Inhibition of Tobacco DNA Methyltransferase in vivo Causes Unequal Hypomethylation of DNA Repeated Sequences
DNA repetitive sequences are characterized with a high content of methylated cytosine (5-methylcytosine, 5-mC). In tobacco genomic DNA about 30% of all cytosine residues are methylated. However, in our previous study it was found that two families of 5S rDNA reiterated sequence have more than 50% of all cytosine residues methylated suggesting that differences in DNA methylation levels between DNA repeated sequences may exist. The aim of this study was to compare the methylation patterns in several repetitive DNA sequences of Nicotiana tabacum nuclear genome as well as their susceptibility to the effect of a hypomethylating drug dihydroxypropyladenine (DHPA). This drug is thought to reduce DNA methyltransferase activity by increasing S-adenosylhomocysteine levels. To detect methylation state in CG and CCG sites in DNA loci studied, methylation-sensitive restriction enzymes (MspI, HapII, Sau3A1 and BamHI) were used.
It was found that within the 5?-CCG-3? triplet, there is almost saturation level of 5-mC at the inner (3?) cytosine position in all DNA repeats studied but variable degrees of methylation at the outer (5?) position. The high copy number repetitive sequences (HRS60, GRS) and 18S-5.8S-25S rDNA showed a significant heterogeneity in methylation of their units. In contrast, the middle repetitive sequences (R8.1, GRD5, 5S rDNA, RPS) were uniformly modified at both cytosine residues. Thus most densily methylated DNA was localised to the middle repetitive fraction of genome rather than to high copy satellites. DHPA treatment resulted in extensive demethylation of outer cytosine in all repeats studied and, at a higher concetration of the drug, partial hypomethylation of cytosines at the inner positions in less densely methylated repeats (HRS60, GRS) occurred. The result suggests that hypomethylation of mCmCG sites with DHPA is a gradual non-random process proceeding in the direction mCmCG -> CmCG -> CCG.
The 18S-5.8S-25S rDNA was remarkably hypomethylated relatively to the 5S rDNA. Fluorescence in situhybridization showed DNA decondensation in both 18S-5.8S-25S and 5S rDNA loci at interphase, but nuclei with maximal decondensed 5S chromatin were still overall more condensed than can be observed for 18S-5.8S-25S rDNA. The 18S-5.8S-25S rDNA chromatin appeared to be more sensitive to digestion with mircococcal nuclease compared to 5Sr rDNA chromatin. Taken together, in tobacco both transcribed ribosomal RNA gene loci differ remarkably in methylation levels and degree of condensation of their chromatin.
Kovarik, A., Koukalova, B., 1Lim, Y.K., Matyasek, R., 1Lichtenstein, C.P., 1Leitch, A.R. and Bezdek, M.
Institute of Biophysics, Academy of Sciences of the Czech Republic,