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 the rat for developmental changes in methylation of cytosine at CpG sequences as a correlate of the onset of GFAP mRNA expression and for the effect of methylation on GFAP promoter activity. The methylation of nine CpG sites in the GFAP promoter and ten sites in exon 1 was analyzed in F344 rats by a quantitative application of ligation-mediated polymerase chain reaction. Whole rat brain poly(A)+ RNA showed an exponential increase of GFAP mRNA after embryo day 14 that reached stable adult levels by postnatal day 10. During development, only the seven CpG sites in the far-upstream promoter showed large changes in methylation; these sites constitute the brain-specific domain of methylation described in adult rats (Teter et al: J Neurosci Res 39:680, 1994). These seven CpG sites showed a cycle of demethylation during the onset of GFAP transcription in the embryo (between embryonic day 14 and postnatal day 10) followed by remethylation at later postnatal ages when GFAP mRNA remains prevalent. The minimum levels of methylation across these CpG sites displayed a gradient with the lowest minima at the 3' sites. This demethylation/remethylation cycle is a novel phenomenon in DNA methylation during perinatal development. The demethylation/remethylation cycle during development was also shown by the opposite-strand cytosines. Two cytosines in this region that are conserved in rat and mouse also undergo the same demethylation/remethylation cycle in the mouse GFAP gene during development, implying evolutionary conservation and functional significance. As a further test of functional significance, a Luciferase reporter gene assay was evaluated in primary cultured astrocytes; the activity of the GFAP promoter was reduced when it was methylated at one or all CpG sites. Therefore, the GFAP promoter may be activated in rodent development by transient demethylation of a conserved brain-specific methylation domain.