substituted DNA and Specific Non-histone Nuclear Proteins

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 7, Issue of April 10, pp. 4059-4068,1985 Printed in U. S . A.

Formation of Highly Stable Complexes between 5-Azacytosine- substituted DNA and Specific Non-histone Nuclear Proteins IMPLICATIONS FOR 5-AZACYTIDINE-MEDIATED EFFECTS ON DNA METHYLATION AND GENE EXPRESSION*

(Received for publication, October 29, 1984)

Judith K. Christman, Natalie Schneiderman, and George Acs From the Departments of Biochemistry and Pediatrics, Mount Sinai School of Medicine, New York, New York 10029

Incubation of 5-azacytosine-substituted DNA ([5- aza-CIDNA) with nuclear proteins leads to the formation of highly stable DNA-protein complexes which remain intact in the presence of 1 M NaCl and/or 0.6% Sarkosyl. The proteins involved in binding double- stranded [5-aza-C]DNA in these stable complexes com- prise a specific subset of non-histone nuclear proteins that includes DNA methyltransferase. Complex formation does not require S-adenosylmethionine and does not involve covalent linkage of protein to DNA or modification of 5-azacytosine residues. Non-histone nuclear proteins do not form complexes with double- stranded unsubstituted DNA that are resistant to dissociation with NaCl and Sarkosyl but are capable of forming such complexes with single-stranded DNA regardless of whether it contains 5-azacytosine residues or not. However, it can be demonstrated 1) that single- stranded regions do not account for stable binding of proteins to native [5-aza-C]DNA and 2) that many nuclear proteins which form stable complexes with single-stranded DNA are incapable of forming such complexes with double-stranded [5-aza-C]DNA.

Synthesis of [5-aza-C]DNA by cells growing in the presence of either 5-azacytidine or 5-aza-2’-deoxycytidine leads to rapid loss of extractable DNA methyltransferase (Creusot, F., ACS, G., and Christman, J. K. (1982) J. Biol. Chern. 257, 2041-2048). Analogous depletion of non-histone nuclear proteins capable of forming stable complexes with [5-aza-C]DNA in vitro is observed, suggesting that the same proteins can form highly stable complexes with [5-aza-CIDNA in vitro and in uiuo. Formation of stable complexes between non-histone nuclear proteins and [5-aza-C]DNA could potentially affect not only the activity of DNA methyltransferase but the action of other regulatory proteins or enzymes that interact with DNA. Such interactions could explain effects of 5-azacytidine on gene expression that cannot be directly linked to loss of methyl groups from DNA.

fi-Azacytidine, a cytidine analog in which the 5 carbon atom is replaced by a nitrogen atom, is taken up by cells and

* This work was supported by Public Health Service Grants CA 16890 and CA 25985. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

phosphorylated by uridine:cytidine kinase (1). 5-aza-CMP’ then serves as a precursor for both 5-aza-CTP and 5-aza- dCTP. Thus, 5-aza-Cyt residues can be incorporated into and substitute for Cyt residues in both DNA and RNA (2). In RNA, 5-aza-Cyt substitution has been linked to interference with normal processing of ribosomal RNA (3) and inhibition of 5m-Cyt:tRNA methyltransferase (4) while incorporation of 5-aza-Cyt into DNA has been associated with inhibition of methylation of Cyt residues in DNA (5-8), changes in DNA conformation (g), chromatin condensation (101, and chro- mosome breakage (11). Because of their toxicity for rapidly proliferating cells, initial interest in 5-aza-Cyd and 5-aza- dCyd centered on their chemotherapeutic potential and they are currently used for treatment of acute myelogenous leukemia (12). More recently, however, research has focused on the ability of subtoxic doses of these analogs to activate specific genes or developmental programs in treated cells (see Refs. 13-15 for recent reviews).

When doses of the analogs are chosen, such that reasonable numbers of cells survive treatment, heritable changes that result in altered phenotype or increased expression of a specific gene can be demonstrated to occur in some fraction of the population. Depending on the gene(s) studied and the cell type, the number of affected cells may range from 1 in lo6 to as high as 50-70/100 (14). One of the earliest changes noted in 5-aza-Cyd- and 5-aza-dCyd-treated mammalian cells is a loss of active 5m-Cyt:DNA methyltransferase (8). Because DNA synthesis continues in the treated cells, methylation of newly synthesized DNA may be inhibited by more than 90% (16). Since methylation of Cyt residues in specific regions of DNA can inhibit gene expression (17) and since methylation represents a heritable modification of the genome (18), it is generally assumed that 5-aza-Cyd-mediated inhibition of DNA methylation leads to stable changes in the pattern of methylation that allow previously inactive genes to be ex- pressed.

Earlier results from our laboratory demonstrated that 5- aza-Cyd must be incorporated into DNA in order to mediate loss of active DNA methyltransferase and suggested that loss of enzyme activity resulted either from tight binding of the

The abbreviations used are: 5-aza-CMP, 5-aza-CTP, 5”mOnO- and triphosphates of 5-aza-Cyd; 5-aza-Cyd, 5-azacytidine; 5-aza-Cyt, 5-azacytosine; 5-aza-dCyd, 5-aza-2’-deoxycytidine; Fj-aza-dCTP, 5’- triphosphate of 5-aza-dCyd; Cyt, cytosine; [5-aza-C]DNA, 5-aza-Cyt- substituted DNA, i.e. DNA in which some Cyt residues have been replaced by 5-aza-Cyt residues; 5m-Cyt, 5-methylcytosine; ds DNA, double-stranded (Sl nuclease resistant) DNA; ss DNA, single- stranded DNA SSRC, salt- and Sarkosyl-resistant DNA.protein complex; SDS, sodium dodecyl sulfate; PBS, phosphate-buffered sa- line; HPLC, high-performance liquid chromatography; AdoMet, S- adenosylmethionine; AdoHcy, S-adenosylhomocysteine.

4059

4060 Azacytosine in DNA and Binding of Specific Nuclear Proteins

enzyme to 5-aza-Cyt residues in the substituted DNA or to irreversible inactivation and release of enzyme interacting with 5-aza-Cyt residues in DNA (8). It was also shown that, under appropriate in vitro conditions (i.e. limiting enzyme), DNA methyltransferase could be inhibited by incubation with 5-aza-Cyt-substituted DNA (19-21) even though such DNA is hypomethylated and an excellent methyl acceptor under conditions of DNA methyltransferase excess (8, 19). This finding supported the hypothesis that 5-aza-Cyt residues in DNA are directly responsible for inactivation of DNA methyltransferase but did not elucidate the mechanism of inactivation. To investigate further, we compared the interaction between DNA methyltransferase and unsubstituted or 5-aza- Cyt-substituted DNAs. Here, we describe results indicating that a specific group of non-histone nuclear proteins that includes DNA methyltransferase is capable of forming much more stable complexes with 5-aza-Cyt-substituted DNA than with normal DNA both in vivo and in vitro. The implications of these results with regard to the effects of 5-aza-Cyd and 5- aza-dCyd on gene expression are discussed.

EXPERIMENTAL PROCEDURES

Cell Culture-Cells used in these studies were kindly provided by Dr. Charlotte Friend (Friend erythroleukemia cells (22), strain 745A), Mt. Sinai Medical Center, New York, and Drs. S. J. Collins and R. C. Gallo (HL-60 cells) (23), National Cancer Institute, Bethesda, MD. Both cell types were grown in suspension culture as previously described (8, 16). 5-aza-Cyd (Sigma) was dissolved in PBS at 1-4 mg/ ml and filter-sterilized immediately before dilution into the culture medium of cells in the logarithmic growth stage (6-8 X 106/ml).

Rudiochernicals-[’H]Methyl AdoMet (specific activity, 10-30 Ci/ mmol) was obtained from ICN Pharmaceuticals; [3H]methyl thymidine (specific activity, 6.7 Ci/mmol) from New England Nuclear; and [4-l4CJ5-aza-Cyd (specific activity, 50 mCi/mmol) from Moravek Chemicals, City of Industry, CA.

Preparation of 0.3 M NaCl Nuclear Extracts and Assay of DNA Methyltransferme-These procedures were performed as described in Ref. 8. All nuclear extracts were dialyzed prior to use. Substrate DNA for DNA methyltransferase assays was isolated from Friend erythroleukemia cells grown for 4 days in the presence of 4 mM L-ethionine (24).

Preparation of DNAs-Cells washed in PBS were pelleted and lysed with 4 volumes of 10 mM NaCl, 10 mM Tris-HC1, pH 7.4,3 mM MgCl,, 0.5% Nonidet P-40 (BDH Chemicals, Poole, England). Nuclei, collected by centrifugation at 2000 X g for 5-10 min were suspended in 5-10 volumes of 150 mM NaCl, 100 mM EDTA, pH 8.0. Proteinase K (400 pglml) and SDS (1% final concentration) were added and the mixture was incubated at 37 ‘C. When the DNA was well dispersed, NaCIOl was added to a final concentration of 1 M. DNA was further purified as described in Ref. 25 and dissolved in 10 mM Tris-HC1, pH 7.5, 0.1 M EDTA. For S1 nuclease digestion, the DNA solution was brought to a final concentration of 30 mM sodium acetate, pH 4.5, 0.3 M NaCl, 3 mM ZnClp. S1 nuclease (Sigma) was added (25-30 units/pg of DNA) and the mixture was incubated at 23 “C for 30 min. EDTA was added to 5 mM and the solution was dialyzed against 10 mM Tris-HC1, pH 7.4, 0.1 mM EDTA prior to use. No residual s1 nuclease activity was detected in these preparations during binding or methyltransferase assays. For radiolabeled DNA, the amount of trichloroacetic acid-precipitable DNA remaining after digestion was quantitated by chilling the digestion mixture on ice and bringing it to a final concentration of 10% trichloroacetic acid. Precipitated DNA was collected on 0.45-pm nitrocellulose filters and washed with 3 5-ml aliquots of ice-cold 5% trichloroacetic acid. After drying, precipitated radioactivity on the filters was quantitated in a liquid scintillation counter using nonaqueous fluor (Betafluor, National Diagnostics).

Filter Binding Assays-Unless otherwise indicated, each assay tube contained 5 pg of radiolabeled DNA and 1.5-150 pg of 0.3 M NaCl nuclear extract protein in a final volume of 200 p1 of buffer A (100 mM imidazole, pH 7.5, 20 mM EDTA, and 0.5 mM dithiothreitol). When included (see “Results”), the concentration of AdoMet was 10 p ~ . After incubation for 20 min at 37 “C, the reaction was terminated by addition of Sarkosyl (ICN Pharmaceuticals) and NaCl to final

concentrations of 0.6% and 0.5 M, respectively. After thorough mixing and 10-min incubation at 4 “C, the reaction mixture was filtered onto a Millipore HA 0.45-pm filter and washed with 3 10-ml aliquota of dissociation buffer (0.6% Sarkosyl, 0.5 M NaCl, 10 mM Tris-HC1, pH 7.4, 0.1 mM EDTA). The filters were dried, and bound radiolabeled DNA was quantitated in a liquid scintillation counter using nonaqueous fluor.

Preparation of Proteins Tested in Filter Binding Assay-Friend erythroleukemia cell histones were prepared from nuclei by acid extraction as described in Ref. 26. Calf thymus histones were obtained from Sigma. Cytoplasmic proteins were prepared by freeze-thawing (4 cycles) packed Friend erythroleukemia cells in an equal volume of 10 mM NaC1, 10 mM Tris-HC1, pH 7.4, 3 mM MgCl,. Nuclei, unlysed cells, and large membrane fragments were removed by centrifugation at 2500 X g for 5 min.

Density Gradient Centrifugation-Solutions of DNA or DNA incubated with 0.3 M NaCl nuclear extract protein (assay mixture described above) were brought to a final concentration of 0.6% Sarkosyl, 0.5 M NaCl and layered on preformed discontinuous CsCl/ Cs&30, gradients prepared as described in Ref. 27 with the exception that NH,SCN was omitted from and 0.6% Sarkosyl and 0.5 M NaCl were added to the solutions used for gradient preparation. After centrifugation for 18 h at 30,000 rpm in an SW 50.1 rotor, the gradients were fractionated and the acid-precipitable radiolabel and optical density at 260 nm of each fraction were determined.

RESULTS

Stability of Binding of Nuclear Proteins to 5-ma-Cyt-substituted DNA and Unsubstituted DNA-As is typical of mammalian cells, the DNA methyltransferase of Friend erthroleu- kemia cells is tightly bound to nuclear DNA. It can be quantitatively extracted from isolated nuclei or chromatin along with a variety of other non-histone nuclear proteins by treatment with 0.3 M NaCl (14). When incubated with either native unsubstituted or native 5-aza-Cyt-substituted Friend erythroleukemia cell DNA ([5-aza-C]DNA) in vitro under the conditions used to assay for DNA methyltransferase activity, the proteins in 0.3 M NaCl extracts of Friend erythroleukemia cell nuclei were able to bind to the DNAs and cause their quantitative retention on nitrocellulose filters (detected as filter binding of radiolabeled DNA; see “Experimental Pro- cedures”). Addition of 0.3-1 M NaCl to the reaction mixture after complexes had formed and to the buffer used to wash the DNA. protein complexes onto filters reduced the amount of native unsubstituted DNA retained by 50-70%. With addition of 0.6% Sarkosyl to the NaCl dissociation buffers or even with addition of 0.6% Sarkosyl alone, the amount of native unsubstituted DNA retained on filters by nuclear proteins was even further reduced (to 4 0 % of input DNA). In contrast, the amount of native [5-aza-C]DNA retained on filters by nuclear proteins was unaffected by treatment of the complexes with 0.6% Sarkosyl even when 1 M NaCl was added (Table I, A). Despite the fact that salt and Sarkosyl had no effect in reducing filter binding of native [5-aza-C]DNA, Sarkosyl (0.6%) alone was able to prevent binding of nuclear proteins to both unsubstituted and [5-aza-C]DNAs (Table I, B). This indicates that even though complex formation with unsubstituted and [5-aza-C]DNA was equally sensitive to inhibition by dissociating agents, once formed, the stability of complexes of nuclear protein with native [5-aza-C]DNA was much greater than the stability of complexes with native unsubstituted DNA. Despite this high degree of stability, native [5-aza-C]DNA .protein complexes could be dissociated with SDS (Table I, A), This indicated that complex stability was unlikely be a result of formation of covalent linkages between protein and DNA.

Evidence that stable complexes are specifically formed between proteins and DNA regions enriched in 5-aza-Cyt residues is detailed below. However, it should be noted here that

Azacytosine in DNA and Binding of Specific Nuclear Proteins 4061

TABLE I Stability of DNA. nuclear protein complexes

DNA was isolated from Friend erythroleukemia cells incubated for 4 h with [3H]methyl thymidine in the presence or absence of 10 pM 5-aza-Cyd. Each reaction mixture (see "Experimental Procedures") contained 5 pg of DNA (specific activity, 10.5 X 10' cpm/mg for DNA from untreated cells, 12 X 10' cpm/mg for DNA from 5-aza-Cyd- treated cells), dialyzed 0.3 M NaCl nuclear extract (150 pg of protein) and 2 nmol of AdoMet. Each value shown is an average of at least 4 determinations f S.E. A. Effect of salt and detergent treatment on stability of preformed DNA.

protein complexes DNA bound to filter

Additions" Native Native [B-aza-C]DNA.protein DNA.protein

None 0.3 M NaCl 0.5 M NaCl 1.0 M NaCl 0.6% Sarkosyl 0.6% Sarkosyl +

0.5 M NaCl 0.6% Sarkosyl +

1.0 M NaCl 0.05% SDSb 0.005% SDSb

cpm 55,000 f 1,950 52,000 f 2,090 60,000 f 2,380 52,900 f 2,020 54,500 f 2,150 52,900 f 1,300

53,200 f 1,370

790 f 150 52,000 f 2,430

50,000 f 1,930 23,000 f 1,670 17,500 f 1,530 16,700 f 1,490 7,500 f 890 4,000 f 460

5,100 f 540

ND' ND'

B. Factors affecting formation of salt and sarkosyl stable complexes DNA bound to filter

Variation from standard assay [5-aza-C]DNA Unsubsti-

tuted DNA

cpm None 55,400 f 2,140 5,200 f 420 Addition of 0.6% 890 f 90 800 f 110

DNA digested with 52,000 f 1,530 190 f 20

DNA heated to 100 "C and 56,500 f 1,810 51,200 f 1,970

Sarkosyld

S1 nuclease'

quick-chilled Additions to the assay mixture after 20-min incubation at 37 "C.

The assay mixtures were brought to the final NaCl and Sarkosyl concentrations indicated prior to filtration on nitrocellulose. The wash fluids contained the same concentration of NaCl and Sarkosyl as the assay mixture.

The incubated assay mixture was brought 0.05% SDS and allowed to sit for 10 min at 25 "C. After a 10-fold dilution (to 0.005% SDS), the sample was filtered and washed with 0.005% SDS. 0.005% SDS was not sufficient to disrupt preformed complexes regardless of whether the assay mixture was diluted or not.

E ND, not determined. Sarkosyl was added to the incubation mixture before 0.3 M NaCl

nuclear extract protein. e Conditions given under "Experimental Procedures."

the 5m-Cyt content of DNA does not appear to be a critical factor in determining complex stability. Protein complexes formed with native hypomethylated DNA isolated from Friend erythroleukemia cells grown in the presence of L- ethionine (3.15 & 0.1% 5m-Cyt (28)) or native Escherichia coli DNA ((0.3% 5m-Cyt (29)) were as sensitive to dissociation with 0.5 M NaCl and 0.6% Sarkosyl as were complexes with native unsubstituted Friend erythroleukemia cell DNA (3.7 f 0.05% 5m-Cyt-8), suggesting that the low level of methylation of DNA synthesized during 5-aza-Cyd treatment of Friend erythroleukemia cells (8) does not account for its ability to form stable complexes.

The marked difference in stability of protein binding to [5- aza-CIDNA and unsubstituted DNA was abolished by heat denaturation of DNA. Denatured ss DNA, regardless of whether it contained 5-aza-Cyt residues or not, remained

bound to nuclear proteins in the presence of 1 M NaCl and 0.6% Sarkosyl (Table I, B). This result raised the possibility that nuclear proteins formed more stable complexes with native [5-aza-C]DNA than with native unsubstituted DNA because the [5-aza-C]DNA contained more single-stranded regions than the native unsubstituted DNA. However, several lines of evidence argued against this. First, 5-aza-Cyt-enriched regions of DNA were no more susceptible to digestion with S1 nuclease than unsubstituted DNA (Table 11). Second, digestion of native [5-aza-C]DNA with S1 nuclease had little or no effect on its capacity to form salt- and Sarkosyl-resistant complexes (SSRC) with nuclear proteins, while S1 nuclease digestion of native, unsubstituted DNA essentially abolished its residual capability for forming SSRC with nuclear proteins (Table I, B). Third, the kinetics of SSRC formation with nuclear proteins differed for ss DNA and native [5-aza-C] DNA. This was most obvious when SSRC formation was slowed at low temperature; ss DNAs rapidly formed SSRC at 0 "C while SSRC formation with native [5-aza-C]DNA at 0 "C was barely detectable (Table 111). No significant differences occurred between ss unsubstituted and ss [5-aza-C]DNAs with regard to temperature dependence of SSRC formation indicating that 5-aza-Cyt residues in single-stranded regions, in contrast to those in native DNA, made, at best, a minor contribution to stability of protein binding.

On the basis of the results shown in Tables 1-111, all native DNAs utilized in subsequent studies of protein complex formation were treated with S1 nuclease prior to assay to remove any ss DNA regions and, unless otherwise stated, 0.5 M NaCl and 0.6% Sarkosyl was added to all dissociation buffers.

Evidence That NaC1- and Sarkosyl-resistant Binding of Nuclear Proteins to ds DNA Is Dependent on the Presence of 5-aza-Cyt Residues-If, as indicated by the results described above, SSRC formation is dependent on the presence of 5- aza-Cyt residues per se and not on s,ome other alteration of DNA occurring during 5-aza-Cyd treatment, then it should be possible to demonstrate that SSRC formed by incubation of nonuniformly 5-aza-Cyt-substituted ds DNA with nuclear proteins have specifically sequestered a fraction of DNA enriched in 5-aza-Cyt residues. Conversely, the fraction of DNA

TABLE I1 Susceptibility of [5-aza-C]DNA to digestion with SI nuclease

DNAs were digested with S1 nuclease and radiolabel in acid- precipitable DNA determined as described under "Experimental Pro- cedures." For heat denaturation, DNA solutions were incubated at 100 "C for 15 min and quick-chilled on ice. Values are the average of quadruplicate determinations f S.E.

Acid-insoluble material DNA

[4-"C]5-aza-Cyd0 thymidineb ['HIMethyl

CPm Native 5-aza-Cyt-

substituted No additions 7,800 f 150 31,350 f 500 S1 nuclease 8,000 f 190 30,550 f 520

Heat-denatured 5-aza-Cyt- substituted

No additions 7,980 f 140 29,500 f 510 S1 nuclease 650 f 100 1,400 f 100

No additions 35,400 f 650 S1 nuclease 34,600 f 530

a DNA isolated from Friend erythroleukemia cells grown for 4 h in medium containing 10 p~ [4-"C]5-aza-Cyd.

DNA isolated from Friend erythroleukemia cells grown for 4 h in medium containing 10 p~ 5-aza-Cyd and [3H]methyl thymidine or 13H]methyl thymidine alone.

Native unsubstituted


TABLE I11 Effect of temperature on formation of salt- and Sarkosyl-resistant

complexes with native and heat-denatured DNA Assay conditions are described in Table I. Values represent an

average of triplicate determinations in three separate experiments. Identical results were obtained when AdoMet was omitted from the reaction mixture.

Fraction radiolabeled DNA retained on filtef

0 "C 37 "C

5min 30min 5 min 30 min

Native DNA 5-aza-Cyt-substituted 0.03 0.058 0.87 0.93 Unsubstituted 0.005 0.013 0.04 0.12

5-aza-Cyt-substituted 0.36 0.76 0.74 0.86 Unsubstituted 0.37 0.62 0.62 0.82

Heat-denatured DNA

Fraction DNA in SSRC after incubation with 0.3 M NaCl extract = (cpm retained on filter after treatment with NaCl and Sarkosyl/ cpm of DNA retained on filter without addition of NaCl and Sarko- syl). The amount of extract protein used was sufficient to mediate filter binding of all of the radiolabeled DNA in the absence of NaCl and Sarkosyl treatment.

which can be freed of protein by NaCl and Sarkosyl treatment should be relatively low in 5-aza-Cyt content. This assump- tion could be experimentally tested because the ratio of protein to DNA in SSRC was high enough to allow separation of protein.DNA complexes from free DNA by sedimentation through preformed, discontinuous CsCl/Cs2S04 gradients. Nonuniformly substituted ds [5-aza-C]DNA, specifically radiolabeled in 5-aza-Cyt-rich regions, was isolated from Friend erythroleukemia cells cultured for 4 h (-25% of a cell cycle) in medium containing either [4-14C]5-aza-Cyd or [3H]methyl thymidine and unlabeled 5-aza-Cyd. As can be seen in Fig. 1A, after incubation with nuclear extract protein and subsequent treatment with 0.5 M NaC1,0.6% Sarkosyl, 85-90% of this DNA (detected and quantitated by its absorbance at 260,,) sedimented into the bottom half of a CsCl/Cs2S04 gradient (fractions 6-8) at the same position as ds DNA which had not been incubated with nuclear protein. However, incubation with nuclear proteins led to a reduction of approximately 85% in the specific activity of the DNA recovered in Fractions 6-8 which could be accounted for by the finding that more than 80% of the radiolabeled DNA was bound in SSRC which floated near the top of the gradient? In contrast, when ds DNA from cells cultured for 4 h with [3H]methyl thymidine in the absence of 5-aza-Cyd was incubated with nuclear extract protein and treated with 0.5 M NaC1, 0.6% Sarkosyl, more than 95% of radiolabel sedimented to Frac- tions 6-8 (Fig. 1B) and the DNA in these gradient fractions showed no significant change in specific activity as a result of incubation with nuclear extract protein.

Evidence That Formation of NaC1- and Sarkosyl-resistant ds 5-aza-Cyt DNA. Protein Complexes Does Not Involve Mod- ification or Degradation of 5-aza-Cyt Residues-Since our aim was to study binding of DNA methyltransferase to [5-aza-C] DNA, the initial incubation conditions chosen for binding assays were identical to those employed for studies of DNA methyltransferase activity including addition of AdoMet at

After deproteinization, radiolabeled [5-aza-C]DNA isolated from the top of the gradient shown in Fig. 1A co-sedimented with native DNA on 5-20% sucrose gradients and coelectrophoresed with native DNA on 1% agarose gels (data not shown). This indicates that the failure of radiolabeled [5-aza-C]DNA to sediment into CsCl/Cs2SOI gradients was not the result of fragmentation during incubation with nuclear extract proteins. See also Table IV.

F R A C T I O N

FIG. 1. Selective formation of SSRC with ds [5-aza-C]DNA demonstrated by CsCl/Cs2SO, density gradient analysis. Panel A, DNA radiolabeled by incubating Friend erythroleukemia cells for 4 h with 10 p~ [4-14C]5-aza-Cyd (O-----O) or 10 p~ 5-aza-Cyd and [3H]methyl thymidine (A-A). Panel B, DNA radiolabeled by incubating Friend erythroleukemia cells for 4 h with [3H]methyl thymidine (A-A). Each DNA sample (25 pg) was incubated with 0.3 M NaCl nuclear extract protein (1 mg) and treated with 0.5 M NaCl and 0.6% Sarkosyl prior to centrifugation on a CsCl/Cs2SO4 gradient containing 0.5 M NaCl and 0.6% Sarkosyl. Details as given under "Experimental Procedures." Results of a typical experiment are illustrated. Values shown are direct reading of A2wnm and cpm/lOO-pl aliquot of a 500-pl fraction. Recovery of input DNA in Fractions 6-8 as determined by absorbance at 260 nm (0- - -0) was 85% for 5-aza- Cyt DNA and 89% for unsubstituted DNA. Specific activity of [3H] methyl thymidine-labeled DNA from 5-aza-Cyd-treated cells was 1.1 x lo6 cpm/A2w., and from untreated cells, 0.99 X lo6 cpm/AZmnm. Specific activity of DNA in from Fraction 7, Panel A, 173,000 cpm/ Am,,,; and from Fraction 7, Panel B; 880,000 cpm/Azw,.

10 p ~ . Under these conditions, binding of nuclear proteins to ds [5-aza-C]DNA was shown to be dependent on temperature (Table 111), on amount of nuclear extract protein added (Fig. 2A), and on time of incubation (Fig. 2B). However, no dependence of binding on AdoMet could be demonstrated. Bind- ing of ds [5-aza-C]DNA to nuclear proteins occurred at essentially the same rate and to the same extent whether or not AdoMet was added (Fig. 2). To rule out the possibility that low levels of AdoMet present in the nuclear extract were sufficient to account for binding, AdoHcy, the product of AdoMet after methyl transfer and a competitive inhibitor of AdoMet binding to methyltransferases (reviewed in Ref. 30), was added to the reaction mixture. At the highest concen- trated tested, 10 mM, AdoHcy had no inhibitory effect on binding. The finding that neither AdoMet nor AdoHcy affect the kinetics of SSRC formation indicated that neither binding of AdoMet to nuclear proteins nor enzymatic transfer of methyl groups were required for formation and stabilization of SSRC.

To determine whether any other modifications occurred in 5-aza-Cyt-rich regions of DNA during incubation with nuclear extract proteins that might account for their ability to form SSRC, such as ring scission of 5-aza-Cyt residues or removal of 5-aza-Cyt (by N-glycosylases or endonucleases), the fate of radiolabeled ds [5-aza-C]DNA in SSRC was examined. As shown in Table IV, neither [3H]methyl thymidine in 5-aZa- Cyt-rich regions of DNA nor [4-'4C]5-aza-Cyt residues them- selves were released in a trichloroacetic acid-soluble form when ds [5-aza-C]DNA was incubated in the presence of nuclear extract proteins with or without added AdoMet. Thus, neither specific loss of 5-aza-Cyt residues nor specific degradation of 5-aza-Cyt-rich regions in DNA occurred during

Azacytosine in DNA and Binding of Specific Nuclear Proteins

F” 300

00

80 z - 0 3

s 0

60

40

20

Time (min) FIG. 2. Dependence of SSRC formation with ds [5-aza-C]DNA on concentration of 0.3 M NaCl

nuclear extract protein (A) and time of incubation ( B ) in the presence of 10 WM AdoMet (A), 10 mM AdoHcy (0), or without these additions (0). All incubation mixtures contained 5 pg of ds [5-aza-C]DNA. For A, incubation time was 20 min at 37 “C. At 300 pg of protein, the amount of DNA bound with AdoMet orAdoHcy present was the same as that bound in their absence. For B, ds [5-aza-C]DNA was incubated with 150 pg of extract protein for the indicated times. All other details as under “Experimental Procedures.”

TABLE IV Comparative stability of [5-aza-C]DNA and unsubstituted DNA

during incubation with 0.3 M NaCl nuclear extract protein 5 pg of the indicated DNA was incubated with 150 pg of nuclear

extract protein with or with addition of 10 p~ AdoMet. After 20-min incubation at 37 “C, the incubation mixture was chilled and brought to a final concentration of 10% with trichloroacetic acid. Incubation conditions and determination of radiolabel in trichloroacetic acid- insoluble material is as described under “Experimental Procedures.” Values are average f S.E. for triplicate determinations in two experiments. 3H-labeled ds [5-aza-C]DNA was prepared from cells grown in the Dresence of both 5-aza-Cvd and r3H1methy1 thymidine.

Acid-insoluble material DNA radiolabeled with

[4-14C]5.aza-Cyd [3H]Methy’ thvmidine

ds [5-aza-C]DNA No additions + Nuclear protein + Nuclear protein + AdoMet

ds unsubstituted DNA No additions + Nuclear protein + Nuclear protein + AdoMet

7,855 f 190 28,150 f 610 7,960 f 170 29,500 f 690 7,790 f 215 28,000 f 630

33,150 k 920 36,600 f 1,100 34,500 f 980

formation of SSRC. Further, radiolabel incorporated into DNA from 5-aza-Cyd could be recovered as 5-aza-dCyd from DNA that had been involved in SSRC formation. The same amount of radiolabeled 5-aza-dCyd was recovered from [5- aza-CIDNA isolated from SSRC formed in the presence or in the absence of AdoMet and this amount did not differ significantly from that recovered from ds [5-aza-C]DNA incubated in the absence of proteins (Fig. 3). This indicated that exten- sive modification and/or degradation of 5-aza-Cyt residues were not determining factors in SSRC formation. HPLC analysis did reveal the presence of some radiolabeled break-

4063

100

80

z 60

- 5 0 z

4 0 *

20

down products of 5-aza-dCyd among the separated nucleosides derived from digests of [5-aza-C]DNA. The breakdown products comprised 15% or less of the total radiolabeled material when nucleoside digests were analyzed immediately after preparation but increased in proportion as the digests were aged for 24-48 h. Since 5-aza-Cyd, 5-aza-dCyd, and their phosphorylated intermediates are highly unstable in aqueous solution (31-33) and since storage of DNA for 1-2 months at 4 “C did not markedly alter the yield of 5-aza-dCyd from ds [5-aza-C]DNA,3 it is most likely that these products arose during or subsequent to enzymatic hydrolysis of DNA to nucleosides.

Evidence That Specific Proteins Are Involved in Binding of ds [5-aza-C]DNA in SSRC-Proteins present in 0.3 M NaCl nuclear extracts, whether derived from Friend erythroleukemia (murine) or HL-60 (human) cell nuclei, were found to be capable of forming SSRC with ds [5-aza-C]DNA (Table V). However, neither histones nor cytoplasmic proteins were able to mediate significant NaCl and Sarkosyl resistant filter binding of radiolabeled ds [5-aza-C]DNA. These results indicate that SSRC formation with ds [5-aza-C]DNA specifically requires non-histone nuclear proteins and, since proteins in extracts from human cells can bind DNA from murine cells, that at least some of the proteins involved can bind [5- aza-CIDNA from a heterologous source. Approximately 6 times more HL-60 than Friend erythroleukemia cell nuclear protein is required to bind the same amount of ds [5-aza-C] DNA in SSRC (Table V). Thus, the possibility that some of the binding proteins are species-specific cannot be ruled out. However, 0.3 M NaCl extracts of HL-60 cell nuclei have only 10% the level of active DNA methyltransferase found in Friend erythroleukemia cell nuclear extracts (16), so the ob-

J. K. Christman, N. Schneiderman, and G. Acs, unpublished observation.


? 0 i

E a 0

0 s

VO + 20 -

-

15-

-

10-

5AzoCR 4

SAzoCdR + 1

" IO 20 30 40 FRACTION

FIG. 3. HPLC analysis of radiolabeled derivatives of [4-"C] 6-ma-Cyd recovered from ds 15-aza-CIDNA before (0) and after (0 and X) SSRC formation. ds [5-aza-C]DNA, purified from nuclei of Friend erythroleukemia cells grown for 4 h in the presence of 10 p~ [4-"C]5-aza-Cyd, was enzymatically hydrolyzed to deoxyribonucleosides without further treatment (0) or after purification from SSRC formed by incubating the DNA with 0.3 M NaCl nuclear extract proteins in the presence (0) and absence (X) of 10 p~ AdoMet. Deoxyribonucleosides were separated by chromatography on a Spher- isorb 10-pm octadecylsilane column (4.6 mm, inner diameter, X 50 cm) under isocratic elution conditions with 0.75 M sodium phosphate, pH 5.5 (flow rate, 0.5 ml/min). Half ml fractions were collected directly into scintillation vials; radioactivity was quantitated in a liquid scintillation counter after addition of aqueous fluor (Liquiscint, National Diagnostics). Arrows indicate the retention time (1 fraction = 1 min) of 5-aza-Cyd and 5-aza-dCyd added to the nucleotide samples. No radiolabeled material was detected in fractions 41-125 (not shown). All details of chromatography and sample preparation are as given in Ref. 25 with the exceptions that 1) samples were never exposed to alkali or acid treatment to avoid degradation of 5-aza-Cyt residues in DNA and 2) DNA was hydrolyzed to deoxyribonucleosides by addition of alkaline phosphatase digestion to allow comparison of retention times of radiolabeled products with those of commercially available markers. However, similar elution patterns are obtained by HPLC analysis of deoxyribonucleotides with 5-aza-dCMP eluting in 16 min. 5AzaCR, 5-aza-Cyd; iiAzaCdR, 5-aza-dCyd.

served difference may simply reflect lower levels of specific ds [5-aza-C]DNA binding proteins.

All of the protein preparations tested, including preparations of purified histones, were able to mediate some degree of SSRC formation with ss DNA (Table V) but all failed to form SSRC with ds unsubstituted DNA. These results clearly demonstrated that proteins exist which are capable of forming of SSRC with ss DNA which do not form SSRC with ds [5-

TABLE V Specificity for non-histone nuclear proteins in formation of SSRC 5 pg of DNA was incubated for 20 min at 37 "C with the indicated

proteins prior to treatment with NaCl and Sarkosyl as described under "Experimental Procedures." FL, Friend erythroleukemia.

Additions to assay to filter DNA bound

pg cpm (% input)

Calf thymus histone 70 1,150 (2.8) FL cell histone 70 1,340 (3.3) FL cell cytoplasm 660 427 (1.1) FL cell 0.3 M NaCl nuclear extract 48 26,540 (66) HL-60 cell 0.3 M NaCl nuclear extract 165 12,961 (32) HL-60 cell 0.3 M NaCl nuclear extract 330 25,330 (63) No added protein 260 (0.6)

FL cell histone 70 1,300 (5.1) FL cell cytoplasm 660 182 (0.7) FL cell 0.3 M NaCl nuclear extract 48 540 (2.1) HL-60 cell 0.3 M NaCl nuclear extract 165 1,250 (4.9) No added protein 60 (0.2)

Calf thymus histone 70 49,550 (100) FL cell histone 70 48,950 (99) FL cell histone 25 13,410 (27) FL cell cytoplasm 660 7,170 (14) FL cell nuclear extract 48 27,790 (56) No added protein 40 (0.1)

ds [5-aza-C]DNA and

ds unsubstituted DNA and

ss unsubstituted DNA and

Protein content determined using Bio-Rad protein dye reagent.

aza-CIDNA and served as an additional indication that the altered binding capacity of ds [5-aza-C]DNA is not the result of localized denaturation of DNA in 5-aza-Cyt-rich regions.

To further characterize those proteins capable of forming SSRC with ds [5-aza-C]DNA, the proteins in 0.3 M NaCl extracts of Friend erythroleukemia cell nuclei were fractionated according to their affinity for AdoHcy (Fig. 4). The extracts were dialyzed to remove salts and loaded onto a column of agarose-AdoHcy. Proteins with little or no affinity for AdoHcy were eluted with NaC1-free buffer. This fraction contained 40-50% of the total extract protein and -40% of the protein capable of binding ss DNA in SSRC. Unless the binding capacity of the column was exceeded, neither DNA methyltransferase activity nor proteins capable of forming SSRC with ds [5-aza-C]DNA were detected in this eluant. They remained bound to the column and were only eluted as the ionic strength of the elution buffer was increased by a gradient-wise addition of NaCl. The fractions with the highest concentration of active DNA methyltransferase also had the highest concentration of ds [5-aza-C]DNA SSRC binding protein (Fig. 4). However, not all ds [5-aza-C]DNA SSRC binding proteins coeluted with DNA methyltransferase activity and, depending on the fraction, the ratio of DNA methyltransferase activity to ds [5-aza-C]DNA SSRC binding capacity varied widely. Approximately 20-30% of the total capacity for SSRC formation with ds [5-aza-C]DNA coeluted with a group of proteins with lower affinity for AdoHcy than DNA methyltransferase which had no detectable DNA methyltransferase activity. Thus, with a simple fractionation procedure, it was possible 1) to separate non-histone nuclear proteins lacking the ability to form SSRC with ds [5-aza-C] DNA from those capable of forming SSRC with ds [5-aza-C] DNA and 2) to demonstrate that proteins which are incapable of catalyzing methyl transfer to DNA can still form SSRC with ds [5-aza-C]DNA. Although significant enrichment of particular protein species was accomplished by this procedure, none of the fractions examined was homogeneous. All contained multiple protein species which could be visualized by

Azacytosine in DNA and Binding of Specific Nuclear Proteins 4065 TABLE VI

Competition between ds or ss unsubstituted DNAs and ak [5-ata-C] DNA for binding of nuclear proteins

20

15

U/ml I O

5

0

f ’\

NaCl (mM) FIG. 4. Elution pattern of DNA methyltransferase (M),

ss DNA SSRC binding protein (A-----A), and ds 16-aza-CIDNA SSRC binding protein (0- - -0) from agarose AdoHcy. Di- alyzed 0.3 M NaCl nuclear extract protein (15-20 mg) was loaded onto a 1.5 X 15-cm column of agarose AdoHcy (Bethesda Research Laboratories) equilibrated with imidazole buffer (25 mM imidazole, pH 7.5, 1 mM dithiothreitol, 5 mM EDTA, and 0.1 mM phenylmeth- ylsulfonyl fluoride). The column was washed with this buffer and fractions were collected until all unbound protein was eluted. (Ap- proximately 40-50% of total extract protein was present in these fractions which also contained -40% of the ss DNA SSRC binding activity). No ds [5-aza-C]DNA binding or DNA methyltransferase activity was detected. At this point a linear NaCl gradient elution (from 50 to 300 mM NaCl in imidazole buffer) was initiated and 1-ml fractions were collected. The molarity of NaCl in each fraction was determined with a conductivity meter and found to increase by 5 mM/fraction. After dialysis, fractions were tested for methyltransferase activity, capacity for binding ss DNA in SSRC, and capacity for binding ds [5-aza-C]DNA in SSRC as described under “Experimental Procedures.” No addition binding proteins or DNA methyltransferase activity were recovered by eluting the column with 1 M NaCl in imidazole buffer. One unit of DNA methyltransferase activity is defined as the amount of enzyme which transfers 1 pmol of methyl groups/l5 min to DNA isolated from Friend erythroleukemia cells grown in the presence of L-ethionine (8); one unit of DNA binding capacity is defined as the amount of extract required to give 50% maximal binding of the radiolabeled fraction of the indicated DNA in SSRC. Data obtained from a typical fractionation are illustrated. The DNA methyltransferase activity in this fraction is not separated from proteins that form SSRC with ds [5-aza-C]DNA by molecular sieving on Ultrogel ACA 34A (data not shown).

Coomassie Blue staining after SDS-polyacrylamide gel elec- trophoresis of reduced denatured protein. Thus, a much more rigorous purification procedure will be required before it can be unequivocally determined which proteins are actually involved in SSRC formation with ds [5-aza-C]DNA and what other functions they perform in the nucleus. However, the predominant protein band in the fraction with maximal capacity for forming SSRC with ds [5-aza-C]DNA and with DNA methyltransferase activity was a species of 175,000

For direct competition (experiment I), radiolabeled ds [5-aza-C] DNA and nonradiolabeled competing DNA were mixed prior to addition of 0.3 M NaCl nuclear extract protein and incubated for 20 min at 37 “C. For preincubation experiments, either the nonradiolabeled competing DNA (experiment 2) or the radiolabeled ds [5-aza- CIDNA (experiment 3) was incubated with 0.3 M NaCl nuclear extract protein for 10 min before adding radiolabeled ds [5-aza-C]DNA (experiment 2) or nonradiolabeled competing DNA (experiment 3), respectively. Incubation was then continued for 20 min. Incubation mixtures contained 2.5 pg of radiolabeled ds [5-aza-C]DNA, competitor in the indicated proportion, and an amount of 0.3 M NaCl extract experimentally determined to bind 50% of the ds [5-aza-C]DNA in SSRC in the absence of competitor. Radiolabeled [5-aza-C]DNA in SSRC was quantitated as described under “Experimental Proce- dures.” Data shown are from a typical experiment with values repre-

beled ds [5-aza-C]DNA bound in SSRC did not increase by more senting the average of triplicate determinations. Amount of radiola-

than 10% when incubations were continued for up to 2 h. ds [5-aza-C]DNA bound to filter

Competing DNA Ratio competitor:& [5-aza-C]DNA 0 1 0.51 1:l 2:l

cpm 1. Direct competition

ss unsubstituted 14,300 11,100 6,430 595 ds unsubstituted 14,200 13,200 11,380 9,836

2. Nuclear proteins preincubated with competing DNA

ss unsubstituted 5,640 1,620 325 ds unsubstituted 8,583 5,525 3,918

3. Nuclear proteins preincubated with ds [5-aza-C]DNA

ss unsubstituted 14,200 ds unsubstituted 14,400

daltons, a molecular mass similar to that reported for Friend erythroleukemia cell DNA methyltransferase I1 (34). This 175,000-dalton protein was not detected in fractions which contained proteins with capacity for binding ds [5-aza-C]DNA in SSRC but which lacked DNA methyltransferase activity.’

Interaction of ds [5-aza-C]DNA SSRC Binding Proteins with Unsubstituted DNA-The fractionation procedures described above allowed isolation of non-histone nuclear proteins which could form SSRC with ss DNA but which were incapable of forming SSRC with ds [5-aza-C]DNA. However, none of the fractions that contained proteins able to form SSRC with ds [5-aza-C]DNA were free of ss DNA SSRC binding capacity. This suggested the possibility that proteins involved in formation of SSRC with ds [5-aza-C]DNA might also have a high affinity for ss unsubstituted DNA. The competition experiments summarized in Table VI show that ss unsubstituted DNA could indeed effectively compete with ds [5-aza-C]DNA for binding of proteins that form SSRC with ds [5-aza-C]DNA. Significant reduction of ds [5-aza-C] DNA binding in SSRC (>50%) was observed with direct competition between equimolar amounts of ss unsubstituted DNA and ds [5-aza-C]DNA for limited amounts of nuclear extract protein (Table VI). When nuclear extract proteins were allowed to bind ta ss unsubstituted DNA before ds [5-

‘ J. Christman, N. Schneiderman, and G . Acs, unpublished results. SDS-polyacrylamide gel electrophoretic analysis of proteins from these fractions released from SSRC with [5-aza-C]DNA by SDS treatment reveals the presence of multiple molecular weight species making it seem unlikely that inactivated DNA methyltransferase that has retained its ability to bind to ds [5-aza-C]DNA in SSRC is the major component of this fraction.


aza-CIDNA was added to the reaction mixture, inhibition of SSRC formation with ds [5-aza-C]DNA was even more dra- matic (>85%). Qualitatively similar results were obtained when nuclear proteins were preincubated with ds unsubstituted DNA (Sl nuclease-treated to remove any ss DNA regions), although 2-4 times more ds DNA was required to give an inhibition of ds [5-aza-C]DNA SSRC formation comparable to that mediated by ss DNA. When nuclear proteins were preincubated with ds [5-aza-C]DNA, neither ss nor ds unsubstituted DNA was able to compete off ds [5-aza-C]DNA SSRC binding proteins. These results indicate that the ds [5- aza-CIDNA SSRC binding proteins are capable of binding to both ss and ds unsubstituted DNA and that once bound to any DNA, ss or ds, substituted or unsubstituted, they do not freely exchange under incubation condition^.^ This means that, in contrast to all of the other experiments described here, which measured affinity of proteins for DNA on the basis of their ability to resist dissociation and remain bound to DNA in the presence of NaCl and Sarkosyl, these experiments measured binding affinity of proteins for DNA in terms their ability to associate with DNA. It can be concluded that, under standard incubation conditions, [5-aza-C]DNA SSRC binding proteins associated more readily with ss DNA than with ds DNA. Further, their ability to bind to ds unsubstituted DNA was clearly less than their ability to bind to ds [5-aza- CIDNA. However, it was sufficiently high as to allow ds unsubstituted DNA to compete with ds [5-aza-C]DNA for protein binding. Since the ds [5-aza-C]DNAs utilized in these studies had a low level of 5-aza-Cyt substitution (0.3-1% of Cyt residues), this would indicate a high probability that the bulk of proteins capable of forming SSRC with ds [5-aza-C] DNA became associated with unsubstituted rather than with the small fraction of 5-aza-Cyt-substituted regions of DNA during the initial binding step. Nevertheless, after incubation at 37 "C, 5-aza-Cyt residues were found to be quantitatively bound in SSRC (Fig. 2B) . As will be discussed, this suggests that formation of SSRC with 5-aza-Cyt residues in DNA depends on some step subsequent to the initial binding interaction between DNA and protein.

Effect of 5-aza-Cyd Treatment on ds [5-aza-C]DNA SSRC Binding Proteins-The experiments described here demonstrated that introduction of 5-aza-Cyt residues into DNA produced unique and specific alterations in the interaction between DNA and non-histone nuclear proteins. However, to determine whether these changes could be relevant in terms of 5-aza-Cyd's action in uiuo, it was still necessary to demonstrate that similar binding of proteins to DNA would occur in the nuclei of 5-aza-Cyd-treated cells. For this purpose, 0.3 M NaCl extracts from Friend erythroleukemia cells grown in the presence and absence of 5-aza-Cyd were compared with respect to 1) DNA methyltransferase activity, 2) level of ss DNA SSRC binding protein, and 3) level of ds [5-aza-C]DNA SSRC binding protein. The data presented in Table VI1 confirm our earlier results (8,16) and show that, although the total yield of non-histone nuclear proteins that can be extracted from nuclei with 0.3 M NaCl is not significantly different, the level of active DNA methyltransferase in nuclear extracts from 5-aza-Cyd-treated cells is less than 10% that in extracts from untreated cells. They also show that extracts from nuclei of 5-aza-Cyd-treated cells are markedly depleted with respect to ds [5-aza-C]DNA SSRC binding

In the experiments illustrated in Table VI, the time allowed for proteins to exchange from one DNA to another after prebinding was 20 min. However, when the period allowed for exchange was increased to 2 h, the amount of radioalbeled ds [5-aza-C]DNA bound in SSRC did not change by more than 10%.

TABLE VI1 Effect of in vivo exposure to 5-aza-Cyd on levels of DNA

methyltransferase and DNA bindingproteins in 0.3 M NaCl nuclear extracts

Data shown are from a typical experiment and are calculated from an average of three determinations using protein concentrations in the range of linear response to added nuclear extract. Cells were seeded at 1 X 105/ml. When a cell density of 6-8 X 10' cells/ml was achieved, 5-aza-Cyd was added at 2 PM to half of the culture with the other half serving as control. All cultures were harvested 18 h after addition of 5-aza-Cyd. In three separate experiments, DNA methyltransferase activity was reduced to 4-10% of control and native [5- aza-CIDNA binding capacity to 25-38% of control. The overall yield of nuclear extract protein was not significantly affected by 5-aza-Cyd treatment. In the experiment presented here, extract from 108 control cells and 108 5-aza-Cyd-treated cells contained 960 and 1040 pg of protein, respectively (determined with Bio-Rad protein dye reagent).

unitslmg protein (% control)'

DNA methyltransferase Control (no 5-aza-Cyd) 32.9 5-aza-Cyd-treated cells 1.8 (5.5%)

Control 12.3 5-aza-Cyd-treated cells 3.9 (32%)

Control 9.4 5-aza-Cyd-treated cells 10.1 (107%)

ds [5-aza-C]DNA binding capacity

ss DNA binding capacity

a Units are defined in Fig. 4 legend.

proteins but that the level of proteins capable of forming SSRC with ss unsubstituted DNA is undiminished or even slightly increased. However, it can also be seen (Table VII) that the levels of active DNA methyltransferase and [5-aza- CIDNA binding proteins were not equivalently reduced, with -30% of the [5-aza-C]DNA binding activity remaining in extracts with <6% the DNA methyltransferase activity of untreated cells. These results emphasize, as did the fractionation experiments shown in Fig. 4, that proteins without DNA methyltransferase activity can act as ds [5-aza-C]DNA SSRC binding proteins. They further support the hypothesis that the same proteins that form SSRC with [5-aza-C]DNA in vitro bind tightly to [5-aza-CIDNA in viuo and are thus resistant to extraction?

DISCUSSION

As we have previously documented, the primary mechanism by which 5-aza-Cyt residues in DNA inhibit DNA methylation is by inactivation of DNA methyltransferase and not through an inability to be methylated when substituted for Cyt residues in methylation sites (8). The findings 1) that active DNA methyltransferase cannot be extracted from nuclei of 5-aza- Cyd-treated cells and 2) that DNA methyltransferase can be inactivated by incubation with ds [5-aza-C]DNA in the absence of AdoMet suggested that binding of the enzyme to 5- aza-Cyt residues in DNA might play a role in its inactivation (8, 20). The results reported here demonstrate that incorporation of 5-aza-Cyt residues into DNA does indeed alter its interactions with non-histone nuclear proteins. This alteration has been experimentally defined in vitro by showing that ds [5-aza-C]DNA forms complexes with nuclear proteins that

More rigorous extraction procedures (1 M NaCI) failed to increase the yield of ds [5-aza-C]DNA SSRC bindingproteins, andcytoplasmic proteins from 5-aza-Cyd-treated cells were as incapable of forming SSRC with ds [5-aza-C]DNA as cytoplasmic proteins from untreated cells. Absence of inhibitors was indicated in experiments where 0.3 M NaCl extracts of nuclei from 5-aza-Cyd-treated and untreated Friend erythroleukemia cells were mixed and gave additive binding of ds [5-aza-C]DNA in SSRC.

Azacytosine in DNA and Binding of Specific Nuclear Proteins 4067

are resistant to dissociation with NaCl and Sarkosyl while ds unsubstituted DNA does not. However, the observations 1) that the only proteins with the capacity to form SSRC with ds [5-aza-C]DNA are non-histone nuclear proteins and 2) that the same proteins which form SSRC with [5-aza-C]DNA i n vitro are depleted from the nuclear extracts of cells grown in the presence of 5-aza-Cyd strongly suggest that the altered protein binding capacity of [5-aza-C]DNA plays an important and specific role in mediating in vivo effects of 5-aza-Cyd and 5-aza-dCyd.

That DNA methyltransferase is involved in SSRC formation is indicated by the findings l) that active DNA methyltransferase always copurifies with [5-aza-C]DNA SSRC binding capacity (although the converse is not true), 2) that loss of DNA methyltransferase activity in 5-aza-Cyd-treated cells is always paralleled by loss of [5-aza-C]DNA binding capacity, and 3) that both the level of active DNA methyltransferase and the ds [5-aza-C]DNA SSRC binding capacity of nuclear extract proteins from HL-60 cells is less than those in Friend erythroleukemia cells. However, the finding that non-histone nuclear proteins without detectable DNA methyltransferase activity form SSRC with ds [5-aza-C]DNA further suggests that incorporation of 5-aza-Cyt residues into DNA may affect gene expression not only by inhibiting DNA methyltransferase but also through changing the affinity of interactions between genes and the proteins that regulate their expression.

For example, the activation of the metallothioneine-1 gene in 5-aza-Cyd-treated cultured cells before one round of DNA replication has occurred (35) is difficult to account for on the basis of altered methylation patterns unless one assumes either that loss of methylation in one strand of DNA is sufficient to allow gene activity or that hemimethylated sites already exist in the gene (14). However, rapid activation would be predicted if limiting amounts of proteins involved in re- pressing active expression of this gene were prevented from interacting with its regulatory sequences because they bound with equal or higher affinity to 5-aza-Cyt residues in newly synthesized DNA (without regard to sequence). If, as has recently been proposed (36, 37), heritable changes in gene activity can be mediated through formation of stable complexes between nuclear proteins and regulatory sequences that are preserved during DNA replication, the possibility also arises that 5-aza-Cyt residues in ds DNA could trigger heritable changes in chromatin configuration and gene activity by binding proteins that maintain the “open” or transcriptionally active configuration of genes with an affinity comparable to ss DNA. Such a mode of action might explain why 5-aza-Cyd treatment can induce heritable alteration in phenotype at high frequency in organisms that do not have detectable levels of 5m-Cyt in their DNA (38).

This does not imply that the effect of 5-aza-Cyt residues in DNA on DNA methyltransferase activity and the subsequent specific loss of methyl groups from sites in newly activated genes are irrelevant in establishing heritable changes in gene expression. Rather, it suggests that 5-aza-Cyt can affect more than one of the components in an interdependent hierarchy of modifications in DNA and chromatin structure involved in epigenetic determination of gene activity.

It is clear that as a group the proteins that form SSRC with ds [5-aza-C]DNA are normally bound relatively tightly to DNA in the cell (i.e. extraction with 0.3 M NaCl is required to release them from nuclei of untreated cells), that they bind readily to both ss and ds DNA in vitro, and that they have a higher affinity for ss than ds DNA. Furthermore, in solutions of low ionic strength, once bound to DNA, these proteins remain bound and do not readily exchange from one DNA

molecule to another. With no measurable exchange of proteins between DNA molecules, it is difficult to account for our observation that some non-histone nuclear proteins are able to quantitatively and specifically form SSRC with the low level of 5-aza-Cyt residues in our DNA preparations (>1% of Cyt residues) on the sole basis of differences in the binding constants of the proteins to substituted and unsubstituted regions of DNA. However, if bound proteins diffuse freely along unsubstituted DNA, those with a high affinity for 5- aza-Cyt would be “trapped” when they encountered one of the rare 5-aza-Cyt residues in the DNA. This two-step binding mechanism would be consistent with the time and temperature dependence observed for SSRC formation.

It should be noted that this type of interaction with DNA is characteristic of DNA methyltransferase isolated from mammalian cells. At the low ionic strength required for assay of enzyme activity, DNA methyltransferase binds to ss DNA more readily than to ds DNA and its binding affinity appears independent of the 5m-Cyt content of the DNA (19,39, 40). Under in vitro conditions, DNA methyltransferase remains bound to DNA during the methylation process but can be released at elevated ionic strength (39). Thus, denatured or native fully methylated DNA can compete with unmethylated substrate DNAs for binding of the enzyme and inhibit their methylation. However, when DNA methyltransferase is bound to an unmethylated substrate DNA before competing DNAs are added, no inhibition of its ability to methylate the substrate to which it is bound is detected. This result suggested that DNA methyltransferase moves processively or diffuses along its DNA substrate as it methylates available sites (19, 34, 39).

Although hemimethylated DNA containing 5-aza-Cyt residues is an excellent substrate, preincubation of DNA methyltransferase with ds [5-aza-C]DNA (in the absence of AdoMet) not only prevents the enzyme from methylating other DNAs but leads to a loss of the enzyme’s capacity for methylating the [5-aza-C]DNA to which it is bound (19-21), results which imply that with time its ability to move along the substrate has been inhibited. In view of our results, the simplest interpretation of this phenomenon is that, in diffus- ing along the DNA, the enzyme has encountered and become tightly bound to a 5-aza-Cyt residue or has been blocked from moving because another protein is tightly bound to a 5-aza- Cyt residue in its path. Since we have not yet been able to recover active DNA methyltransferase from its complexes with ds [5-aza-C]DNA, and thus cannot rule out the possibility that an irreversible change in the enzyme has been cata- lyzed by its interaction with 5-aza-Cyt residues, it is prema- ture to conclude that high affinity binding alone is sufficient to account for the observed loss of DNA methyltransferase activity. With recent advances in purification of DNA methyltransferase (34, 40, 41) it should soon be possible to isolate sufficient amounts of pure enzyme protein to allow its reiso- lation after interaction with S-aza-cyt residues in DNA and thus to determine directly what changes have occurred in its structure. However, our findings that AdoMet is not required for either SSRC formation or DNA methyltransferase inactivation and that SSRC can be disrupted with SDS already argue against one proposed mechanism for enzyme inactivation, formation of stable covalent linkages between DNA methyltransferase and 5-aza-Cyt residues (42).

In summation, our results suggest that tight binding of DNA methyltransferase and other non-histone proteins to 5- aza-Cyt residues in DNA plays an important role in mediating the i n vivo effects of 5-aza-Cyd and 5-aza-dCyd. They also pose the question of how cells that survive 5-aza-Cyd treat-


ment deal with proteins that are bound to 5-aza-Cyt residues 18. Stein, R., Gruenbaum, J., Pollack, Y., Razin, A., and Cedar, H. in their DNA. Do most of the proteins remain bound to DNA (1982) P m . Natl. Acad. Sci. U. S. A. 79,61-65

block in DNA and RNA synthesis observed after prolonged treatment with 5-aza-Cyd? Are bound proteins eventually

of S-Adenosylmethionine and Related Compounds (Usdin, E.,

released from the DNA by proteolytic digestion so that normal Borchardt, R., and Creveling, C. R., eds) pp. 223-229, Macmil- lan, London

cell function Can be resumed? It is hoped that ongoing at- 21. Bouchard, J., and Momparler, R. L. (1983) Mol. Phurmucol. 2 4 ,

tempts to purify and raise antibodies to DNA 22. Friend, C., Patuleia, M. C., and deHarven, E. (1966) Natl. Cancer ase and other non-histone proteins that bind to [5-aza-C] DNA will provide the answers. 23. Collins, S. G., Gallo, R. C., and Gallagher, R. E. (1977) Nature

REFERENCES 24. Christman, J. K., Price, P., Pedrinan, L., and Acs, G. (1977) Eur.

during replication? Are bound proteins responsible for the 20. Christman, J. K., creusot, F., and G. (1982) in ~ i o ~ h e ~ k ~ ~ 19. Taylor, S., and Jones, P. A. (1982) J. Mol. Biol. 162,679-692

109-114

Znst. Monogr. 22,505-522

(LO&.) 270,347-349

I. Liacouris, A. S., and Anderson, E. P. (1979) Mol. Pharmacol. 15,

2. Li, L. H., Olin, E. J., Buskirk, H. H., and Reinke, L. M. (1970)

3. Lee, T. T., and Karon, M. R. (1976) Biochem. Phurmacol. 26 ,

4. Lu, L.-J. W., and Randerath, K. (1980) Cancer Res. 40, 2701-

5. Friedman, S. (1979) Biochem. Biophys. Res. Commun. 8 9 , 1328-

6. Jones, P. A., and Taylor, S. M. (1980) Cell 20,85-93 7. Tanaka, M., Hibasami, H., Nagai, J., and Ikeda, T. (1980) Aust.

8. Creusot, F., Acs, G., and Christman, J. K. (1982) J. Biol. Chem.

9. Zadraiil, S., FuEik, V. Bartl, P., Sormova, Z., and Sorm, F. (1965)

10. Viegas-Pequingnot, E., and Dutrillaux, B. (1976) Hum. Genet.

11. Karon, M., and Benedict, W. F. (1972) Science 178 ,62 12. Rivard, G. E., Momparler, R. L., Demers, J., Benoit, P., Raymond,

R., Lin, K., and Momparler, L. F. (1981) Leukemia Res. 6,453- 462

13. Jones, P. A., Taylor, S. M., and Wilson, V. (1983) J. Exp. Zool.

14. Christman, J. K. (1984) Curr. Top. Microbiol. Immunol. 108,49-

15. Hoffman, R. M. (1984) Biochim. Biophys. Acta 738,49-87 16. Christman, J. K., Mendelsohn, N., Herzog, D., and Scheiderman,

17. Busslinger, M., Hurst, J., and Flavell, R. A. (1983) Cell 34, 197-

331-340

Cancer Res. 30,2760-2769

1737-1742

2705

1333

J. Exp. Biol. Med. Sci. 68,390-396

267,2041-2048

Biochim. Biophys. Acta 108 , 701-703

34,247-254

228,287-295

78

N. (1983) Cancer Res. 43 , 763-769

206

J. Biochem. 81,53-61 25. Jeffreys, A. J., and Flaveil, R. A. (1977) Cell 12, 429-439 26. Blankstein, L. A., Stollar, B. D., Franklin, S. G., Zweidler, A.,

27. Shaw, J. L., Blanco, J., and Mueller, J. C. (1975) Anal. Biochem.

28. Christman, J. K. (1982) Anal. Biochem. 119,38-48 29. Shapiro, H. S. (1976) in Handbook of Biochemistry and Molecular

Biology (Fasman, G. D., eds) pp. 268-275, Chemical Rubber Co., Cleveland

30. Borchardt, R. T. (1977) in Biochemistry of Adenosylmethionine (Salvatore, F., Borek, E., Zappia, V., Williams-Ashman, H. G., and Schlenk, F., eds) pp. 151-171, Columbia University Press, New York

31. Notari, R. E., and DeYoung, Y. L. (1975) J. Phurm. Sci. 6 4 ,

32. Lin, K. T., Momparler, R. L., and Rivard, G. E. (1981) J. Phurm.

33. Zeilinski, W. S., and Sprinzl, M. (1984) Nucleic Acids Res. 12 ,

34. Bestor, T. H., and Ingram, V. M. (1983) Proc. Natl. Acad. Sci.

35. Compere, S. J., and Palmiter, R. D. (1981) Cell 2 6 , 233-240 36. Groudine, M., and Weintraub, H. (1982) Cell 3 0 , 131-139 37. Brown, D. D. (1984) Cell 37,359-365 38. Tamame, M., Antequera, F., Villanueva, J. R., and Santos, T.

(1983) Mol. Cell. Biol. 3 , 2287-2295 39. Drahovsy, D., and Morris, N. R. (1971) J. Mol. Biol. 6 1 , 343-356 40. Wang, R. Y.-H., Huang, L.-H., and Ehrlich, M. (1984) Nucleic

41. Pfeifer, G. P., Griinwald, S., Boehm, T. L. J., and Drahovsky, D.

42. Santi, D. V., Garrett, C. E., and Barr, P. J. (1983) Cett 33,9-10

and Levy, S. B. (1977) Biochemistry 16,4557-4562

6 6 , 125-131

1148-1157

Sci. 70,1228-1232

5025-5036

U. S. A. 80,5559-5563

Acids Res. 12,3473-3490

(1983) Biochim, Biophys. Acta 740,323-330

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