Early studies proposed that DNA methylation could have a role in regulating gene expression during development [Riggs, A.D. (1975) Cytogenet. Cell Genet. 14, 9 -25]. However, some studies of DNA methylation in known tissue-specific genes during development do not support a major role for DNA methylation. In the results presented here, tissue-specific differentially methylated regions (TDMs) were first identified, and then expression of genes associated with these regions correlated with methylation status. Restriction landmark genomic scanning (RLGS) was used in conjunction with virtual RLGS to identify 150 TDMs [Matsuyama, T., Kimura, M.T., Koike, K., Abe, T., Nakao, T., Asami, T., Ebisuzaki, T., here is a history of inverse correlation between DNA methylation of CpG island promoter regions and gene expression. Almost all CpG islands on the inactive X chromosome are methylated, and monoallelic methylation of imprinted genes is associated with monoallelic gene expression (1-3). Moreover, programmed changes in DNA methylation are essential features of development, with disruption frequently resulting in aberrant development (4). Although early studies proposed that DNA methylation could have a role in regulating development (5, 6), more recent studies of DNA methylation in known tissue-specific genes during development did not support a major role for DNA methylation. Studies by Warnecke and Clark (7) found that the tissue-specific expression of the skeletal ␣-actin gene in the adult mouse does not correlate with the methylation state of the promoter. Walsh and Bestor (8) investigated the 5Ј methylation status of seven tissue-specific genes and found no correlation with tissue-specific expression. In the study presented here, rather than examining the methylation status of known tissuespecific genes, tissue-specific differentially methylated regions (TDMs) were first identified, and then genes located near the TDMs were analyzed for tissue-specific expression.Restriction landmark genomic scanning (RLGS) is a method for the two-dimensional display of end-labeled DNA restriction fragments (9-11) and can be used to scan for genomic DNA methylation (11). Because the NotI recognition site contains two CpGs and the great majority of NotI sites are within CpG islands, RLGS (with NotI as the restriction landmark) displays CpG islands and adjacent regions. If a NotI site is methylated, it will not be digested and will not be end-labeled, resulting in the absence of the spot in the RLGS profile. Global analysis of genomic DNA methylation of different tissues by using RLGS indicates that there are a relatively large number of differences in profiles suggesting TDMs (12-14). Numerous differences in the RLGS profiles of ES cells and differentiated tissues, such as kidney and brain, have been identified (13), but the DNA sequence of these genomic regions was not established, and it is not known whether these methylation differences are associated with tissue-specific gene expression.In the results presented here, RLGS was us...

Brassinazole, a synthetic chemical developed in our laboratory, is a triazole-type brassinosteroid biosynthesis inhibitor that induces dwarfism in various plant species. The target sites of brassinazole were investigated by chemical analyses of endogenous brassinosteroids (BRs) in brassinazole-treated Catharanthus roseus cells. The levels of castasterone and brassinolide in brassinazole-treated plant cells were less than 6% of the levels in untreated cells. In contrast, campestanol and 6-oxocampestanol levels were increased, and levels of BR intermediates with hydroxy groups on the side chains were reduced, suggesting that brassinazole treatment reduced BR levels by inhibiting the hydroxylation of the C-22 position. DWF4, which is an Arabidopsis thaliana cytochrome P450 isolated as a putative steroid 22-hydroxylase, was expressed in Escherichia coli, and the binding affinity of brassinazole and its derivatives to the recombinant DWF4 were analyzed. Among several triazole derivatives, brassinazole had both the highest binding affinity to DWF4 and the highest growth inhibitory activity. The binding affinity and the activity for inhibiting hypocotyl growth were well correlated among the derivatives. In brassinazole-treated A. thaliana, the CPD gene involved in BR biosynthesis was induced within 3 h, most likely because of feedback activation caused by the reduced levels of active BRs. These results indicate that brassinazole inhibits the hydroxylation of the C-22 position of the side chain in BRs by direct binding to DWF4 and that DWF4 catalyzes this hydroxylation reaction.

Many CpG islands have tissue-dependent and differentially methylated regions (T-DMRs) in normal cells and tissues. To elucidate how DNA methyltransferases (Dnmts) participate in methylation of the genomic components, we investigated the genome-wide DNA methylation pattern of the T-DMRs with Dnmt1-, Dnmt3b−/− ES cells, the same 236 spots also emerged, and no additional spots appeared differentially. Therefore, Dnmt1 and Dnmt3a/3b share targets in CpG islands. Cloning and virtual image RLGS revealed that 81% of the RLGS spots were associated with genes, and 62% of the loci were in CpG islands. By contrast to the previous reports that demethylation at repeated sequences was severe in Dnmt1 −/− cells compared with Dnmt3a Dnmt3b−/− cells, a complete loss of methylation was observed at RLGS loci in Dnmt3a Dnmt3b−/− cells, whereas methylation levels only decreased to 16% to 48% in the Dnmt1 −/− cells. We concluded that there are CpG islands with T-DMR as targets shared by Dnmt1 and Dnmt3a/3b and that each Dnmt has target preferences depending on the genomic components.