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(The FASEB Journal. 1999;13:1518-1522.)
© 1999 FASEB

Inhibition of poly(ADP-ribosyl)ation introduces an anomalous methylation pattern in transfected foreign DNA

GIUSEPPE ZARDO*, STEFANIA MARENZI{dagger}, MARIAGRAZIA PERILLI* and PAOLA CAIAFA*,{ddagger}1

* Department of Biomedical Sciences and Technologies, University of L'Aquila;
{dagger} Department of Biological Science `A. Rossi-Fanelli', University of Rome `La Sapienza'; and
{ddagger} C.N.R. Centre for Molecular Biology, Rome, Italy

1Correspondence: Dipartimento di Scienze e Tecnologie Biomediche, Università dell'Aquila; Via Vetoio, Loc. Coppito, I-67100 L'Aquila, Italy. E-mail:caiafa{at}axscaq.aquila.infn.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this paper is to verify whether the control played by poly(ADP-ribosyl)ation on genomic DNA methylation, and in particular on CpG islands, can also be seen on foreign DNA transfected in cells where inhibition of the poly(ADP-ribosyl)ation process was obtained by treating them with 2 mM 3-aminobenzamide for 24 h. The CpG island-like pVHCk plasmid containing the bacterial chloramphenicol acyltransferase (CAT) gene under the control of SV40 early promoter was transfected in L929 mouse fibroblast cells. The bisulfite reaction, which is capable of immortalizing the methylation state of cytosine on DNA, was performed before amplification of the plasmid DNA fragment, then used for sequence analysis. Our results have shown that 1) when transfected in control cells, the plasmid maintains its characteristic unmethylated pattern, whereas this pattern is lost when the plasmid is transfected in cells treated with 3-aminobenzamide; and 2) the presence of new methyl groups on plasmid DNA is paralleled by a decrease of CAT reporter gene expression. These data confirm that poly(ADP-ribosyl)ation is a process tightly involved in protecting genomic DNA from full methylation and suggest the use of 3-aminobenzamide as a possible experimental strategy to mime other conditions of DNA hypermethylation in cells.—Zardo, G., Marenzi, S., Perilli, M., Caiafa, P. Inhibition of poly(ADP-ribosyl)ation introduces an anomalous methylation pattern in transfected foreign DNA.


Key Words: poly(ADP • ribose) polymerase • 5 • methylcytosine • 3 • aminobenzamide • transfection


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EXPERIMENTS PERFORMED IN order to examine the possible correlation between DNA methylation and poly(ADP-ribosyl)ation processes, particularly whether or not the inhibitory effect exerted by H1 histone on in vitro enzymatic DNA methylation (1) could essentially be due to the poly(ADP-ribosyl)-ated isoform of this protein, have shown that the poly(ADP-ribose)-free isoform of H1 histone failed to inhibit in vitro DNA methylation when added up to a protein/DNA ratio of 0.25 (w/w), whereas the poly(ADP-ribosyl)ated one was highly inhibitory under the same conditions (2) .

In vivo experiments carried out on L929 mouse fibroblasts preincubated for 24 h with or without 3-aminobenzamide (3-ABA),2 a well-known inhibitor of poly(ADP-ribose) polymerase (3) , have confirmed a significant correlation between poly(ADP-ribosyl)ation and DNA methylation processes. This suggests that the poly(ADP-ribosyl)ation process somehow acts as a protecting agent against full methylation of CpG dinucleotides on genomic DNA. In fact, blocking of the poly(ADP-ribosyl)ation process has caused in isolated nuclei a consistent increase in DNA susceptibility to be methylated by endogenous DNA methyltransferase such that the subsequent DNA methylation by exogenous enzymes was severely reduced (2) . Furthermore, working on single cell nuclei in interphase (using 5-methylcytosine monoclonal antibodies as a probe), we found that some methyl groups insert themselves on DNA even during the incubation period with 3-ABA (4) .

We examined the possibility that the poly(ADP-ribosyl)ation reaction is involved in maintaining the unmethylated state of CpG islands. This was necessary as these DNA regions (5 , 6) , which are found almost exclusively at the 5' end of housekeeping genes, are rich in those CpG dinucleotides, which are the target of mammalian DNA methyltransferase. Two different strategies (7) were used in these experiments: methylation-dependent restriction enzymes on purified genomic DNA and a sequence-dependent restriction enzyme on an aliquot of same DNA, previously modified by bisulfite reaction (8) . In the first approach, we observed that the "HpaII tiny fragments" greatly decrease when the cells are preincubated with 3-ABA. The second approach showed an anomalous methylation pattern on the CpG island of the promoter fragment of Htf9 gene that had been amplified from DNA obtained from fibroblasts preincubated with 3-ABA. These data confirm the hypothesis that at least for the Htf9 promoter region, an active poly(ADP-ribosyl)ation protects the unmethylated state of the CpG island.

To emphasize the role played by the poly(ADP-ribosyl)ation process in maintaining the methylation pattern in genomic DNA and to gain further insight into the mechanisms involved, we conducted parallel experiments in order to verify whether the inhibition of poly(ADP-ribosyl)ation process can also affect the methylation pattern of transfected unmethylated foreign DNA.

Our results show that 1) the plasmid transfected in control cells maintains its characteristic unmethylated pattern, this pattern being lost when the plasmid is transfected in cells treated with 3-aminobenzamide; and 2) the presence of new methyl groups on plasmid DNA is paralleled by a decrease of bacterial chloramphenicol acyltransferase (CAT) reporter gene expression. The use of 3-aminobenzamide can therefore be suggested as an experimental strategy to mime conditions of physiological or pathological DNA hypermethylation in cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transfection and analysis of CAT activity
Lipofection (DOTAP, Boehringer, Mannheim, Germany) was used to transfect 2.5 µg of pVHCk plasmid (constructed by Stefan Kass) into 3 x 105 L929 and NIH/3T3 mouse fibroblast cells, which, when growing exponentially, were divided into subcultures and treated differently with or without 2 mM 3-ABA (Sigma, St. Louis, Mo.) for 24 h (2 , 4 , 7) . Cell extracts were prepared at 24 and 48 h post-transfection and CAT activity was assayed according to Seed and Sheen (9) . The medium with or without 2 mM 3-ABA was replaced 6 h after transfection.

Bisulfite reaction
The DNA was extracted from nuclei (2 , 4 , 7,) , and a Qiagen column [polymerase chain reaction (PCR) purification kit] was used to remove genomic DNA and obtain a partially purified pVHCk DNA. Plasmid DNA was eluted with KpnI digestion buffer (New England BioLabs, Beverly, Mass.) and digested by addition of 10 units of restriction enzyme at 37°C for 3 h. Linearized DNAs were then used for the bisulfite reaction, which was carried out according to Frommer's method (8) , resuspended in 100 µl of previously barren MilliQ H2O, and stored at -80°C.

PCR amplification and DNA sequencing
Ten microliters were used to amplify by PCR the pVHCk bisulfite-modified DNA fragment. This pair of modified primers was used to amplify DNAs purified from cells preincubated either with or without 3-ABA.

The sequence of primers was as follows:

Forward: 5'-ATAGGTATATTGAGTAATTGATTGAAAT-3'

Reverse: 5'-ATCTCAATTCAACAACCAAATATAAAAA-3'

The PCR mixture contained 50 pmol of primer reverse, dNTPs (final concentration was 0.2 mM), and 2.5 U Taq DNA polymerase (Qiagen, Chatsworth, Calif.) in 67 mM Tris-HCl (pH 8.8), 6.7 mM MgCl2, 170 mg/ml bovine serum albumin, 16.6 mM ammonium sulfate, and 0.5 mM tetramethylammoniumchloride. The reaction (50 µl) was carried out under the following conditions: denaturation at 96°C/5 min, 95°C/1 min, 49.5°C/1 min 30 s, 72°C/1 min 30 s for 10 cycles; and a final cycle of 95°C/1 min, 49,5°C/2 min, and 72°C/6 min. Five microliters of this first amplification reaction were subjected to a second amplification cycle in the presence of both primers (40 pmol both in forward and reverse), 0,2 mM dNTPs (final concentration), 1.3 mM MgCl2 in Boehringer buffer plus 2.5 U of Taq DNA polymerase. Now the reaction (50 µl) was carried out as described previously, but for 35 cycles. The reaction product was loaded and electrophoresed on 2% agarose gel and DNA fragment purified by QUIAEX II gel extraction kit (Qiagen). The fragment was sequenced after quantitative densitometric determination by ABI PRISM 377 automated sequencer (Perkin-Elmer, Norwalk, Conn.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To study the role played by the poly(ADP-ribosyl)ation process in maintaining the methylation pattern on genomic DNA, we examined whether a foreign DNA, transfected in L929 mouse fibroblast cells treated for 24 h with 2 mM 3-ABA, changes its characteristic unmethylated pattern as a consequence of the inhibition of the poly(ADP-ribosyl)ation process. Plasmid pVHCk, having SV40 early promoter linked to the CAT gene, was used in our experiments since this prokaryotic vector, which contains a total of 216 CpG pairs, is "CpG island-like" (10) . The DNA fragment used for sequence analysis (Fig. 1 A) had undergone bisulfite reaction before amplification (8) , which is capable of immortalizing the methylation state of cytosines on DNA. As can be seen (Fig. 1B ), inhibition of poly(ADP-ribosyl)ation process inactivated the control mechanism involved in maintaining methylation pattern on DNA. In fact, when the plasmid is transfected in cells where the poly(ADP-ribosyl)ation process was previously inhibited, an anomalous methylation pattern characterizes its DNA. In this new pattern, nearly all cytosines (not only those present in CpG dinucleotides) are now methylated.



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  		Figure 1. Dependence of plasmid DNA methylation level on poly(ADP-ribosyl)ation. A) pVHCk construct with the relative amplified region. B) Sequence of the DNA fragment in its unmethylated transfected form (pVHCk), sequence of DNA fragment amplified from cells untreated with 3-ABA (CT), sequence of DNA fragment amplified from cells treated with 3-ABA for 24 h and analyzed at 24 h after transfection (3ABA-24), and sequence of DNA fragment amplified from cells treated with 3-ABA for 24 h and analyzed at 48 h after transfection (3ABA-48). CT, 3ABA-24, and 3ABA-48 samples were amplified after bisulfite reaction; sequenced region corresponds to position 2333–2442 of plasmid.

We also decided to carry out experiments in which pVHCk plasmid, in its unmethylated form, is transfected in L929 and NIH/3T3 cells in order to verify any change in the efficiency of CAT reporter gene expression at 24 and 48 h after transfection in cells pretreated with or without 2 mM 3-ABA for 24 h. The literature reveals that in vitro methylation of plasmid significantly reduces transcription of reporter gene after transfection (10 11 12 13 14 15 16) .

As shown in Fig. 2 , the expression of CAT reporter gene was decreased by ~30% when CAT activity was measured 24 or 48 h after transfection into cells preincubated with 2 mM 3-ABA for 24 h relative to control cells untreated with 3-ABA, considered as 100%.



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  		Figure 2. Dependence of CAT reporter gene expression on poly(ADP-ribose)polymerase inhibition. The amount of CAT activity is expressed relative to the values obtained for untreated cells (control). Each value is the average value of six different experiments in duplicate


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has recently been shown (17 , 18) and debated (19 , 20) that histone deacetylase, through its association with methyl-CpG binding protein MeCP2 (21 , 22) , reaches methylated DNA, allowing the methylation-dependent chromatin condensation that favors gene silencing. The importance of histone deacetylase in inducing chromatin condensation (23 , 24) would seem to need the presence of methyl groups on DNA to be able to carry out its condensing effect. In this situation, an important part is played by MeCP2, a protein able to transfer the deacetylase onto DNA, since through its methyl-CpG binding domain it recognizes methylated DNA and through its transcriptional repression domain (17) it binds a complex containing Med corepressor mSin3A and histone deacetylase, which are associated in vivo (25) .

We intend to show that along the chain of events that induces methylation-dependent chromatin condensation, another epigenetic modification, i.e., poly(ADP-ribosyl)ation, intervenes in a preliminary step when the methylation pattern is established and needs protection.

Our previous data have shown that poly(ADP-ribosyl)ation protects the methylation pattern on genomic DNA (2 , 4) and, in particular, on CpG islands (7) .

The hypothesis that MeCP2, which is able to recognize the methylated DNA, can carry histone deacetylase onto chromatin regions that need deacetylation to be condensed can be used to explain why the inhibition of CAT reporter gene expression in our experiments is not as evident as would be expected considering data in the literature (10 11 12 13 14 15 16) , where inhibition is considered to be dependent on the extent of methylation and the time after transfection. The latter conclusion is interpreted by the idea that this inhibition is mediated by formation of inactive chromatin spreading from a focus of methylation.

Despite the high methylation level present in plasmid transfected into cells in which there was inhibition of poly(ADP-ribosyl)ation, the inhibition of CAT reporter gene expression was clearly less than we expected. However, when plasmid was transfected into cells in which poly(ADP-ribose) polymerase was inhibited, the scenario was different, as several methyl groups were already inserted on the long genomic DNA (4) . As a consequence, the MeCP2 along with deacetylase could now be associated with the long molecule inducing methylation-dependent chromatin condensation.

Our results indicating that there is a low decrease in expression of CAT reporter gene independent of the methylation level can be explained by taking into account the possibility that the above-mentioned proteins, necessary for methylation dependent-chromatin condensation, are now present in small amounts in the cellular environment. This could either be because they have been attracted by genomic DNA or the cell has started to defend itself from this excess of methylation, thus stopping production of some of these proteins.

Our data on the correlation between DNA methylation and poly(ADP-ribosyl)ation processes take place on a backdrop in which a question yet to be solved is to identify different cis-acting (26 27 28 29 30 31 32 33 34) signals and trans-acting protein factors (21 , 22 , 35 36 37 38 39 40 41 42 43 44 45 46) that may play a key role in defining and/or maintaining the bimodal pattern of methylation (47) involved in cell differentiation and gene expression.

Other hypotheses can be formulated that explain how poly(ADP-ribosyl)ation is involved in maintaining DNA methylation pattern apart from the possibility that it is the enzyme itself that modifies its catalytic activity, whether modified or not.

A possible interpretation of our results is that immediately after transfection the plasmid is recognized by some proteic factor(s) that, in its poly(ADP-ribosyl)ated isoform, is capable of linking itself to plasmid DNA, thus protecting it from DNA methyltransferase action. On the contrary, when the same transfection takes place in cells in which the poly(ADP-ribosyl)ation process is inhibited, the above-mentioned protection from methylation does not occur: the same ADP-ribose free protein is unable to inhibit DNA methyltransferase and therefore the enzyme can now carry out its reaction.

Our in vitro research allowed us to consider H1 histone in its poly(ADP-ribosyl)ated isoform (2) and through its genic somatic variant H1e (48 49 50) as a nuclear proteic trans-acting factor involved in maintaining the unmethylated state of CpG islands. That H1 histone is a good substrate for the poly(ADP-ribose)polymerase and the enzyme is capable of modifying it in both a covalent and an uncovalent way (51) bear this hypothesis out.

Another hypothesis is that this postsynthetic modification does not interact directly with DNA through some proteic factor. We suggest that poly(ADP-ribosyl)ation could be involved in modulating the binding of proliferating cell nuclear antigen (PCNA) and DNA methyltransferase (52) .This hypothesis is based on the fact that PCNA can undergo poly(ADP-ribosyl)ation in cells where it can be found in both its unmodified and modified isoforms (53) . DNA hypermethylation dependent on poly(ADP-ribosyl)ation block could be explained by assuming that the unmodified form of PCNA is the only one capable of binding DNA methyltransferase and transferring the enzyme onto DNA. The absence of modification can also be involved in the competition existing between DNA methyltransferase and p21 for the same domain on PCNA (52) .

Much remains to be done to clarify the mechanism by which this influence takes place and to identify the chromatin protein(s) involved in this role and the mechanism required to allow the cells in which there is the knocking-out of poly(ADP-ribose) polymerase to survive.

Both our present and previous results (2 , 4 , 7) indicate that treatment of cells with 3-ABA represents a valid experimental strategy to mime physiological DNA hypermethylation in cells. Therefore, whereas treatment of cells with 5-azacitidine represents a method to obtain hypomethylated DNA (54) , 3-aminobenzamide can offer a method that induces DNA hypermethylation.


   ACKNOWLEDGMENTS
 
This work was supported by the Italian Ministry of University and Scientific and Technological Research (60% Progetti di Ateneo dell'Aquila and "La Sapienza" Roma and 40% Progetti di Interesse Nazionale) and by National Research Center (C.N.R), Italy.


   FOOTNOTES
 
2 Abbreviations: 3-ABA, 3-aminobenzamide; CAT, bacterial chloramphenicol acyltransferase; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction.

Received for publication November 30, 1998. Accepted for publication April 20, 1999.


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 INTRODUCTION
 MATERIALS AND METHODS
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 DISCUSSION
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