Accessibility to tissue‐specific genes from methylation profiles of mouse brain genomic DNA

The DNA methylation status of a large number of genomic loci is visualized simultaneously and quantitatively as two‐dimensional gel spots in the newly developed restriction landmark genomic scanning with a methylation‐sensitive restriction enzyme (RLGS‐M). Here, we demonstrate that RLGS‐M using NotI as a methylation‐sensitive enzyme could also scan gene loci of mammalian genomes, since almost all of the NotI loci corresponding to randomly chosen RLGS‐M spots were located near or in transcriptional units (6 out of 7 NotI‐linking clones) when mouse brain genomic DNA was used. This supports the previous prediction that most NotI sites are located in CpG islands (Lindsay and Bird, Nature 1987, 327, 336–338). Furthermore, beginning with RLGS‐M spots we examined how to approach their corresponding RNA messages, whose expression may be associated with methylation. We compared RLGS‐M patterns among various developmental stages of the mouse brain from embryonic day 9.5 to postnatal 8 weeks or among in vitro cell lines, and detected alterations of RLGS‐M spots which were due to methylation of NotI sites. Two experiments using NotI‐linking clones or polymerase chain reaction (PCR) were carried out to approach to their corresponding RNA messages. Consequently, we isolated two PCR‐amplified clones (# 15 and # 91) which corresponded to methylatable loci and gave positive signals to mRNA from the adult brain. Furthermore, we identified two NotI‐linking clones (C211 and C198) whose corresponding NotI loci localized near or at transcriptional units and were methylated in cell lines. Thus, RLGS‐M is expected to be widely applicable for the isolation of tissue‐specific genes whose genomic loci are associated with DNA methylation.