METHYLATION OF THE GLIAL FIBRILLARY ACIDIC PROTEIN GENE SHOWS NOVEL BIPHASIC CHANGES DURING BRAIN-DEVELOPMENT

The gene for glial fibrillary acidic protein (GFAP) was analyzed in th e rat for developmental changes in methylation of cytosine at CpG sequ ences as a correlate of the onset of GFAP mRNA expression and for the effect of methylation on GFAP promoter activity. The methylation of ni ne CpG sites in the GFAP promoter and ten sites in exon 1 was analyzed in F344 rats by a quantitative application of ligation-mediated polym erase chain reaction. Whole rat brain poly(A)+ RNA showed an exponenti al increase of GFAP mRNA after embryo day 14 that reached stable adult levels by postnatal day 10. During development, only the seven CpG si tes in the far-upstream promoter showed large changes in methylation; these sites constitute the brain-specific domain of methylation descri bed in adult rats (Teter et al: J Neurosci Res 39:680, 1994). These se ven CpG sites showed a cycle of demethylation during the onset of GFAP transcription in the embryo (between embryonic day 14 and postnatal d ay 10) followed by remethylation at later postnatal ages when GFAP mRN A remains prevalent. The minimum levels of methylation across these Cp G sites displayed a gradient with the lowest minima at the 3' sites. T his demethylation/remethylation cycle is a novel phenomenon in DNA met hylation during perinatal development. The demethylation/remethylation cycle during development was also shown by the opposite-strand cytosi nes. Two cytosines in this region that are conserved in rat and mouse also undergo the same demethylation/remethylation cycle in the mouse G FAP gene during development, implying evolutionary conservation and fu nctional significance. As a further test of functional significance, a Luciferase reporter gene assay was evaluated in primary cultured astr ocytes; the activity of the GFAP promoter was reduced when it was meth ylated at one or all CpG sites. Therefore, the GFAP promoter may be ac tivated in rodent development by transient demethylation of a conserve d brain-specific methylation domain. (C) 1996 Wiley-Liss, Inc.