Gene Therapy and Molecular Biology Volume 4, page 291
Gene Ther Mol Biol Vol 4, 291-296. December 1999.
3-aminobenzamide: a novel drug to induce in vivo
Giuseppe Zardo1, Anna Reale2, Mariagrazia Perilli1, Adriana de Capoa3 and Paola
Department of Biomedical Sciences and Technologies1, University of LâAquila, Italy.
Department of Cellular Biotechnologies and Haematology2 and of Genetics and Molecular Biology3, University of Rome âLa
Correspondence: Prof. Paola Caiafa, Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Biochimica Clinica, FacoltÃ di Medicina e Chirurgia, UniversitÃ di Roma "La Sapienza", Viale Regina Elena, 324 (Policlinico), 00161, Roma, Italia. Tel: +39-06-49910900, Fax: +39-06 4440062, E-mail firstname.lastname@example.org
Key Words: histone hypoacetylation, gene silencing, histone deacetylation, 5-azacytidine, trichostatin A, chromatin
Received: 9 August 1999; accepted: 19 August 1999
Both DNA methylation and core histone hypoacetylation are associated with gene silencing but only recent experiments allowed the interlocking of these two processes. Through such experiments it was shown that the two processes are united in inducing gene silencing through a âshuttle-systemâ involving the methyl CpG binding protein (MeCP2). In this scenario, it is not clear whether DNA methylation or histone deacetylation is the leader in inducing down regulation of gene expression. Trichostatin A (TSA), a potent inhibitor of histone deacetylase, is usually used to clarify this point. As far as DNA methylation is concerned, only the 5-azacytidine (5-AzaC), able to induce hypomethylation, has been described until now. The aim of this paper is to suggest the use of 3-aminobenzamide (3- ABA) as a method capable of inducing in vivo DNA hypermethylation, so that new experiments could be performed in both directions to clarify the chronology by which the influence on gene expression takes place and to pinpoint the structure of methylated condensed chromatin.
DNA methyltransferase (EC 188.8.131.52) is a nuclear enzyme that, by transferring methyl groups from S-adenosyl methionine (S-AdoMet) to cytosine (C) converts these residues into 5-methylcytosine (5mC) (Bestor and Ingram,
1983), the best substrate being the cytosine located in the CpG dinucleotide (Gruenbaum et al, 1981). This epigenetic modification is proposed to have an active role in the modulation of gene expression (Keshet et al, 1985; Boyes
and Bird, 1992; Li et al, 1993; Hsiet, 1994). This role was confirmed by experiments in which an anomalous methylation, caused by targeted mutation of DNA methyltransferase gene in mice, results in embryonic lethality (Li et al, 1992). The DNA methylation pattern, which is defined during embryonic development (Brandeis et al, 1993), is very important since its characteristic is that some DNA regions, located in the 5â promoter region of
housekeeping genes - termed CpG islands (Bird et al, 1985; Bird, 1986; Bird, 1987) - are present in their unmethylated state, this condition being essential for the expression of related genes (Keshet et al, 1985).
A second mechanism by which DNA methylation may be involved in down regulation of gene expression has recently been shown (Jones et al, 1998; Nan, et al, 1998) and debated (Bestor, 1998; Razin, 1998). This mechanism foresees that histone deacetylase, via its association with methyl-CpG binding protein (MeCP2) (Boyes and Bird,
1991; Meehan et al, 1992) reaches methylated DNA allowing the methylation-dependent chromatin condensation favoring gene silencing. Until now it is not clear how cytosine methylation might affect chromatin structure and
much still has to be done to clarify the mechanism by which this influence takes place and to identify whether DNA methylation or histone deacetylation is the post-synthetic modification âleaderâ in inducing gene silencing (Selker,
Zardo et al: 3-aminobenzamide induces in vivo DNA hypermethylation
1998; Eden et al, 1998; Cameron et al, 1999).
Trichostatin A (TSA), a potent inhibitor of histone deacetylase (Yoshida et al, 1995), is usually used to clarify this point. As far as DNA methylation is concerned, only the treatment of cells with 5-azacytidine (5-AzaC), able to induce hypomethylation, has been described until now (Adams and Burdon, 1985). The aim of this paper is to propose the treatment of cells with 3-aminobenzamide as a method to induce in vivo DNA hypermethylation so that new experiments can be performed in two directions in order to both clarify the order by which the influence on gene expression takes place and to pinpoint the structure of methylated condensed chromatin.
II. Treatment of cells with 3- aminobenzamide induces in vivo DNA hypermethylation
The 3-aminobenzamide is a specific inhibitor (Huletsky et al, 1989; Rankin et al, 1989) of the poly(ADP-ribose) polymerase (EC 184.108.40.206), an enzyme able to build and/or transfer ADP-ribose polymers onto chromatin proteins (Jacobson and Jacobson, 1989; de Murcia, et al, 1995).
The statement that following inhibition of poly(ADP- ribose) polymerase DNA methyltransferase becomes able to methylate the unmethylable cytosines on DNA is based on our experiments showing that a block of poly(ADP- ribosyl)ation introduces an anomalous hypermethylated pattern in genomic DNA (Zardo et al, 1997; de Capoa et al,
1999). Although our research was performed in order to
demonstrate that poly(ADP-ribosyl)ation is an important process involved in controlling the expression of housekeeping genes (Zardo and Caiafa, 1998), the aim of this review is to point out that the 3-ABA induced block of this enzymatic process introduces an anomalous hypermethylated pattern on genomic DNA. All experiments were carried out on L929 and NIH\3T3 mouse fibroblast cells and poly(ADP-ribose) polymerase was inhibited by treatment of cells with 2 and/or 8 mM 3-aminobenzamide for 24 hours.
The first evidence came from experiments on endogenous DNA methyl-accepting ability (Zardo et al.,
1997). In these experiments the methyl-accepting ability of
isolated nuclei, obtained from 6.5 x 105 L929 mouse fibroblasts, previously preincubated with or without 3-ABA for 24 hours, was performed in the presence of [3H]-S-
AdoMet. After one hour of incubation at 37Â° C, we compared the ability to incorporate labeled methyl groups in their DNA in the absence of any exogenous DNA methyltransferase (i.e. by a process catalyzed by the endogenous enzyme). The level of incorporated methyl groups, evaluated on the total DNA purified from cells, was found to be 60% higher in the DNA from 3-ABA treated cells than in DNA from control cells whose methyl- acceptance value was taken as 100%, Figure 1.
Figure 1. Methyl-accepting ability experiment. The endogenous methyl-accepting ability of native nuclei, obtained from 6.5 x 106
L929 fibroblasts preincubated for 24 h without (control) and with 8 mM 3-ABA, was performed in the presence of 16 M [3H]-S- AdoMet. The level of methyl groups has been evaluated on the total DNA purified from cells Control DNA, whose incorporation was 2.8 0.1 pmol of [3H]-S-AdoMet, was considered as 100%. Zardo et al. (1997) Biochemistry 36, 7937-7943.
More recently (de Capoa et al, 1999) we have been able to show that during the 24 hours of 3-ABA treatment, interphase nuclei had already incorporated some methyl groups. The cells were indirectly immunolabeled with anti-
5-methylcytosine (anti-5mC) monoclonal antibodies (de Capoa et al, 1996), microscope analysis was performed on a cell-by-cell basis and the images of the nuclei were recorded by a b/w CCD camera. A computer-assisted quantitative analysis of the methylation state of individual interphase nuclei was performed by dedicated software (de Capoa et al,
1998). Cells preincubated with 3-ABA consistently showed
increased levels of anti-5mC antibody binding to heterochromatic regions, Figures 2 and 3.
Thus, both the DNA methyl-accepting assay and monoclonal anti 5-methylcytosine antibodies allowed us to show that reduced levels of poly(ADP-ribosyl)ation result in DNA hypermethylation.
III. Possible interpretation of the mechanism by which poly(ADP- ribosyl)ation controls DNA methylation
These results indicate that poly(ADP-ribosyl)ation protects in some way genomic DNA from full methylation although much still has to be done to explain the molecular mechanism(s). As for the CpG islands, our recent research (Zardo and Caiafa, 1998) has shown that, at least for the Htf9 promoter region, the inhibition of poly(ADP- ribosyl)ation allows the new methyl groups to position
Gene Therapy and Molecular Biology Vol 4, page 293
themselves on DNA. Further experiments have shown that this inhibition also changes the methylation pattern of plasmid transfected in its unmethylated form (Zardo et al,
1999 in press).
Poly(ADP-ribose) polymerase that is dimeric in its catalytic form (Mendoza-Alvarez and Alvarez-Gonzales,
1993), has three domains which play specific roles in the poly(ADP-ribosyl)ation process. The N-terminal domain contains the zinc-finger motifs which are responsible for binding to DNA (Gradwohl et al, 1990), the C-terminal domain contains the catalytic site (de Murcia and Menissier de Murcia, 1994; Rolli et al, 1997) and the central domain is the domain that undergoes automodification (Desmarais et al, 1991). The enzyme starts its automodification following binding of the enzyme to DNA and needs breaks on DNA strands to be activated (de Murcia and Menissier de Murcia,
1994). During the automodification process the ADP-ribose
polymers - up to 200 residues â are built in the 28 automodification sites (Kawaichi et al, 1981; Desmarais et al, 1991) located in this domain. Following automodification, the enzyme can start heteromodification reactions allowing interactions between ADP-ribose polymers and chromatin proteins (Boulikas, 1989; Scovassi et al, 1993). Several proteins (Wesierska-Gadek et al, 1996;
Malanga et al, 1998) can be modified both in a covalent and non-covalent way, the best substrate being H1 histone (Poirier and Savard, 1980; D`Erme et al, 1996; Panzeter et al, 1992; Panzeter et al, 1993; Malanga et al, 1998).
Our in vitro findings show that the H1 histone, in its poly(ADP-ribosyl)ated isoform (Zardo et al, 1997) and through its genic variant H1e (Santoro et al, 1995; Zardo et al, 1996) could be a nuclear proteic trans-acting factor involved in maintaining the unmethylated state of CpG islands. We cannot exclude that other presently unknown proteic factor(s) could also play a regulatory role in the control of DNA methylation by means of this post-synthetic modification.
This work was supported by the Italian Ministry of University and Scientific and Technological Research (40% Progetti di Interesse Nazionale, 60% Ricerca Scientifica UniversitÃ di LâAquila e di Roma, âLa Sapienzaâ) and by the Consiglio Nazionale delle Ricerche (CNR). We thank Alessandra SpanÃ² for technical assistance.
Figure 2. Increased levels of heterochromatin methylation in 3-ABA treated nuclei from mouse fibroblast cell lines as shown by indirect immunolabeling with anti-5MeC antibodies. de Capoa et al. (1999) The FASEB J. 13, 89-93; b/w CCD camera images of control ( a,b) and treated (c,d) nuclei from L929 (left) and NIH/3T3 cell lines (right).
Zardo et al: 3-aminobenzamide induces in vivo DNA hypermethylation
Figure 3. Methylation levels of control and 3-ABA treated mouse fibroblasts in samples of 20 nuclei each. Left: L929 and, right: NIH/3T3 cells. Upper rows: Examples of pseudocolored heterochromatic regions in some control (a, b) and treated (c, d) nuclei. A computer-assisted quantitative analysis of methylation levels in b/w CCD camera images of control and treated nuclei was performed. For each nucleus the methylation state was expressed as area of the methylated regions ( 2) and different levels of optical densities (ODs, 0-185 in the gray scale range). Blue, yellow and red staining indicate, respectively, the heavily, medium and lightly methylated regions per nucleus. Lower rows : Percentages of differentially labeled areas and different optical densities in control and treated nuclei from each sample. Blue, yellow and red staining indicate, respectively, the heavily, medium and lightly methylated regions per sample.
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(From left) Anna Reale, Paola Caiafa & Giuseppe Zardo