completion of methylation assa),ed as above, Methylases (New Englaltd Uiolabs) .~sI and tllmt meth)'latcd to complexion but M//Fall was incompletely effective, 2.4. Trn#~f¢ctlml , f tlSSUe¢uhur~d cells m~d mmlysis uf CA T net,riO" Mcthylatcd or unmethylatcd plasmid DNA (5 fig) was introduced lion method [Itt]. Cells were grown in ~ cm dish~ in Eagle's medium (Glasgow modifi~ttion), supplemented with 10~ newborn call,cram. All transfcctions were perl'ormcd in duplicate. $outharn blot analysis of trunsl'ected cells indicated the presence of comparable amounts of plmsmid DNA whether or not it was methytated prior to transfcction. Cell extracts were prepared and a ~ y c d for CAT activity by following the transfer of butywl groups to v'C-labelled chlorampbeni¢ol a~ordinlt to the method of ~eed and Sheen [19]. Protein assays were per. formed on the r~'~mcextract using the method of Bradford ['2-0]. 2.S. Gr! ~l~bilit.v sh~[t .ssa.vs Oligonucleotides were prepared on an Applied Biosystems apl~tratug, The s ~ u c n ~ and Iocaxion of oligo 22, m22 and m40 i~ shown in Fig. Ib. Oligo 62 h,,': the unrdated sequea~ CAA GCT TGG CGT AAT CA, When required, oligonucleotides were end.labelled with [¥J"P]ATP using polynuclcotide kinase (Bochrinsar) under conditions recommended by the manuracturcr. HeLa ¢¢11nuclear extracts were prepared according to the method of Dignam et at. [21 ]. Protein-DNA complexes were formed in a reaction containing 0.S-l.0 ng of end. labelled oligonucleotid¢, lO/ag of HeLa cell nuclear extract, 2/Ji~ pnly dl-dC. 20 m M H E P E S pH 7.9,20% vlv glycerol.0.I M KCI, 0,2 m M EDTA. S mM DTT in a total volume of 10/al. Complexes were ~parutcd on a 7~ non-denaturinlt polyncrylamidc gel and detected by autoradlollraph)'.
3. R E S U L T S 3.1. Met/w/ation of p VH CI with re,rise DNA methy ',asc
reduces expresa'iot~fram the SV40 early promoter The plasmid pVHCI, containing the SV40 promoter linked to the chloramphenicol acctyl transferase (CAT) gone, was methylated in vitro using murine DNA moth. yla~. All ho',,'3h . . . . . "---" ' methylation were obtained (measured by tritiated methyl group transfer), more typical values after a 5-6 h incubation ranged from 20-30%. This is partly the 98
result of formation of unproductive, covalent complexes between tha mouse enzyme and substrata DNA [22]. Moreover. Hpull digestion of the population of methylated plasmid molecules (Fig. 2:0 showed some molecul~ that were larl~ely resistant to digestion, whilst others were completely dighted by/-/pail. Maxam and Gilbert sequencing on the plasmid which had been methylatcd in vitro showed little evidence for methylslion of promoter or adjacent resionn (Fig. 2b). It is not cl~ar from thc~ r~.~ult5 "'" - -.d~.LUl~. ' . . . . . . .u,,-~..tt., w , i c"". . . r .~ I , - . 3 ii~¢.|~lylated and unmcthylated molecules in present or whether plnsmid molecul~ with a range of methylation have bccn produced, although the latter is probably the CaSe.
When this DNA was introduced into mouse L929 cells transcription was prollressively inhibited (Fig. 3). $0% inhibition was achieved with plasmid that clearly showed less than ~",% methylation (Fig. 2). p~/HCI contains a total of 216 CpG pairs (,~laown in Fig. Is), g of which arc contained within the SV40 encoded promotor DNA. If mcthylation were random, then at this level of saturation fewer than 0.1% of plasmid molecules would have all the GC boxes in the promoter methylated and g0% of molecules would still have four unmethylated OC boxes. It seems unlikely that in the present situation inhibition of transcription can be m,'Aiated via promoter mcthyhttion. 3.2. Methylation of p VHCI with bacterial DNA ntethy-
lases The methylase SssI has the same specificity as the marine enzyme° methylatinB all cytosines in CG dinu. clone,des, but it acts in a de novo manner which results in a much faster transfer of methyl groups to unmethylatcd DNA. Complete methylation ofpVHC1 by M.Sssl (as assessed by resistance to HpalI and by Maxam and Gilbert sequencing; Fig. 2) led to almo.~t complete inhibition of transcription from the SV40 early promoter wl=n t ~ ~ z s m ~ w=s i n t r e d t = ~ in'~ mo-.~-. ~ L929 cet~ (Fig. 3). With the fully methylated plasmid, the percent inhibition of transcription was dependent on the time after transfection (Fig. 4): less than 50% inhibition being
Volume 309, numl~r I
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2
FEBS LETTERS
4
5
AuBust 1992
D
•,,,,-- VI
.,,,-.-V W
.,,,---.IV
W
•
--.,
•ql..-- I I I
4---11 Fill. 2..~_~_~__tm~ntof the ~tcnt or m~thylation, tramples m~thylatcd for $ h a, ¢Jczcribcd in the h:liend to Fill, 3 w~r¢ ~ith~r diip~te.d with ~Vpall (u) or subj~ctQd to partial r,equen¢.inll by flu; Maxam Gilbert method (b), In (a) the lan~ reprezent marker (I); undil~tea plat,mid (2); plasmid methylntr.d with M,S.rs] mM dil~tcd with llpul| (3); plasmid m¢thldatcd with routine methylene and dilleltcd with//'pall (4); mock methylat~ plasmid dill~itc.d with #pall ($). In (b) the lancet r~prcrcnt the G.rca¢lion; th~ A~G re.action; the C+T r~tetion 'and the C r~¢tion on the/-/it~dll l/Kbu~I liniment from th~ unm~thylated plat. mid and th~ C r~¢tion emrricd out on th~ fraBmgnt from tl~ plebe'aid methylat~ wi|h M.~td (S) OF the murir~ m~thyla~ (M). The frallm~nt was fndlaballfd at the Hindl[l end u~inB Klfnow polym¢~ts¢ and [s-nP]dCTP. The ~trro~ indicate the position of the ¢'/totin¢$ in CG dinu¢l~otid¢,t in the ~ix GC box~ of the SV40 promoter,
•, , . - - . l
..
3.3,
Inhibition af transcription is not mediated through GC box methylatian
The SV40 promoter contains 6 GC box motifs which influm~_¢¢t-r~u'~.r__"_xp_ti¢_n_to varying deg,r~es and are capable of binding the tran.~eription factor SpI [23]. We confirm here that $pl binding is unaffectcd by mcthylation.
,
.
.
,....,.
G found at 24 h whilst over 90% inhibition was observed at 48 h. This implies that formation of the inactive .~tructur¢ does not occur immediately following transfeetion. In contrast methylation with blpalI and Hhal mothyloses had no effect on transcription (results not shown), There are only 33 sites for these enzymes distributed evenly but spnrsely over the prokaryotic section of the plasmid (Fil~. la). None of these sites lie in the $V40 early promoter re,ion.
.
A
C
q-
.4-
G
T
C
S
M
3.3.1. Transfection exper#tzents. Oligonu¢leotides 22 or 40 residues Ions, containing one or three $pl binding sites resp~tively, were synthesized (Fi& I b), Cotransfcction of mouse L929 ~lls with pVHCI and the double stranded oligonuclcotides resulted in inhibition of gone expression, an effect which was independent of the methylation state of the synthetic oligonucleotides (Fig. 5). Both thQ rnethylated and unmethylatcd 22mers resulted in a 5-fold reduction in 8one expression while the methylated 40m=r inhibited ~xpression by 90%. A non-specific oligon_u_cl¢_otide_ (oligo62) or small restriction fragments of pAT153 had no effect. This indicates that oligonucleotides contain. inB $pl binding sites are equally effective at .~uester99
'Volume 309. number l
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Aujmt 1992 1001
~
re°sit mtthylst~
/
.ol -°.'+..'°+ /
m
Mouse rnethylase 6-
40
20 0
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12
24
36
46
time post trantfectlon (h} Fig, 4, Time e..ounm of expression of CAT activity. Cells were trans-
o 0
2
4
6
8
24
Time of methylatlon {h) Fig. 3. Time coarse of mcthylation of pVHCI and the ell'tot on tran=¢ription, gpg ~mplcti ofpVHCI were rnethylatcd as dmmribcd in the ,~ectien 2 ulin$110 units of the marine methyltmt or I .S units of M.S.ul (note that th~ unit definitions are different), After the indicated time, the DNA was r=iwlated and uutd to t rand'~:t mouu: I..929cells, Other ~mples wcr¢ used to ass~ss the cxtont of mcthylation. CAT activity
was measured 411h after transl'¢ction or the ¢¢11s.
ing transcription factor whether or not they arc methylatcd. 3.3.2. Gel retardation assa2s Fig, 6 shows that protein factors present in Hela cell nuclear extract bind equally well to mcthylat¢d or unmcthylated oli$o22 in $,1 retardation assays. While we offer no formal proof that the protein involved is Spl. this would be the expectation from previous workers" findings [23] and from the competition experiments reported below. Both the m=thylatcd and non.mcthylattd non-labelled oligonucleotides compete equally well for Spl binding to labelled probe (Table I and [23.24]). Addition of an excess of the promc,eriess plasmid, p200. had no effect on binding while oligonucl¢otides including unrelated promoters had only a small competitive effect, These results confirm that the ubiquitous transcription factor Spl can bind to its target sequence independently of the state of methylation of that sequence.
fectH with the platmid, methylatH (or mock methylated) usitlg M,$=I and CAT activity was determined at 6. 24 and 411 h.
sin#c, key methylcytosines in the promoter of a gcn¢ may prevent the binding of an ¢sscntial transt;ription factor and hence reduce transcription. However, it has bccn shown previously [24.2S] and we have confirmed here that Spl can bind to its target sequence independently of its methylation status. This is despite the fact that each Spl binding site contains a CG dinucleotide. A.~Spl is involved in the activation of many hous, keepin$ genes it would be detrimental if these genes were to be inactivated by random methylstics, however infrequently this were to occur. On such grounds one would not expect promoters of housekeeping genes to be controlled via cytosine mcthylation. It is also pertinent that the SV40 enhancer is free of CO dinucleotid,s and so is not susceptible to inactivation by methylation. A previous study on the effect of methylatics on the SV40 early promoter in its natural environmeat, showed thai mcthylation of the 27 pairs of CpG dinucl=otidcs in the SV40 genome had no effecton early gent expression when the viral D N A was introduced into cells [26]. However, the $V40 gcnome is very deficient in CpGs and their distribution is very uneven; 19 Table l Competition in bandshift experiments, 2 nil cnd-lal~llcd 22rncr (double-strand=d) was incub;ttH with 10/.tg nud,ar oxtract (as tar Fig. fi}, in the pre~n~ of increasing concentrations of ¢omlmting oligonucieotidc., n:t indicntH. The retarded band watt cxcisH and counted Competitor DNA
4, DISCUSSION
p200 22mcr
D N A methylation controls the level of expression of differentgenes by differentmechanisms. In some cases,
22m~r.mcthyl,'ttcd 40m~r-m~thylatH
I00
Amount (n$) required to give 50% inhibition of binding
8 6
3
'Volume 3 0 % n u m b e r
FEB$ LETTERS
1
120
:Ion
i
lOO
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62 ) 22
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m22 mqO
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Fill. s. Colran~cction of pVHC[ with ollilonuclcotidcs. Cells were translated with 5 p$ pVHCI in the prc~ncc of 5 ~ugo1"the indicated oli$onuclcotid¢ and CAT activitymeasured48 h tater.
of the 2"1 CpGs occurring within a region of 350 bp spanning the promoter region and the origin of replication [27I. These CpGs may be functionally important (e.g. 6 arc present in Spl binding yit~) but arc not normally methylated in viva. Nevertheless. we show her, that methylation does
H.Ext.(H0)
August Ig92
cause inhibition of transcription when the promoter is in a plasmid and this ctT~t is seen at quite low levels o f methylation. Greater inhibition is sccn the greater the extent of methylation and inhibition may be dependent on the presence of a critical number of methylcytosines in the D N A , 70~ inhibition is seen when pla~'nid molccul~s contain an average of about 50 methylcytosines, We propose that this inhibition is independent o f promater methylation but is mediated by the formation of inactive chromatin characteristic of that found in nonexpressing cells [13], However, formation of this inactive structure does not occur immediately and. even with the fully m~thylated plasmid. 50% of the control enzymic activity is found 24 h after tran~fcction (Fig. 4). Meehan et al. [l l] have shown that a methylcymsinebinding, protein binds to DNA only when 15 or more methylcytosines are present within a short region and the eft'cot we see may be mediated by the bindin$ of this, or a similar, protein, In the fully methylated plasmid, taking the distribution of CG dinucleotidcs into account, methyl groups will be present over most of the molecule (excluding the terminator) every 13 bp; i.e. the vector resembles a CpO island. However, as there are only 9 CGs in the promoter region, such inhibition cannot be mediated through the binding of thi~ protein to the promoter region of the playmid and must rely on binding to vector sequen~s. These results indicate a constraint that is imposed by the vector on the expression of recombinant genes in higher cukaryoten, Clearly G+C rich vectors may not be the most satisfactory for use in c¢11 transformation studies as there is evidenc¢ that CpG island DNA is progressively methylated in transformed cells [28.29].
[ 22 J[~m22 I 0 S10 ~S20 0 S ~0~S20
., " . . . .
c0mDlex
Free d.s, 2~me,
Fij,6, Gel retardationanalysis:2 n$ ofend.lab~|led22mer or m22mer (double-stranded) were incubated with 0 to 20aB nuclear extrdctas descrilmd
in the section 2. The productswere~eparaled on a 7% polyacrylamidegel whichwas dried and autoradiographcd. lOl
Volume )09. number I
FEDS LETTERS
Funh©rmor¢, tallowing pn= it=nit'or..n iniroduccd gone c~[n inc¢ln~t¢ at r~ndom into the host chromosome and may become located in ~l G+C.rich ~nvironrr~ni, The Wcsenl iludy shows ih~i rneihyl~don o£ fhmkinB sequel.s cnn h~v~ zz dr-m~tic ¢1"r¢c~on ~ub:mqu~nl ~¢n¢ ¢xpr~ion. Arknmvlrd~¢mrmx: We would like I~ (hank the M¢ir~l K~-q~ar~h Council and lhi Welicorr~. Trust I'¢r funds thai r.upimrlid Ihls ~ork. We wo~uldIbm like to tll~nk Profit.sot Hourly and the Univcrs~l)~O£ Glasllow For the provision or t',cililics.
REFERENCES [I} '~'alhF, and Molloy. P.L. (19~ GCn~ Dcv. 2, 1136-I143, [-~JK©v~ski. 1,,Riichil,R. ~nd N~vins. J,R.{19117iPr~. Natl.Acad. ~i. USA iS4. 21ti0-2184. [:~] Hcnnann. R.. H~acvclcr. A. and [~mrllcr. %V.(19t~9).1. Moi. Lliol. .~10. 411-4lS. {4] lluchi.Ari|a.$,M.M, and SehulTner, W, (19B9} Genes D=~,, ~l. 61~.-619. {5] Comb. h4. ~nd Goodman. H.M. (1990) Nucleic Acids ges. ll~. 3915-3982, [6} Lanier. K.D., Vardiman, L., Rent, U. and Do©rflcr. W. (1984) Proc. Nail. Acid. Set. USA El. 2950-29S4. [?] Hoivctcr. A. ~nd Docrfl~r. W. (19117)DNA 6. 449-460. (~J /3¢n.Haitcr. L. Dcard. P. and Jiricny..1. (19~) I~uclci¢ Acids R~. ]?. 101?9-10190. [9} Grmessmann. A. aild Gr~c'ssmann, M, (19¢B)Gin¢ 74. 135-137, [10} l:~ob~skar. D.D.. Li~bllr. M.. Gnscum~.nn. M, ~nd Grncssrr~nn. A. (1990) Prec. Natl. Acid, $ci. USA ~?, i691-1~%, [[ [] Mcchan. R,R,. Lewis, J,D,. McKay. $., Klcinir. E.L. and Bird, A,t*. (1~69) C¢1[ 58. 499-50?. [12] ]~y~s. J. and Bird. A. (iggl) Cell 64. 1123-1134.
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A u l u i | 1992
(13] K~hth 1,, L~man-Hurwilx. J, and Cedar, H. {1916) Crdl, 44. [ 141 Yisracli. J,, Adiisilin, R,S,. Mdloni, D,. Nud©l, U,, YalTl, D, and Ccd~r, H, (19~) Cell 46. 409~15, [15| Pirouih, ~,,, Kcsh¢l, l.. Ylsraclir J. and Cedar, H. (1990) Call 6), [16| Adams, R.I_P,, Gardirmr. K., Rinaldi. A,, Bryam, M.. hlcGarv~, M. and Butdon. R,H, (191~) I~iochim, Biopll~, Aciil ~ , 9-16, liT] Marzh, J,L,, Erlll, M, and W~tk~6 E.I, (1984) Gcnl 3~. 41H-4ZiS, [ I ill Gorrnan. C, M.. MolTal, L,F. and Howard. B, H. (19112) Mol. Coil. Bi~l. 2. 1044-105l. [19] S~cd. B. and Sheen. J..Y. (19M) Gone 67, 271-277, [-sO} Bradford, M.M. (1976) Anat. 5io~hcm. 72, 241$-2~, [21] Di~am. $,D,, Lctmvi,~, R,M. ~,nd Rocdcr, R,G, {19153)Nucleic Acidl Rts. i 1. 14T$-141~9, (22] Adams. R,L,P., Lind~y. H.. R~I~> A.. Scivwrilhz, C,. Kau, S., Cumminp. M. and HouLslon.C',E, [n DNA mcthylation: biol~lilll iiinil~an~e (].P. JOSl and H,P, ~aluz. Eds.) Birkhauser, Basel, in press. [23i Kudonala, J.T,. Jones. K.A. and Tjian. R. (19~) Trends Bio, chore, Sci. Ih 20-23, [24} l'[arrinllon, M,A,, Jon¢~. P,A., Imall~wa. M, ~nd Karin, M, (19~ti) Pr~. Nod. Acad. Sci, USA 85, 20~6-2070. [25] l-[ollir, M,, Wcslin, G., Jiricny. J, and SchulTnir, W, (Igt[~) Gcncs I~v, 2, 1127-1135. [-~6| Gmc~'nann. M,, Gr~ssmann. A,, W-slncr, H.. Wern©r. E, and Simon. D. {19113)Pro~, Nazi, ~,~d, Sci, USA ~ , 6470-~74, ['27] Uuchman. A,R,, Burnltt. L. and Bit|. P, in: DNA turnout viruses (J. Tooze. Ed,). Cold Sprinl Harl~ur Pr~u. 19Z~O,N~w York, pp, 7g~8~,
[21~] J~ncs. P.A.. Wolkowiix. M..I.. ~idiout. W.M.. Gonxalct. F.A.. Marzia~. CM., Co¢1x¢¢.G.A. ~nd Tap~:o(¢. S.J. (1990) Proc. Nail. Acad. SoL USA. !~?. 8117-612L [29] Antcqucra. F.. Boy,s..[. ~nd Bird. A. (1990) Cell. 62. 503-$14.
