Condorelli D F, Nicoletti V G, Barresi V, Caruso A, Conticello S, de Vellis J, Giuffrida Stella A M
Institute of Biochemistry, Faculty of Medicine, University of Catania, Italy.
J Neurosci Res. 1994 Dec 15;39(6):694-707. doi: 10.1002/jnr.490390610.
The glial fibrillary acidic protein (GFAP) is an intermediate filament protein, specific of the cytoskeleton of astrocytes in the central nervous system. In the present work, as a preliminary step to the study of glial-specific gene expression, we cloned the rat GFAP gene, and we report the sequence of 1.9 kb of the 5' flanking region, exon 1, and the majority of the first intron. By digestion with methylation-sensitive restriction enzymes followed by Southern blot analysis, the methylation status of various CpG sites was examined in this genomic segment. We tested whether structural modification of the GFAP gene, such as DNA methylation, could be related to its tissue-specific transcriptional activity. Therefore, we compared a GFAP-expressing cell population (primary culture of astroglial cells), a mixed population of GFAP-expressing and -nonexpressing cells (adult rat cerebral hemispheres), and a GFAP-nonexpressing tissue (liver). In the 5' flanking region we identified a CpG site at position -1176 whose level of methylation is inversely correlated to GFAP expression. In primary cultured astrocytes, 75% of the GFAP gene alleles were demethylated at this site, while the corresponding value obtained for the cerebral hemispheres was 45%, and for liver only 9%. On the basis of the sequence data, a CpG-rich region (putative CpG island) was identified extending from -38 to +347 and overlapping 80% of the first exon. HhaI and HpaII sites located in the putative CpG island showed a relatively high level of methylation in all the cell populations examined, and did not show any clear correlation with the level of GFAP gene expression or with the methylation status of the -1176 site. Further in vivo developmental studies and in vitro differentiation studies are necessary to better understand the functional differences of the various methylatable CpG sites in the 5' end of the GFAP gene.
胶质纤维酸性蛋白（GFAP）是一种中间丝蛋白，是中枢神经系统星形胶质细胞细胞骨架所特有的。在本研究中，作为研究胶质细胞特异性基因表达的初步步骤，我们克隆了大鼠GFAP基因，并报告了其5'侧翼区1.9 kb、外显子1以及大部分第一内含子的序列。通过用甲基化敏感限制性内切酶消化，随后进行Southern印迹分析，检测了该基因组片段中各个CpG位点的甲基化状态。我们测试了GFAP基因的结构修饰（如DNA甲基化）是否与其组织特异性转录活性相关。因此，我们比较了表达GFAP的细胞群体（星形胶质细胞原代培养物）、表达和不表达GFAP的细胞混合群体（成年大鼠脑半球）以及不表达GFAP的组织（肝脏）。在5'侧翼区，我们在-1176位置鉴定出一个CpG位点，其甲基化水平与GFAP表达呈负相关。在原代培养的星形胶质细胞中，该位点75%的GFAP基因等位基因去甲基化，而脑半球的相应值为45%，肝脏仅为9%。根据序列数据，鉴定出一个富含CpG的区域（假定的CpG岛），从-38延伸至+347，与第一外显子的80%重叠。位于假定CpG岛内的HhaI和HpaII位点在所有检测的细胞群体中显示出相对较高的甲基化水平，并且与GFAP基因表达水平或-1176位点的甲基化状态没有明显相关性。需要进一步的体内发育研究和体外分化研究，以更好地了解GFAP基因5'端各种可甲基化CpG位点的功能差异。
