METHYLATION OF THE RAT GLIAL FIBRILLARY ACIDIC PROTEIN GENE SHOWS TISSUE-SPECIFIC DOMAINS

The gene for glial fibrillary acidic protein (GFAP) was compared for C pG sites that are potential locations of methylated cytosine (C-m). GF AP sequences in the 5'-upstream promoter and in exon 1 of rat, mouse, and human showed extensive similarity in the locations of CpG sites in the promoter and in exon 1, implying conservation, The methylation of C-m at 9 CPG sites in the promoter and 10 sites in exon 1 was analyze d in F344 male rats by a quantitative application of ligation-mediated polymerase chain reaction (LMPCR). CpG sites with varying C-m in diff erent tissues were found in the GFAP promoter and in a CpG island in e xon 1, In the brain, the promoter had about 40 % less C-m than in test is and liver, The degree of methylation varied strikingly between adja cent sites within and between tissues, Testis GFAP exon 1 had a gradie nt of C-m from 5' to 3' across the exon that was absent in liver, brai n, and cultured neurons and astrocytes. Among brain regions, the hippo campus had 10-40 % less C-m at 12 CpG sites than in hypothalamus; the other sites (7/19) showed smaller differences between these brain regi ons, In DNA from primary cultures, astrocytes had slightly less C-m th an neurons at all sites, Because neuron-rich hippocampal subregions an d primary neuron cultures had less methylation than nonneural tissues, we hypothesize that neuroectodermal derivatives tend to be less methy lated, whether or not GFAP is expressed, Four domains of methylated Cp G sites are proposed on the basis of tissue and cell-type distribution : I) a constitutively methylated domain in the mid-upstream promoter; II) a testis-specific gradient of methylation in exon 1; III) a hypome thylated domain found in neuroectodermal derivatives; and IV) subsets of sites in the promoter and in exon 1 that have the least methylation in astrocytes, and therefore may be astrocyte-specific domains. (C) 1 994 Wiley-Liss, Inc.