Planta (1991)184:487~90

P l a n t a 9 Springer-Verlag1991

5-Azacytidine-induced hypomethylation of tobacco HRS60 tandem DNA repeats in tissue culture Milan Bezd~k, Bla~ena Koukalovd, Bietislav Brzobohat~, and Boris Vyskot Institute of Biophysics, CzechoslovakAcademyof Sciences, CS-612 65 Brno, Czechoslovakia Received 6 October 1990; accepted 28 January 1991

Abstract. The methylation status and 5-azacytidineinduced hypomethylation of CCGG sites within a family of tandemly organized, highly repeated DNA sequences of the Nicotiana tabacum L. nuclear genome (HRS60 family) were studied. As shown by in-situ hybridization experiments, the HRS60 family is clustered in a few regions of some tobacco chromosomes. The DNAs of leafderived calli, leaf-derived calli cultured on media with 5-azacytidine, and leaves were cleaved with restriction endonucleases differing in the sensitivity to the methylation of cytosine. After electrophoresis and Southern blotting they were hybridized with the HRS60 probe. We show that (i) CpG dinucleotides, and partially also CpCpG trinucleotides, of the HRS60 family are methylated in DNAs of the non-treated calli and leaves, and (ii) that these DNA repeats are sensitive to the action of a hypomethylating drug, 5-azacytidine. Key words: 5-Azacytidine - DNA methylation - DNA repeat (tandem) - Hypomethylation - Nicotiana (hypomethylation of DNA) - Tissue culture (DNA methylation)

Introduction Plant DNAs are highly methylated at cytosine residues. In contrast to mammalian DNAs, where 5-methylcytosine is the sole modified base and is found exclusively in the deoxycytidine-phosphate-deoxyguanosine (CpG) dinucleotide, in plant DNAs methylated cytosine (mC) can exist in CpG and CpXpG (X stands for any deoxyribonucleoside) sequences (Gruenbaum et al. 1981). Many recent studies have documented a negative correlation between the level of methylation and gene expression (Cedar 1988; Riggs and Chrispeels 1990). There are several studies indicating differential methylation of rDNA repeats and some evidence that hypomethylation Abbreviations: azaC=azacytidine; bp=basepairs; mC=methyl-

ated cytosine; CpG=deoxycytidine-phosphate-deoxyguanosine; X (in CpXpG)= any deoxyribonucleoside

of a specific region in the intergenic spacer could regulate the expression of the rDNA genes (Ellis et al. 1983; von Kalm et al. 1986; Blundy et al. 1987; Jupe and Zimmer 1990). There may also be differences in the methylation status of the DNA of developed plant organs and dedifferentiated callus (Reddy et al. 1988; LoSchiavo et al. 1989; Brown 1989; Brown et al. 1989; Vergara et al. 1990). Silent genes can be reactivated if the level of DNA methylation is reduced, for example by the treatment of cells with the methylation inhibitor, 5-azacytidine (azaC). Examples of the reactivation of foreign genes integrated into the plant genome by azaC treatment were recently published (Amasino et al. 1984; Van Slogteren et al. 1984; Hepburn et al. 1987; Vyskot et al. 1989). The results concerning the demethylating effects of azaC on inherent plant genes are very limited and essentially negative (Brown et al. 1989). Repeated DNA sequences, including rDNA genes (Scott et al. 1984) and, in particular, satellite DNAs, are usually heavily methylated (Adams and Burdon 1985). Actually nothing is known about the possibility of inducing hypomethylation in any type of DNA repeat in plants. To examine this problem we used the HRS60 family of DNA repeats of Nicotiana tabacum in which the 182 to 184-bp monomeric units share a high degree of mutual homology. The HRS60 units are tandemly organized and comprise about 2% of the tobacco nuclear genome (Koukalovfi et al. 1989; Matyfigek et al. 1989). They are present almost exclusively in the N. sylvestris component of the N, tabacum additive genome (Koukalov~ et al. 1990). This paper describes the methylation pattern of the HRS60 family of DNA repeats and reports, for the first time to our best knowledge, that the methylation pattern of this type of repeat can be changed by culturing the plant tissues on media containing azaC.

Materials and methods Plant material. Leaves and leaf-derivedcalli of N. tabacum L. cv.

Vielblgttriger (kindly purchased from the Tobacco Research Institute, Bfib, Czechoslovakia)plants were used.

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Callus cultures and azaC treatment. Leaf disks were removed and callus growth was induced on MS media (Murashige and Skoog 1962) supplemented with 0.2 rag" 1-1 benzylaminopurine (BAP) and 2 mg. 1-1 u-naphthalene-acetic acid (NAA). For the azaC treatments ofcalli, 50 mg- 1-1 5-azacytidine was added. The DNAs of calli formed in these conditions were subjected to analyses. Preparation of DNA. Total DNA from leaves was isolated according to Dellaporta et al. (1983) and total DNA from freeze-dried calli was extracted as described by Saghai-Maroof et al. (1984). Digestion by restriction endonuclease. The DNAs were digested with an excess of restriction enzymes to obtain complete digests. A control of digestion completeness was made using a chloroplastDNA probe as an internal standard. Gel electrophoresis and DNA/DNA hybridization were carried out according to standard procedures (Maniatis et al. 1982). HRS60.1 DNA probe. A 182-bp member of the HRS60 family of DNA repeats (Koukalovfi et al. 1989) was used. The probe was labelled using a multiprime DNA labelling system with 32p (for hybridizations on membranes) or with 3H (for in-situ hybridizations). In-situ DNA/DNA hybridization. Squashes of cellulase- and pectinase-treated root tips were digested with RNase and chromosomal DNA denatured in 0.15 N NaOH in 70% ethanol at room temperature. The 3H-labelled, heat-denatured HRS60.1 DNA sequence (1.7 MBq - gg-i DNA) was used as a DNA probe. The hybridization was run in the mixture according to Ambros et al. (1986) for 16 h at 37~ C. The post-hybridization treatment consisted of washings with 2 • SSC, 2 x 2 min at room temperature, 50% formamide in 2x SSC, 2x 5 min, 37~ C, and 2x SSC, 1 x 2 min at room temperature, (1 • SSC=0.15 M NaC1, 0.015 M trisodium citrate, pH 7.0). The slides were covered with the Ilford L4 autoradiographic emulsion and, after a one-week exposure and developing, stained with aceto-orcein.

Results Induction o f hypomethylation o f H R S 6 0 D N A repeats in azaC-treated calli. Two restriction endonucleases, MspI and H p a I I , were used to examine the methylation of C p G dinucleotides in the HRS60 repeats. Both have the c o m m o n recognition C C G G sequence, but they differ in the sensitivity to methylation: C m C G G can be digested with MspI, but not with HpaII, while the sequences m C C G G and m C m C G G cannot be digested with either o f these enzymes. The C C G G sequence has been found in all the members o f the HRS60 family of D N A repeats sequenced till now (Koukalovfi et al. 1989; Maty~i~ek et al. 1989). Deoxyribonucleic acids were isolated from untreated tobacco calli, from the calli maintained on media with azaC, and from leaves. The D N A s were digested with MspI or HpaII, size separated by electrophoresis on 0.7% agarose gels and, after Southern blotting, hybridized with the HRS60.1 probe. The result obtained with the D N A o f the untreated calli is given in Fig. 1, where the MspI cleavage o f the HRS60 repeats was apparent and a characteristic ladder of hybridization bands with 182to 184-bp monomeric units and their multiples could be seen (lane 2). However, upon H p a I I digestion, the HRS60 repeats remained in a fraction of uncleaved "rel-

Fig. 1. Hypomethylation of the HSR60 DNA repeats induced by 5-azacytidine. The DNA of the untreated tobacco callus (lanes 1-3) and DNA of the callus held on azaC (lanes 4-6) were digested with restriction endonucleases, size-separated on 0.7% agarose gels, Southern-blotted and probed with the HRS60.1 DNA repeat. The restriction endonucleases used were: lanes 1, 6, BamHI; lanes 2, 4, Mspl; lanes 3, 5, HpaII. The sizes (in kilobases, kb) of HindIII-cleaved )~-DNA standard markers are shown to the right

ic" D N A (lane 3). This result proved that cytosines in C p G dinucleotides, in most C C G G sites, were methylated in the HRS60 repeats. In the case of the MspI restrict (lane 2), aside from a ladder, a prominent smear of an uncleaved D N A was visible. This suggests that either the first cytosine in the C C G G sequence is also methylated in a fraction of HRS60 repeats, or some C C G G cleavage sites were lost in the course of the HRS60 evolution. A direct p r o o f could not be provided as no enzyme digesting the m C C G G sequence was available. Some discrimination of these possibilities could be provided using azaC treatment (see below). Just for comparison, a B a m H I digestion pattern is shown. B a m H I also cleaved HRS60 repeats into monomeric units and their multiples. Only a small proportion of HRS60 repeats remained uncleaved (Fig. 1, lanes 1 and 6). The D N A of leaves, cleaved with MspI or H p a I I and probed with the HRS60.1 probe, gave hybridization patterns very similar to those of the D N A of untreated calli (Fig. 2). Thus, the methylation pattern of the C p G dinucleotides (and perhaps also C p C p G trinucleotides) in C C G G sequences of HRS60 repeats was essentially equal both in differentiated and in dedifferentiated tissues. In contrast to the previous results, the D N A isolated from calli held on the medium containing azaC became partially sensitive to the digestion with HpaII. Hybridization with the HRS60.1 probe shows the characteristic ladder of hybridization bands up to monomers (Fig. 1, lane 5) revealing the azaC-induced hypomethylation of CpG dinucleotides at C C G G sites, though a substantial part of the HRS60 remained uncleaved. Yet a comparison of the MspI restrict of the D N A from

M. Bezd6ket al.: 5-Azacytidine-inducedhypomethylationof DNA repeats

489

residues at the first position in the CCGG sequences of the HRS60 repeats is methylated. N o n - r a n d o m distribution o f H R S 6 0 repeats. According to

Fig. 2. Comparison of the methylation patterns of DNA isolated from tobacco leaves and calli. The DNAs of leaves (lanes 1 and 2) and calli (lanes 3 and 4) were digested with restriction endonucleases and further treated as given in the legend to Fig. 1. The restriction endonucleasesused were: lanes 1, 3, MspI; lanes 2, 4, HpaII. The sizes (in kb) of HindIII-cleaved E-DNA standard markers are shown to the right

Fig. 3a, b. In-situ hybridization with the 3H-labelled HRS60.1 probe of N. tabacum cv. Vielblfittriger.a Interphase nuclei; b rectaphase chromosomes. Bars= 10 Bm azaC-treated calli with that of untreated calli (Fig. 1, lanes 4 and 2, respectively) shows that in the azaCtreated callus the relative amount of uncleaved DNA is smaller than that in the untreated callus. This result indicates that in untreated calli a fraction of cytosine

our previous results, the HRS60 repeats are clustered in the tobacco nuclear genome (Koukalovfi et al. 1989). The clustering of HRS60 was further confirmed here by the results of in-situ hybridizations with the 3H-labelled HRS60.1 probe (Fig. 3). A majority of hybridization signals were found in the telomeric regions of some chromosomes. Discussion

Highly reiterated satellite DNAs have, as a rule, evolved from short DNA sequences by amplification and are usually located in constitutive heterochromatin regions at or around centromeres and telomeres (Bostock 1980). Numerous reports indicate that satellite-DNA sequences are more highly methylated than moderately repeated and unique sequences (Adams and Burdon 1985). Spontaneous demethylation of various plant genomic sequences in tissue culture (LoSchiavo 1989; Brown et al. 1989) including satellite DNA (Reddy et al. 1988) has been reported. Little is known about the methylation process itself, namely what concerns the recognition of the particular DNA sequences and discrimination of methylated and non-methylated genomic domains. There could be various kinds of methylation targets. One group of them could be foreign genes of transgenic organisms and crown-gall tumours. Plant transgenes and transferred T DNA genes are frequently inactivated by methylation, but azaC treatment can restore their activity (Amasino et al. 1984, van Slogteren 1984; Hepburn et al. 1987; Vyskot et al 1989). Another group of DNA methylation targets could be inherent structural genes, their regulatory sequences and other genomic domains, for example various repeated DNA sequences. The results presented in this paper show that HRS60 tandem repeats, preferentially clustered in telomeric regions of some chromosomes, are methylated at CpG dinucleOtides and CpCpG trinucleotides of CCGG sites in the DNA of both differentiated (leaves) and dedifferentiated (callus) tissues. After the treatment of calli with azaC, CCGG sites within the HRS60 family became partially sensitive to cleavage by methylationsensitive restriction endonuclease HpaII and more sensitive to MspI. However, the yield of hybridization bands was significantly lower for HpaII (Fig. 1, lane 5), as compared with the DNA digested by the isoschisomer MspI (Fig. 1, lane 4). The simplest explanation of this result would be that CmCGG sequences prevail over the mCCGG sequences, and that demethylations of both sequences in HRS60 repeats occur randomly, perhaps only in a fraction of the cells. It is thought that both the methylation status of tandemly repeated sequences and their location within the genome could be a part of the chromatin-folding code (Vogt 1990). Our finding that azaC influence the level of

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m e t h y l a t i o n o f the well-defined t a n d e m r e p e a t s H R S 6 0 , c l u s t e r e d o n l y in a few r e g i o n s o f s o m e t o b a c c o c h r o m o s o m e s , m a y c o n t r i b u t e to the a s s e s s m e n t o f the biological role o f these sequences. We would like to thank Dr. J. Fajkus and Dr. J. Reich (Institute of Biophysics, Brno) for their suggestion of a method of checking the completeness of plant DNA digestion, Professor J. Schell (Max~ Planck Institute, Cologne, FRG), Dr. J. So~ka and Dr. J. Kypr (Institute of Biophysics, Brno) for critical reading of the manuscript, and H. Such/mkov/t and L. Jedli6kov~ for technical assistance.

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