Epigenetic regulation of glial fibrillary acidic protein by DNA methylation in human malignant gliomas

Abstract

Glial fibrillary acidic protein (GFAP) is an intermediate filament expressed in glial cells that stabilizes and maintains the cytoskeleton of normal astrocytes. In glial tumors, GFAP expression is frequently lost with increasing grade of malignancy, suggesting that GFAP is important for maintaining glial cell morphology or regulating astrocytoma cell growth. Most permanent human glioma cell lines are GFAP negative by immunocytochemistry. Given that the GFAP gene is not mutated in human glioma specimens or glioma cell lines, we considered epigenetic mechanisms, such as promoter methylation, as a cause of silencing of GFAP in these tumors. In this study, we treated known GFAP-negative glioma cell lines with 5-aza-2'-deoxycytidine to examine GFAP promoter hypermethylation. Additionally, we performed bisulfite sequencing on primary glioma samples and glioma cell lines and showed an inverse relationship between GFAP promoter methylation status and GFAP expression. Using a gene reporter assay with the GFAP promoter cloned upstream of a luciferase gene, we showed that methylation of the GFAP promoter downregulates the expression of the luciferase gene. Our results suggest that epigenetic silencing of the GFAP gene through DNA methylation of its promoter region may be one mechanism by which GFAP is downregulated in human gliomas and glioma cell lines.

Fig. 1.

Analysis of endogenous GFAP expression in glioma cell lines. qRT–PCR for GFAP in a panel of glioma cell lines and a HeLa-negative control. Relative expression is plotted on a log scale and demonstrates that the GFAP-positive cell line U251 has several-fold higher expression of GFAP when compared with GFAP-negative glioma cell lines and the known GFAP-negative control HeLa cell line.

Fig. 2.

5-aza-dC treatment of GFAP-negative glioma cell lines restores GFAP expression. Glioma cell lines identified as negative for GFAP expression were treated with 5-aza-dC and analyzed by qRT–PCR for GFAP expression. Following treatment, all cell lines were observed to have increased expression of GFAP with U87 and T98 glioma cells exhibiting the highest levels of re-expression.

Fig. 3.

GFAP expression is induced in GFAP-negative cell lines following 5-aza-dC treatment. (A) Immunofluorescence of GFAP-positive U251 glioma cell line with anti-GFAP (green) and counterstained with DAPI nuclear stain (blue). (B) Cell lines previously identified as being negative for GFAP expression were either untreated (left panels) or treated with 5-aza-dC (right panels) and immunostained with anti-GFAP as above. Cells that had been treated with 5-aza-dC for 72 hours demonstrated positive immunostaining for GFAP, which was not present in the untreated cells. GFAP expression was quantified by enumerating positive cells in 5 random fields of view under low magnification, and the data are represented as a percentage of cells demonstrating GFAP-positive staining. DAPI = 4',6-diamidino-2-phenylindole.

Fig. 4.

(A) Bisulfite sequencing of GFAP promoter in glioma cell lines and (B) 10 primary patient GBMs (A–J). The promoter region, first exon, and transcription start site (arrow) are depicted. The vertical black ticks represent CG dinucleotides present within the promoter region of the GFAP gene. Star denotes the CpG site that corresponds to the STAT3 binding site identified by Takizawa et al. The horizontal grey bar depicts the region of the GFAP promoter used in promoter studies. The horizontal black bar denotes the region sequenced and expanded below. Each circle represents a single CG site. Open circles denote the unmethylated sites, and filled circles represent the methylated sites. Each row represents replicate data for each respective sample.

Fig. 5.

Immunohistochemistry of primary GBMs demonstrates an inverse relationship between expression and GFAP promoter methylation status. Immunohistochemical staining for GFAP shows abundant staining in normal brain control and in primary GBM samples C, E–H, and J. In contrast, GBM samples A, D, and I stain negative for the GFAP. GBM B demonstrates a weak amount of GFAP staining. As shown in the chart at the bottom, the high GFAP expression in GBM C, GBM E–H, and GBM J correlates with low GFAP promoter methylation in contrast to the GFAP-negative primary tumors that had high GFAP promoter methylation (with the exception of GBM I, see text).

Fig. 6.

In vitro methylation assay of the GFAP promoter demonstrates that GFAP expression is controlled by methylation. U251 glioma cells were cotransfected with luciferase constructs under the control of the GFAP promoter and a control renilla expression construct that was used to normalize the samples. As a negative control, the empty vector lacking an upstream promoter element was cotransfected with the renilla construct. In addition, the vector containing an upstream CMV promoter was used as a positive control in this study. The samples in which the GFAP promoter was left untreated by an in vitro methylation reaction prior to ligation into the luciferase construct demonstrated robust luciferase expression in contrast to the samples in which the GFAP promoter had been incubated with the M.SssI methyltransferase that demonstrated completely abrogated expression.