Methylation of the rat glial fibrillary acidic protein gene shows tissue‐specific domains

Abstract

The gene for glial fibrillary acidic protein (GFAP) was compared for CpG sites that are potential locations of methylated cytosine (mC). GFAP 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 mC at 9 CpG sites in the promoter and 10 sites in exon 1 was analyzed in F344 male rats by a quantitative application of ligation-mediated polymerase chain reaction (LMPCR). CpG sites with varying mC in different tissues were found in the GFAP promoter and in a CpG island in exon 1. In the brain, the promoter had about 40% less mC than in testis and liver. The degree of methylation varied strikingly between adjacent sites within and between tissues. Testis GFAP exon 1 had a gradient of mC from 5' to 3' across the exon that was absent in liver, brain, and cultured neurons and astrocytes. Among brain regions, the hippocampus had 10-40% less mC at 12 CpG sites than in hypothalamus; the other sites (7/19) showed smaller differences between these brain regions. In DNA from primary cultures, astrocytes had slightly less mC than neurons at all sites. Because neuron-rich hippocampal subregions and primary neurons cultures had less methylation than nonneural tissues, we hypothesize that neuroectodermal derivatives tend to be less methylated, whether or not GFAP is expressed. Four domains of methylated CpG 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 hypomethylated 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.

Methylation of CpG Island Transcription Factor Binding Sites Is Unnecessary for Aberrant Silencing of the Human MGMT Gene

“…Restriction enzyme digestion in combination with LMPCR allows for more sensitive detection of low levels of methylation, but, like Southern blot analysis, can only study methylation at restriction enzyme recognition sequences. Analysis by LMPCR genomic sequencing is not limited to restriction enzyme recognition sequences, but the data derived are less reproducible, especially at highly methylated sites (39). Nonetheless, the data presented here using all three techniques are in good agreement internally, and with that previously published (29).…”

“…5-Methylcytosines appear as gaps in the sequence ladder generated by amplification of products of the sequencing reactions, with the decrease in intensity corresponding directly to the degree of methylation of the nucleotide in the population (39). Representative autoradiographs from these studies are presented in Fig.…”

Epigenetic Regulation of Neural Gene Expression and Neuronal Function

“…Maintaining proper control of gene expression is necessary for these transitions. Teter et al (27) and Takizawa et al (28) have shown that developmental demethylation of the GFAP gene promoter is correlated with the activation of GFAP gene transcription during astrogliogenesis. Furthermore, methylation of the CpG within the STAT3 binding site can block the association of activated STAT3 with the GFAP promoter, consistent with the model that DNA methylation represses GFAP expression (28).…”

DNA Modifications and Neurological Disorders

“…For example, changes in DNA methylation states are concomitant with fate choices of NPCs during embryonic cortical development [68]. In this scenario, glial fibrillary acidic protein (GFAP), a well-known astrocytic marker, is highly expressed during development of the astrocyte.…”